Antibacterial Effects of Monoglycerol Fatty Acid Esters and Sucrose Fatty Acid Esters on Bacillus spp.

Abstract

Fatty acid esters are food additives with strong antibacterial activity against spore-forming bacteria. The antibacterial activity of monoglycerol fatty acid esters (MGs) and sucrose fatty acid esters (SEs) with various fatty acid chain lengths was systematically investigated on four typical Bacillus species: B. cereus, B. subtilis, B. megaterium, and B. coagulans. Monoglycerol monolaurate (MG12) and monoglycerol monomyristate (MG14) showed relatively strong bactericidal effects on vegetative cells of these species among the MGs tested at both pH 6.0 and pH 8.0. Different SEs showed bactericidal effects on different species at pH 6.0, while the SEs had antibacterial effects only on B. coagulans at pH 8.0. SEs showed antibacterial effects on vegetative cells of these species, as was the case with MGs. In addition, it was found that MGs and SEs showed strong antibacterial effects on both B. cereus and B. subtilis in the logarithmic phase of growth, while the antibacterial activities of MGs persisted longer than those of SEs.

Introduction

Some bacteria, including those of the Bacillus and Clostridium genera, form spores with high tolerance to various stresses, such as heat, antibacterial agents, and drying, that are lethal to vegetative cells. Because of the high heat tolerance, bacterial spores in foods may cause spoilage of food or food poisoning since these bacterial spores survive heat sterilization, and germinate and grow during distribution or storage of the contaminated food (Brown, 2000; Carlin, 2011; Granum, 1995). The control of spore-forming bacteria is crucially important in the food industry. Some fatty acid esters are used to control sporulating bacteria in various foods. For example, sucrose fatty acid ester is added to canned cafe au lait to control the growth of B. coagulans and Geobacillus steatothermophilus, which cause flat-sour spoilage of the drink (Conley and Kabar, 1973; Nakayama et al., 1977; Nakayama and Samo, 1980; Nakayama and Shinya, 1981; Nakayama et al., 1982; Piao et al., 2006; Shearer et al., 2000). Monoglycerol, diglycerol, and polyglycerol fatty acid esters also are reported to show antibacterial activity (Kato and Shibasaki, 1975; Miyamoto and Matsushita, 1988). These fatty acid esters show different antibacterial spectra depending on the backbone of their hydrophilic groups and the lengths of their fatty acids, but the reason for these differences in spectra remains unclear (Blaszyk and Holley, 1998; Kabara et al., 1972; Kato and Shibasaki, 1975; Koga and Watanabe 1968; Miyamoto and Matsushita, 1988; Nakayama et al., 2003), since there has been no single report directly comparing the antibacterial action of fatty acid esters with different structures on different bacterial species under the same condition.

In the present study, we systematically investigated the antibacterial activity of monoglycerol fatty acid esters (MGs) and sucrose fatty acid esters (SEs) with various fatty acid chain lengths when tested at pH 6.0 and pH 8.0 against four Bacillus species. Furthermore, to elucidate the mechanisms of the antibacterial action of these fatty acid esters, effects of fatty acid esters on spore germination and growth after germination were investigated in B. cereus and B. subtilis.

Materials and Methods

Strains and spore preparation    Bacillus cereus JCM2152 and B. subtilis JCM1465 were purchased from RIKEN BioResource Center, Tsukuba, Ibaraki, Japan. B. megaterium DSM 319 was purchased from DSMZ, Braunschweig, Germany. B. coagulans No. 1180 was purchased from the Japan Canners Association, Tokyo, Japan. (No. 1180 is the number for the culture collection of the Research Laboratory of the Japan Canners Association.)

