Effect of Lactose Monolaurate on Pathogenic and Nonpathogenic Bacteria

Abstract

The antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM for Listeria monocytogenes isolates and 0.2 to 2 mM for Mycobacterium isolates.

TEXT

Sugar esters are available commercially and are used in a variety of applications in the food, pharmaceutical, and personal care industries. They are classified as nonionic surfactants, and examples include sorbitan monostearate (polysorbate 80), sucrose palmitate, and sucrose oleate. We have recently synthesized a novel sugar ester, lactose monolaurate (LML) (12), and this research investigated the antimicrobial activity of LML and commercially available sucrose monolaurate (SML) against Gram-positive and Gram-negative bacteria.

Sugar esters have been shown to inhibit microbial growth, yet there are conflicting data on the microbes effected. Some reports show inhibition of Gram-negative bacteria (4, 8, 15), while others report inhibition of only Gram-positive bacteria (3, 9, 13). The inhibitory activity of sugar esters is dependent on the sugar, the number and type of fatty acids esterified, and the degree of esterification. Smith et al. (11) synthesized various carbohydrate esters and found that the carbohydrate moiety can markedly influence the microbial inhibitory activity of the fatty acid. The activity of sugar esters has been suggested to be antimicrobial (2), while others have suggested they have just bacteriostatic activity (10). All published studies investigating the activity of sugar esters only provide the MICs; there are no publications that determined the minimal bactericidal concentrations (MBCs) of sugar esters.

Here we investigated the antimicrobial activity of SML, which has been shown to have microbial inhibitory properties (2, 3, 4, 5, 15), and LML against Gram-positive and Gram-negative bacteria. We then determined the MBCs of LML with susceptible bacteria, four isolates of L. monocytogenes and three isolates of Mycobacterium. Scanning electron microscopy (SEM) was also conducted to identify changes in the cell surfaces after LML treatment (7).

The organisms used in these studies (Table 1) were grown to exponential phase in medium at 220 rpm at 37°C. The Listeria strains were initially grown and plated on Palcam agar with supplements (polymyxin B, acriflavine, and ceftazidime, all from Neogen, Lansing, MI) to confirm purity, and individual isolates were used for the remainder of the study and cultured in brain heart infusion (BHI) medium.

Table 1.

Growth media and microorganisms used in this study

No.	Microorganism	ATCC no./serovar	Gram reactiona	Growth medium
1	Escherichia coli H7:O157	35150	−	LB
2	Salmonella enterica serotype Typhimurium	700720	−	LB
3	Klebsiella pneumoniae subsp. pneumoniae	700721	−	LB
4	Enterococcus faecalis	700802	+	BHI
5	Streptococcus suis	89/1591	+	BHI
6	Listeria monocytogenes	EGDe	+	BHI
7	Listeria monocytogenes	FSL/J1-177	+	BHI
8	Listeria monocytogenes	FSL/N3-013	+	BHI
9	Listeria monocytogenes	FSL/N1-227	+	BHI
10	Listeria monocytogenes	FSL/R2-499	+	BHI
11	Mycobacterium smegmatis	14468	+	LB
12	Mycobacterium sp. strain JLS	NAb	+	LB
13	Mycobacterium sp. strain KMS	NA	+	LB

^a+, positive; −, negative.

^bNA, not available.

LML was synthesized as described previously (12) and purified to 98% via differential solubility of the reactants (lactose and vinyl laurate) from the product in 50% ethanol-water. SML was obtained from Sisterna (Roosendaal, The Netherlands) and diluted into 50% ethanol-water before use. The effect of each ester was tested on six different food pathogens (Table 1, no. 1 to 6) with appropriate controls. The bacteria were grown under the conditions specified in Table 1 and diluted to 10⁵ CFU/ml before the addition of esters at concentrations of 1, 0.1, 0.05, and 0.01 mg/ml. For better solubility and dispersion of LML and SML, 0.1% Tween 80 was used in the growth medium. It was observed that the addition of 0.1% Tween 80 to cultures had no marked effect on cell growth compared to that of controls (data not shown). The turbidity of samples was measured in a microtiter well plate at 595 nm after 24 h, and the experiments were replicated. A paired t test was used to compare treatments with controls at each treatment concentration. The MIC of each compound with each bacterium was determined as the lowest concentration of the compound at which there was no increase in the optical density (OD) at 595 nm after 24 h (the molecular mass of both esters is 525 g/mol [12]).

