GML quantified in human and bovine milk samples and commercial formula

Four human milk, 6 bovine whole milk from cows, and 2 store-purchased whole bovine milk samples were quantified for GML (Fig. 1). All milk samples had been pasteurized. Whether compared to whole bovine milk obtained from individual cows or to whole bovine milk purchased from a local grocery store, the four tested human milk samples had approximately 20 times more GML than the 8 bovine samples. There was minimal variation in GML concentrations in the human milk samples. Commercial infant formula (Similac® Advance) contained no detectable GML.

Figure 1 Glycerol monolaurate (GML) concentrations in 6 individual pasteurized whole bovine, 2 whole bovine pasteurized samples purchased from a local grocery store, and 4 pasteurized whole human milk samples. Data are recorded as mean ± SD. Student’s t test was used to assess differences in means. P ≪ 0.001 indicates mean differences much greater than p < 0.001. Full size image

Effect of whole human versus bovine milk and formula on growth of bacteria

We tested the human milk, bovine milk samples, and the commercial infant formula sample for ability to inhibit the growth of selected Gram-positive aerobes S. aureus (a potential gastrointestinal pathogen known for its roles in food poisoning31 and enterocolitis32,33; Fig. 2A) and B. subtilis (highly susceptible to killing by GML34 and essentially a positive control; Fig. 2B); we tested human and bovine milk for effect on the Gram-positive anaerobe C. perfringens (anaerobe; potential gastrointestinal pathogen and microflora; Fig. 2C); and we tested human and bovine milk and commercial infant formula for effect on the Gram-negative Escherichia coli (potential gastrointestinal and urinary tract pathogen, microflora, representative of Enterobacteriaceae; Fig. 2D). For all three Gram-positive bacteria, the 4 human milk samples comparably inhibited the growth of the strains compared to the bovine milk samples and commercial formula. B. subtilis was more susceptible to killing by the human milk samples than either of the other two organisms; we previously observed the same differential susceptibility when we studied the effects of purified GML on Gram-positive bacteria21,34. Both aerobes and obligate anaerobes were significantly inhibited by human milk samples. There were only minimal differences in inhibitory activity among the human milk samples. The Gram-negative organism, Escherichia coli, which is clearly both a commensal and one of the leading causes of gastrointestinal and urinary tract infections worldwide, was likewise inhibited by human milk but not whole bovine milk or infant formula. It is likely that the effect on E. coli, and likely on other organisms depended on synergy among GML and other antimicrobial compounds in human milk; E. coli is typically not susceptible to GML alone, even at 3000 µg/ml35.

Figure 2 Effect of whole pasteurized human milk and bovine milk samples, and commercial infant formula on growth of (A) S. aureus, (B) B. subtilis, (C) C. perfringens, and (D) E. coli. Log colony-forming units (CFUs)/ml are shown for each treatment. Starting inoculum was 1–2 × 105/ml. Samples were cultured to stationary phase for 24 hours with aeration (200 RPM; A,B,D) and stationary in an anaerobic chamber (C). Data represent means ± SD. Student’s t test was used to determine significant differences in means. P ≪ 0.001 indicates mean differences much greater than p < 0.001. Full size image

The stationary phases of all tested organisms, for comparison to milk and formula, in excellent growth medium (Todd Hewitt broth, Difco Laboratories, Detroit, MI) were: S. aureus MN8 7 × 109/ml; B. subtilis 1 × 109/ml; C. perfringens 5 × 108/ml; and E. coli 2 × 109/ml. Thus, the stationary phases of these organisms were generally 0.5–1 log higher in Todd Hewitt medium than whole bovine milk and infant formula.

We next tested the effect of whole human and bovine milk and infant formula on the growth of an Enterococcus faecalis strain, essentially a negative control for inhibitory activity, (Fig. 3) obtained from a vaginal swab from a woman with pure culture22 of this organism. The strain produces reutericyclin, which may be an analogue of GML22. The growth of this organism was not inhibited by human or bovine milk, or infant formula. The stationary phase of growth for this organism in excellent growth medium (Todd Hewitt) was 5.0 × 108/ml. Based on the data obtained, the failure to inhibit the growth of Enterococcus faecalis by the milk sample provides evidence that GML in human milk is contributing to the antimicrobial activity against some but not all bacteria, and additionally shows that human milk can support the growth of some bacteria. This demonstrates that human milk is not nutritionally-deficient, but instead is actively antimicrobial.