The bacterial strains were cultured with shaking (130 rpm) in 100 mL BBL™ Trypticase™ Soy Broth (TSB) medium (Becton, Dickinson & Company, Franklin Lakes, NJ, USA) for 18 h at 30°C for B. cereus JCM2152, B. subtilis JCM1465, and B. megaterium DSM 319, and at 50°C for B. coagulans No. 1180. Aliquots of the cultures then were plated on Standard Method Agar medium (Nissui, Japan) and incubated at the respective temperature for 1 to 2 weeks to produce spores. When the ratio of free spores to whole cells was >80% (as determined by phase contrast microscopy), the colonies were harvested, suspended in cold sterilized water, and centrifuged at 4°C and 6000 × g for 15 min. The pellets were washed three times with cold sterilized water, resuspended in sterilized water, and heated at 80°C for 10 min to kill all of the vegetative cells. Spore suspensions with spore concentrations of 1.0×10⁷ cells/mL (counted by hemocytometer) were stored at 4°C and used for experiments within 2 weeks.

Preparation of fatty acid ester solution    Monoglycerol fatty acid esters with C8, C10, C12, C14, C16, and C18 saturated fatty acid chains were provided by Taiyo Kagaku Co., Ltd., Japan. These compounds are referred to as MG8, MG10, MG12, MG14, MG16, and MG18, respectively. Sucrose fatty acid esters with C8, C10, C12, C14, and C16 saturated fatty acid chains were provided by Mitsubishi-Kagaku Foods Co., Japan. Sucrose stearic acid (saturated C18) ester was provided by Daiichi Kogyo, Co., Ltd., Japan. These compounds are referred to as SE8, SE10, SE12, SE14, SE16, and SE18, respectively. Each fatty acid ester was dissolved in distilled water at a concentration of 1.0 mg/mL, autoclaved at 121°C for 15 min, and stored pending use in the following experiments.

Measurement of viable cell counts    To determine viable counts, samples (100 µL each) including bacterial cells were plated on BBL™ Trypticase™ Soy Agar (TSA) medium (Becton, Dickinson & Company) after dilution with phosphate-buffered saline (PBS) if needed. After the plates were incubated at their optimal growth temperature (described above) for 48 h, viable cell counts were calculated from the number of colonies formed. For measurement of viable cell counts of samples including MG or SE, samples were 10-fold serially diluted with diluent with lecithin & polysorbate 80 “DAIGO” (LP solution, Wako Pure Chemical Industries, Ltd., Japan) to avoid the effects of fatty acid esters on the damaged bacterial cells. One hundred µL of the dilution was plated. For measurement of spore counts, samples were heated at 80°C (in a hot water bath) for 10 min before plating. Spores were considered to have germinated when spore counts decreased over time without a corresponding decrease in the total viable counts.

Antibacterial activity test    The fatty acid ester solutions were diluted with 50% Luria Broth (LB) medium (Nihon Pharmaceutical Co., Ltd, Japan) to obtain 4 mL of two-fold dilution series with concentrations ranging from 2 ng/mL to 500 ng/mL. Spore suspensions (20 µL each) were inoculated to 50% LB broth with or without fatty acid esters, and then cultivated statically at their optimal growth temperature (described above; and also at 37°C for B. coagulans No. 1180) for 48 h. Viable cell counts were measured by plating at 0 and 48 h of cultivation. In the present paper, we defined a decrease in the viable cell count to <10% of the initial count as a “bactericidal effect”, and a change in the viable cell count to ≥10% to <1000% of the initial count as a “bacteriostatic effect”. The minimum concentrations at which a bactericidal effect and a bacteriostatic effect were observed were defined as the minimum bactericidal concentration (MBC) and the minimum inhibitory concentration (MIC), respectively.

Effects of the timing of addition of fatty acid esters on growth after start of incubation of spores    The spore suspension of B. cereus JCM2152 or B. subtilis JCM1465 (20 µL) was inoculated into 2 mL of 50% LB broth in test tubes and mixed thoroughly. Viable cell and spore counts were measured after cultivation for 0, 1, 2, 3, 4, 5, 6, and 48 h. MG12 and SE16 were added to the culture of B. cereus JCM2152 to a final concentration of 16 ng/mL. MG12 or SE12 were added to the culture of B. subtilis JCM1465 to final concentrations of 16 ng/mL or 250 ng/mL, respectively. The fatty acid esters that showed the highest antibacterial activity against the test bacteria were used, and were added at MBC.