LML and SML were tested on three Gram-positive bacteria (Enterococcus faecalis, Listeria monocytogenes EGDe, Streptococcus suis) and three Gram-negative bacteria (Escherichia coli H7:O157, Klebsiella pneumoniae, Salmonella enterica serotype Typhimurium). Gram-negative bacteria did not show a change in growth in either SML or LML compared to that of the controls. SML did not show any effect on the growth of E. faecalis at any concentration; however, LML-treated cells showed a significant decrease in OD at 1.0 mg/ml which corresponds to a MIC of 2 mM (Table 2). For L. monocytogenes EGDe, both SML and LML were effective, with a MIC of each ester of 0.2 mM. Interestingly, the MIC of SML for S. suis was 20 μM, as opposed to the effect of LML, which had an MIC of 0.2 mM.

Table 2.

MICs of sucrose or lactose monolaurate against Gram-positive bacteria as determined by optical density measurements

Microorganism	MIC [mM (mg/ml)] of:
	Sucrose monolaurate	Lactose monolaurate
Enterococcus faecalis	>2 (1)	2 (1)
Streptococcus suis	<0.02 (0.01)	0.2 (0.1)
Listeria monocytogenes (EGDe)	0.2 (0.1)	0.2 (0.1)

We focused on determining the MBCs of LML with Listeria and Mycobacterium strains since preliminary data for the mycobacteria (data not shown) showed that their growth in medium was also reduced with LML (determined by plate counts) but OD measurements were difficult to obtain due to cell aggregation during growth. L. monocytogenes (Table 1, no. 6 to 10) and Mycobacterium (Table 1, no. 11 to 13) strains were grown and treated with LML as described above and in Table 1 with concentrations of 0.1 to 1 or 5 mg/ml. Samples were plated on appropriate agar after 24 h to enumerate viable cells. Each experiment was conducted in replicate, and paired t tests were used to determine significance. The MBC was defined as the minimum concentration of LML in medium that does not display any colonies on BHI agar plates after 24 h of incubation at 37°C.

The starting CFU count for each Listeria isolate in Fig. 1 was approximately 5 × 10⁵ CFU/ml. The Listeria isolates (Fig. 1A to E) were able to grow in the presence of LML at concentrations of less than 1 mg/ml LML. At concentrations of 1 mg/ml and greater, the growth of each Listeria isolate was less than that of controls. Figure 1A to E show that each Listeria isolate (A, L. monocytogenes EGDe; B, L. monocytogenes N1-227; C, L. monocytogenes R2-499; D, L. monocytogenes J1-177; E, L. monocytogenes N3-013) has an MBC of 5.7 to 9.5 mM (3 to 5 mg/ml). L. monocytogenes R2-499 (Fig. 1C) and N3-013 (Fig. 1E) appear to be the most susceptible to LML, since they show a significant growth difference from the control at 0.05 mg/ml, yet these isolates have the same MBC as the other Listeria isolates. LML MBC results for three Mycobacterium strains are shown in Fig. 1F to H (F, Mycobacterium sp. strain JLS; G, M. smegmatis; H, Mycobacterium sp. strain KMS). Each Mycobacterium strain showed an MBC of 0.1 to 1 mg/ml (0.2 to 2 mM). Mycobacterium sp. strains JLS and KMS showed decreased growth at LML concentrations of ≥0.05 mg/ml.

Fig 1.

Average counts (log CFU/ml) of five isolates of L. monocytogenes treated with 0.01, 0.05, 0.1, 1.0, 3.0, or 5.0 mg/ml LML and Mycobacterium strains treated with 0.01, 0.05, 0.1, or 1.0 mg/ml LML after 24 h. The black bars are controls, and the light bars are treatments. The error bars represent the standard deviations, and the asterisks indicate significant difference from the control. A, L. monocytogenes EGDe; B, L. monocytogenes N1-227; C, L. monocytogenes R2-499; D, L. monocytogenes J1-177; E, L. monocytogenes N3-013; F, Mycobacterium sp. strain JLS; G, M. smegmatis; H, Mycobacterium sp. strain KMS.