Figure 3 Effect of whole human and bovine milk and commercial infant formula on the growth of a reutericyclin-producing Enterococcus faecalis. Log colony-forming units (CFU)/ml are shown for each treatment. Starting inoculum was 105/ml. Samples were cultured with shaking at 200 RPM for 24 hours. Data represent mean ± SD. Student’s t test was used to determine significant differences in means; NS = Not Significant. Full size image

Effect of removal of GML and other lipids by ethanol extraction

We treated two human milk samples with 80% ethanol to precipitate molecules >10,000 molecular weight and leave GML and other soluble lipophilic molecules in the ethanol fraction. The precipitates were resolubilized with distilled water. We then tested the GML-free milk samples, containing 0.03 M glucose, an amount below the standard amount present in human milk in the form of lactose36. When GML and other lipophilic molecules were removed from the whole human milk samples, the antimicrobial activity against S. aureus was also removed (Fig. 4). We then added back 500 or 3000 µg/ml, amounts of highly-pure, food-grade GML that has been used by us in multiple studies on humans17,25; the 3000 µg/ml amount was designed in this experiment to overcome GML reduction in activity by human albumin29. Antimicrobial activity was restored by addition of 3000 µg/ml (Fig. 4) but not by addition of 500 µg/ml (data not shown). The fact that 3000 µg/ml GML add-back was more strongly bactericidal after ethanol extraction of human milk, suggests that additional nutrients besides glucose or antimicrobial factors were removed by the extraction process.

Figure 4 Effect of GML removal from two whole human milk samples (human 1, human 2) on growth of Staphylococcus aureus MN8, and effect of re-addition of 3000 µg/ml of GML. Extracted + GML refers to the resolubilized precipitates +3000 µg/ml GML. Log colony-forming units (CFUs)/ml are shown for each treatment. Starting bacterial inoculum was 4.9 × 104/ml. Data represent means ± SD. Student’s t test was used to determine significant differences in means. P ≪ 0.001 indicates mean differences much greater than p < 0.001. Full size image

S. aureus inhibitory activity of bovine milk after addition of GML

In Fig. 5, we examined the antimicrobial activity of bovine milk after addition of 0–5000 µg/ml of highly-pure GML. Whole bovine milk without GML addition was not antimicrobial. However, successive increases in GML amounts added resulted in antimicrobial activity, with milk containing 3000 and 5000 µg/ml having potent bactericidal activity.

Figure 5 Dose response of GML (0–5000 µg/ml) added to bovine milk samples. Log colony-forming units/ml are shown for all samples. Starting inoculum was approximately 105/ml of bacteria. Samples incubated with shaking (200 RPM) for 24 hour at 37 °C. Data represent means ± SD. Student’s t test was used to determine significant differences in means. P ≪ 0.001 indicates mean differences much greater than p < 0.001. Full size image

Anti-inflammatory properties of human milk but not bovine milk

In addition to its antimicrobial activity, GML prevents harmful inflammation initiated by pathogens on mucosal surfaces16,18,19,20,21,25. Also, human milk has been used topically to help in the treatment of atopic dermatitis in children37. We thus hypothesized that whole human milk samples, but not whole bovine milk samples, have anti-inflammatory activities against epithelial cells. This was tested on confluent layers of human squamous epithelial cells (HSECs) used only as the model cell system (Fig. 6). In this model test system, as expected the superantigen toxic shock syndrome toxin-1 (TSST-1) caused significant production (in 6 hours) of the chemokine IL-8, as only one of TSST-1 induced cytokines from HSECs38. The two representative human milk samples alone added to the HSECs did not induce production of IL-8, but the bovine samples alone caused low-level IL-8 production, but not to the extent of TSST-1. When the two separate human milk samples were added to HSECs in the presence of TSST-1, there was minimal IL-8 produced, indicating both samples were anti-inflammatory. The HSECs alone were not killed by the human or bovine milk samples. Neither bovine sample reduced IL-8 production caused by TSST-1. This experiment was repeated one additional time with similar results. The ethanol-extracted milk samples shown in Fig. 4 lost ability to reduce IL-8 production by TSST-1 (data not shown). We did not fractionate the ethanol-extracted fraction to determine which component(s) were removed that resulted in loss of activity. However, it is likely that GML and other ethanol-soluble molecules contributed to anti-inflammatory activity of human milk samples.

Figure 6 Anti-inflammatory activity of two whole human milk samples versus two whole bovine milk samples during stimulation of human squamous epithelial cells with the superantigen toxic shock syndrome toxin-1 (TSST). Data represent means ± SD of secreted chemokine IL-8, as the representative cytokine, after 6 hours incubation. Assays performed in triplicate in 96 well microtiter plates with confluent human epithelial cells + 200 µl keratinocyte serum-free medium with or without 20 µl milk samples. Student’s t test was used to determine significant differences in means. P ≪ 0.001 indicates mean differences much greater than p < 0.001; NS = Not Significant. Full size image

Human milk samples were also tested for ability to reduce IL-8 production from HSECs by the studied bacteria. These bacteria had previously been shown in our laboratory, some published and some unpublished, to stimulate IL-8 production from the HSECs over the 6 hour time period16,22,30. For all microbes, human milk reduced the IL-8 production to or near to background.

Collectively, our data suggest that human milk is both inhibitory to pathogen growth and exhibits anti-inflammatory activity with both activities in part dependent on GML. In contrast, bovine milk and commercial formula lacked or had greatly reduced GML and exhibited neither antimicrobial nor anti-inflammatory properties.