Electron microscopy B. cereus    JCM2152 spore suspension (100 µL) was dropped on an ISOPORE membrane filter (pore size 0.2 µm; Merck Millipore, Billerica, MA, USA), and spores were collected onto the filter under vacuum using a filtration bottle (NALGENE 115-mL Filter Unit, Thermo Fisher Scientific Inc., Waltham, MA, USA). Then, 100 µL of 50% LB broth was placed on the filter and incubated at 30°C for from 6 to 12 h to induce spore germination. Subsequently, the cells were washed continuously with 10 mM HEPES buffer (pH 7.0) at 4°C, preventing the cells from drying. The filter was then soaked in 10 mM HEPES buffer (pH 7.0) containing 2.5% glutaraldehyde and incubated at 4°C for 24 h. After sample fixation, cells on the collection filter were dehydrated by continuous drop-wise addition of a graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%, and 100%). Substitution was performed with the drop-wise addition of t-butyl alcohol to the filter at 30°C. Following substitution, the filters were removed from the filtration system and frozen at −20°C for 15 min. The frozen samples on filters were dried in a freeze dryer (ES-2030; Hitachi Ltd. Tokyo, Japan) at −20°C, and each filter was cut to a suitable size and affixed to the aluminum specimen holder of a scanning electron microscope (SEM; S-4300SE/N; Hitachi Ltd. Tokyo, Japan) using double-sided carbon tape for SEM observations.

The procedures for preparing samples for transmission electron microscopy (TEM) were the same up to the dehydration step. Aliquots of 20 µL dehydrated cell suspension were distributed into BEEM embedding capsules (Nissin EM Co., Japan) and the ethanol was substituted with propylene oxide (Nissin EM Co., Japan). Following propylene oxide substitution, 0.05 mL resin (Q-615; Nissin EM Co., Japan) was added and the samples were degassed in a sonicator (Branson 5200; Yamato Scientific Co., Japan) for 30 s. The samples then were infiltrated with resin in a desiccator under negative pressure at room temperature for 48 h. After resin substitution was complete, the BEEM capsules were heated in a block heater (Cool Thermo Unit CTU-N Neo; Taitec Co., Saitama, Japan) at 40°C for 24 h, and then at 70°C for 48 h to harden the resin. Ultra-thin sections (70 – 90 nm) were cut from the capsules with a diamond knife (Sumi Knife; Okenshoji Co., Tokyo, Japan) using a microtome (Ultracut-S; Leica Microsystems, Tokyo, Japan) and collected on copper mesh (Sheet Mesh 150-A; Nissin EM Co., Tokyo, Japan) for TEM examination. After drying, the mesh containing the collected sample was examined by TEM (H-7650; Hitachi Ltd. Tokyo, Japan).

Results

Effects of fatty acid esters on viability of Bacillus spp.    The antibacterial effects of the MGs and SEs on the four Bacillus species are shown in Tables 1–5.

Table 1. The MBC of the MGs and SEs on B. cereus JCM2152 spores

Ester	pH	MBC of fatty acid esters (ng/mL)
		C8	C10	C12	C14	C16	C18
MG	6	125	125	16	-	-	-
	8	-	125	16	-	-	-
SE	6	-	-	500	32	16	-
	8	-	-	-	-	-	-

-, MBC is over 500 ng/mL; MG, Monogrycerol ester; SE, Sucrose ester

Table 2. The MBC of the MGs and SEs on B. subtilis JCM1465 spores

Ester	pH	MBC of fatty acid esters (ng/mL)
		C8	C10	C12	C14	C16	C18
MG	6	500	125	16	-	-	-
	8	500	63	16	500	-	-
SE	6	-	-	250	-	-	-
	8	-	-	-	-	-	-