L. monocytogenes N3-013 (Fig. 2) and Mycobacterium sp. strain JLS (Fig. 3) were observed by SEM. The cells were grown as stated above and included 1 mg/ml LML treatment and controls. We initially used an LML treatment concentration of 5 mg/ml for L. monocytogenes, but light microscopy revealed that there were very few cells present, presumably due to cell death and/or lysis. Interestingly, the use of 1 mg/ml LML with Mycobacterium sp. strain JLS did result in visible, but not culturable, cells. The control Listeria cells are normal rod shaped, although some cocci are observed (Fig. 2A). Listeria organisms in the treated sample (Fig. 2B) are observed as cocci in chains, yet no change in cell surface was observed. The change in the morphology of Listeria from a rod to a coccus has been shown to be based on the medium type and the presence of stress (temperature or pressure) (14). Figure 3 shows SEM of Mycobacterium sp. strain JLS, and in Fig. 3B, the cell surface appears wrinkled compared to that of the control (Fig. 3A).

Fig 2.

(A) SEM of L. monocytogenes N3-013 control (magnification, ×3,500). (B) SEM of L. monocytogenes N3-013 after treatment with 1 mg/ml LML for 24 h (magnification, ×4,000).

Fig 3.

(A) SEM of M. tuberculosis JLS control (magnification, ×20,000). (B) SEM of M. tuberculosis JLS after treatment with 1 mg/ml LML for 24 h (magnification, ×15,000).

One recent study showed that that E. coli O157:H7 is affected by SML, especially in the presence of sodium hypochlorite (15). These findings are in contrast to some studies that showed that sugar esters have limited effects (0 to 25% decrease in growth) on E. coli (4, 5, 9). Commercially available sugar esters are used in foods and cosmetics as emulsifiers at concentrations of up to 10 mg/ml (1% or 20 mM) (3), which is twice as high as the MBC determined here for Listeria isolates and 10 times as high as the MBC for the mycobacteria. Our results are in agreement with other studies that investigated the effects of various esters on Gram-positive bacteria. Streptococcus mutans was shown to be inhibited by galactose and fructose laurate at a concentration of 0.5 mg/ml (13). Other studies showed that laurate esters of sucrose, glucose, maltose, galactose, or fructose inhibited the growth Streptococcus mutans and Streptococcus sobrinus at a concentration of 20 mg/ml (3), inhibited that of Bacillus species at 0.8 mg/ml, and inhibited that of Lactobacillus plantarum at 4 mg/ml (4). Yang et al. (16) showed the inhibition of Zygosaccharomyces bailii and Lactobacillus fructivorans in salad dressing with 10 mg/ml esters of sucrose laurate and sucrose palmitate. Recently Nobmann et al. (8) found that the lauric ether of methyl α-d-glucopyranoside and the lauric ester of methyl-α-d-mannopyranoside had the greatest growth-inhibitory effect, with a MIC of 0.04 mM against L. monocytogenes.

The mycobacteria tested here were more sensitive than L. monocytogenes to LML, with MBCs of between 0.2 and 2 mM. Previous research has shown that Mycobacterium rubrum is susceptible to some biosurfactants (e.g., mannoylerythritol lipids from Candida antarctica) but not to sucrose decanoate (6).

The MIC values for Listeria and Mycobacterium are within the range of the proposed critical micelle concentration (CMC) for LML based on the CMC for SML, which is 0.2 to 0.8 mM (1). The MBCs for these organisms are greater than the CMC value, suggesting that concentrations greater than the CMC are needed to affect cell growth.

From our studies it can be concluded that both SML and LML are most effective against the growth of Gram-positive bacteria. SML greatly inhibited S. suis, and both sugar esters inhibited Listeria. Since there are no data on the MBCs of sugar esters in the literature, we continued to test our novel LML on L. monocytogenes and Mycobacterium isolates. In general, for Listeria, a concentration of 1 mg/ml showed some growth inhibition but the MBC was between 3 and 5 mg/ml. The mycobacteria tested were more sensitive to LML, with MBCs between 0.1 and 1 mg/ml. The mechanism of action of these esters will be interesting to study since it has not been previously reported.