-, MBC is over 500 ng/mL; MG, Monogrycerol ester; SE, Sucrose ester

Table 3. The MBC (a) and MIC (b) of the MGs and SEs on B. megaterium DSM 319 spores

(a)
Ester	pH	MBC of fatty acid esters (ng/ml)
		C8	C10	C12	C14	C16	C18
MG	6	-	-	-	500	-	-
	8	-	-	-	-	-	-
SE	6	-	-	-	≤32	-	-
	8	-	-	-	-	-	-
-, MBC is over 500 ng/mL; MG, Monogrycerol ester; SE, Sucrose ester

(b)
Ester	pH	MIC of fatty acid esters (ng/ml)
		C8	C10	C12	C14	C16	C18
MG	6	250	63	≤32	≤32	-	-
	8	250	63	<32	500	-	-
SE	6	-	-	125	≤32	-	-
	8	-	-	-	-	-	-

-, MIC is over 500 ng/mL; MG, Monogrycerol ester; SE, Sucrose ester

Table 4. The MBC of the MGs and SEs on B. coagulans No. 1180 spores at 37°C

Ester	pH	MBC of fatty acid esters (ng/mL)
		C8	C10	C12	C14	C16	C18
MG	6	-	-	8	4	-	-
	8	125	16	16	4	125	-
SE	6	-	-	250	250	16	500
	8	-	250	63	63	63	250

-, MBC is over 500 ng/mL; MG, Monogrycerol ester; SE, Sucrose ester

Table 5. The MBC of the MGs and SEs on B. coagulans No. 1180 spores at 50°C

Ester	pH	MBC of fatty acid esters (ng/mL)
		C8	C10	C12	C14	C16	C18
MG	6	-	250	63	63	-	-
	8	-	-	-	250	-	-
SE	6	-	-	-	-	-	-
	8	-	-	-	250	250	63

-, MBC is over 500 ng/mL; MG, Monogrycerol ester; SE, Sucrose ester

Against B. cereus JCM2152, MG12 (MBC: 16 ng/mL) showed the strongest effect among the MGs tested at both pH 6 and 8 (Table 1). At both pH 6 and 8, the esters with longer fatty acid chain length (MG 14, 16, and 18), showed no effects on viability of the bacterium. In the case of SE, the antibacterial activity differed between pH 6 and 8. SE16 showed the strongest bactericidal effects (16 ng/mL), but at pH 8.0, none of the SEs showed bactericidal effects.

B. subtilis    JCM1465 also was most susceptible to MG12 (MBC: 16 ng/mL) among MGs tested at both pH 6 and 8 (Table 2). MG16 and 18 showed no effects on B. subtilis. Among SEs tested, only SE12 at pH 6 showed bactericidal effect on the bacterium at 250 ng/mL.

B. megaterium    DSM 319 was resistant to the bactericidal action of MGs and SEs; MG14 and SE14 showed bactericidal effects at 500 and ≤32 ng/mL, respectively, at pH 6 (Table 3(a)). Since most of the fatty acid esters tested did not show bactericidality (except for MG14 and SE14 at pH 6.0), MICs were determined(Table 3(b)). MG12 and 14 showed the strongest bacteriostatic effect on B. megaterium at ≤32 ng/mL at pH 6 and MG12 at pH 8. Among SEs tested, SE14 showed the strongest bacteriostatic action at ≤32 ng/mL at pH 6. On the other hand, none of the SEs showed bacteriostatic effects at pH 8.0.

The MBCs of the fatty acids were determined at both 37 and 50°C against B. coagulans No. 1180. At 37°C, MG14 showed the strongest bactericidal effect at 4 ng/mL at both pH 6 and 8 (Table 4). In the case of SEs, SE16 showed the strongest effect at pH 6 among SEs tested; MBC was determined to be 16 ng/mL. At pH 8, SE12, 14, and 16 showed strong bactericidal action at 63 ng/mL. As shown in Table 5, at 50°C, MG12 and 14 showed the strongest bactericidal effect at 63 ng/mL at pH 6. In contrast, only MG14 at pH 8 showed bactericidal effect at 250 ng/mL. Among SEs tested, SE16 showed the strongest effect at pH 6; MBC was determined to be 16 ng/mL. Among SEs tested at pH 8, SE18 showed the strongest bactericidal effect at 63 ng/mL. None of the SEs had bactericidal effects at pH 6.0.

Throughout these experiments, MG12 and MG14 exhibited stronger antibacterial effects on the four Bacillus strains than did the other MGs examined. On the other hand, the bactericidal effects on each strain differed between SEs with different fatty acid chains. SEs exhibited greater bactericidal effects on B. coagulans No. 1180 than on the other strains, especially at 37°C.

Washing with LP solution did not change the viable cell count in almost all the experiments (data not shown).

Effect of fatty acid ester on growth after start of incubation of spores of B. cereus and B. subtilis    Fatty acid esters were added hourly to the culture of each spore in 50% LB broth from 0 to 6 h to evaluate the effect of each fatty acid ester on germination and growth after germination. The results are shown in Fig. 1 for MG and Fig. 2 for SE.

Fig. 1.

Effects of MG12 on growth after start of incubation of spores of B. cereus and B. subtilis

Spores were incubated in 50% LB broth (●) and MG12 was added at 0(■), 1(◆), 2(▲), 3(▶), 4(◀), 5(▬), and 6(▃) h after the start of incubation. Total viable (closed symbols) and spore (open symbols) counts were determined before and immediately after the addition, and after incubation for 48 h. Total viable count is the sum of counts of vegetative cells and spores. MG12 was added to the cultures of B. cereus JCM2152 (A) and B. subtilis JCM1465 (B) at a final concentration of 16 ng/mL.

Fig. 2.

Effect s of SE on growth after start of incubation of spores of B. cereus and B. subtilis

Spores were incubated in 50% LB broth (●) and SE was added at 0(■), 1(◆), 2(▲), 3(▶), 4(◀), 5(▬), and 6(▃) h after the start of incubation. Total viable (closed symbols) and spore (open symbols) counts were determined before and immediately after the addition, and after incubation for 48 h. Total viable count is the sum of counts of vegetative cells and spores. SEs used for B. cereus JCM2152 (A) and B. subtilis JCM1465 (B) were 16 ng/mL SE16 and 250 ng/mL SE12, respectively.

In 50% LB broth free of either MG or SE, the spore counts largely decreased at 1 h of incubation though the viable counts did not decrease (Fig. 1 and Fig. 2). The spore counts further decreased until 3 to 5 h of incubation without increase in the total viable counts, indicating germination of B. cereus JCM2152 and B. subtilis JCM1465 spores continued until 3 to 5 h of incubation in the broth. Thereafter, the total viable counts increased gradually for both bacteria, indicating growth of vegetative cells.

In the case of B. cereus cultured in 50% LB broth supplemented with MG12 at 16 ng/mL (Fig. 1(A)), total viable counts decreased at 48 h when MG12 was added at 0, 1, 2, or 3 h. However, the total viable counts did not decrease to lower than one-tenth of the initial counts at 48 h, when added at 4, 5, or 6 h of incubation. On the other hand, the spore counts showed a large decrease immediately after start of incubation, and the counts did not increase in the presence of MG12. Both total viable and spore counts did not change immediately after the addition of MG12, regardless of the timing of addition of MG12.

Both total viable and spore counts of B. subtilis showed large decreases (to less than 100 CFU/mL at 48 h), regardless of the timing of addition of 16 ng/mL MG12 (Fig. 1 (B)). Although total viable counts decreased immediately after the addition of MG12 at 2, 3, 4, 5, or 6 h of incubation, the spore count did not change.

As shown in Fig. 2 (A), total viable counts of B. cereus at 48 h decreased when SE16 was added at 16 ng/mL and 0 h of incubation. On the other hand, the total viable counts were essentially the same as those of a control at 48 h when SE16 was added at 1, 2, 3, 4, 5, or 6 h of incubation. The spore counts showed large decreases at 48 h when added at 0, 1, 2, or 3 h of incubation, but not when added at 4, 5, or 6 h. Although total viable counts decreased immediately after the addition of SE16 at 4, 5, or 6 h of incubation, the spore count did not change.

Spore counts of B. subtilis at 48 h showed large decreases (to about 100 CFU/mL) regardless of the timing of addition of SE12 at 250 ng/mL (Fig. 2(B)). In contrast, total viable counts did not decrease immediately after addition of SE12 at 0, 1, or 2 h of incubation and increased to the same level as that of the control at 48 h. Although total viable counts decreased immediately after the addition of SE12 at 3 or 4 h of incubation, the viable counts increased at 48 h. When added at 5 or 6 h, total viable counts decreased immediately after the addition; the counts in these cultures either did not increase (5 h) or decreased (6 h) after incubation for 48 h.

Electron micrographs of B. cereus spores after incubation for 6 or 12h    Electron micrographs of B. cereus JCM2152 spores after incubation of for 6 or 12 h are shown in Fig. 3(A)(B) and Fig. 3(C) (D), respectively. The thickness of the cell wall was estimated to be 25 – 30 nm for the spores at 6 h of incubation. The thickness was half of that of vegetative cells (50 – 70 nm) after incubation for 12 h. The surface of the cells at 6 h showed a smooth structure, whereas that of the cells at 12 h showed a scale-like and rough structure compared to that observed at 6 h.

Fig. 3.

Electron micrographs of B. cereus spores after incubation in the absence of fatty acid esters for 6 or 12 h

The cells were observed under both SEM (A and C) and TEM (B and D) after incubation of B. cereus JCM2152 spores for 6 h (A and B) or 12 h (C and D) in 50% LB broth free of fatty acid esters. Arrows in (B) and (D) show the thickness of the cell walls, which were estimated to be 25 – 30 nm (B) and 50 – 70 nm (D).

Discussion

Here we investigated the antibacterial effects of various MGs and SEs on bacterial spores just after the start of incubation. The spore counts of B. cereus JCM2152 and B. subtilis JCM1465 in 50% LB broth just after addition of MG and SE at the concentration with an antibacterial activity decreased with increase in the incubation time, as did those of the control (Fig. 1 and Fig. 2), indicating that these fatty acid esters did not inhibit spore germination. These results are consistent with previous reports (Buńková et al., 2011; Kuwana et al., 2011).

TEM revealed that the cell wall of cells at 6 h after the start of incubation was thinner than that at 12 h. In the case of B. subtilis, when MG or SE was added at 3 to 6 h after the start of incubation, the viable cell count decreased immediately after the addition (Fig. 1(B) and Fig. 2(B)). This phenomenon indicated that the fatty acid esters act strongly on cells with thin cell wall just after germination. On the other hand, the same phenomenon was observed on B. cereus with SE (Fig. 2(A)) but not with MG (Fig. 1(A)). Although the antibacterial action of MG12 was not exhibited on B. cereus immediately after the addition, the total viable counts decreased after incubation for 48 h. This result suggests a difference in the affinity of the fatty acid esters for vegetative cells. Compared with SE, MG appeared to take a longer time to bind stably to vegetative cells of B. cereus.

Thus, the antibacterial action of MG was not exhibited immediately on germinating B. cereus. In contrast, MG caused large decreases in the B. subtilis total viable counts at 48 h when MG was added at 0 to 3 h of incubation. On the other hand, although SE decreased viability of both B. cereus and B. subtilis immediately after the addition at 3 to 6 h from the start of incubation of the spores, the total viable counts largely decreased at 48 h only when SE was added at the start of incubation. SE seemed to be inactivated by some enzyme(s) produced by the bacteria; we postulated that this activity is an esterase, given that Bacillus spp. exhibit high esterase activity (Sugimoto, et al., 1998). In contrast, it seems that MGs were stable and not inactivated by an activity produced by the bacteria. Although further detailed investigation is required, it seems likely that SE reduced total viable count immediately after induction of germination only when SE was present prior to the induction of spore germination; antibacterial activity was not seen when SE was added after the initiation of germination. Assessment of the esterase activities of various strains on MGs and SEs would help to clarify the lack of persistence of SE's antibacterial effect.

Most microorganisms have some enzymes for using fatty acids as an energy source, and obtain energy by degrading fatty acids into water and carbon gas (Sugimoto, et al., 1998). When excess amounts of fatty acids are present around microorganism cells, the cells cannot metabolize all of the fatty acids, and intermediate metabolites accumulate inside the cells. This accumulation inhibits the activities of enzymes and growth of the microorganism. This process is reported to be one of the mechanisms underlying the antibacterial effects of fatty acids and their esters (Kato and Shibasaki, 1975). The uptake speed of fatty acids partly relies on their affinity with the lipid bilayer membrane of the microorganism. Among monoglycerol short-chain fatty acid esters, the longer the fatty acid chain, the higher the hydrophobicity of the fatty acid ester. These hydrophobic fatty acid esters that have high affinity for the lipid bilayer membranes of microorganisms are well incorporated by microorganisms, and show strong antibacterial effects (Buńková et al. 2011). Fatty acid esters also may adsorb to the bacterial surface, where the esters induce damage and the leakage of intracellular substances (Kuwana et al., 2011). These facts indicate that interactions with the bacterial surface are crucial for the antibacterial effects of the fatty acid esters. Among food additives, non-ionic fatty acid esters, including MGs and SEs, have antibacterial effects at lower concentrations than cationic or anionic fatty acid esters. In this study, MG12 and MG14 showed the strongest effect (among the examined MGs) on the four Bacillus strains at both pH 6.0 and pH 8.0 (Tables 1 to 5). In addition, whereas MGs had bactericidal effects on B. cereus JCM2152, B. subtilis JCM1465, and B. coagulans No. 1180, MGs exhibited bacteriostatic effects on B. megaterium DSM 319. This distinction may reflect the high affinity of MG12 and MG14 for the surfaces of the four strains, which therefore show high adsorption of MG12 and MG14. On the other hand, different SEs showed bactericidal effects on different species. We presume that this pattern is due to the chemical characteristics of non-ionic fatty acid esters (e.g., lower critical micelle concentration than cationic or anionic fatty acid esters), thereby facilitating their absorption to bacterial surfaces.

Interestingly, SEs exhibited weak antibacterial activity against mesophiles (B. cereus JCM2152, B. subtilis JCM1465, and B. megaterium DSM 319), but strong antibacterial activity against B. coagulans No. 1180 at 37°C. Antibacterial effects of SEs on B. coagulans No. 1180 at 50°C were weaker than those at 37°C. Further studies to investigate the antibacterial mechanisms of fatty acid esters by their interaction with the bacterial membranes are planned. Proposed studies would include measuring the composition of bacterial lipid bilayers, and determining the critical micelle concentrations and hydrophilic lipophilic balance values of fatty acid esters. A deeper understanding of the antibacterial mechanism of fatty acid esters, and of the interaction between the esters and food ingredients (which interfere with the antibacterial action of the esters) will facilitate effective application of MGs and SEs as safe food additives in foods and beverages.

Acknowledgments    We thank members of Research and Development Center of MITSUBISHI-KAGAKU FOODS CO. for preparing and providing sucrose fatty acid esters with C8, C10, C12, C14, and C16 saturated fatty acid chains.

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