The Effect of Tannin-Rich Witch Hazel on Growth of Probiotic Lactobacillus plantarum

Abstract

Probiotic bacteria help maintain microbiome homeostasis and promote gut health. Maintaining the competitive advantage of the probiotics over pathogenic bacteria is a challenge, as they are part of the gut microbiome that is continuously exposed to digestive and nutritional changes and various stressors. Witch hazel that is rich in hamamelitannin (WH, whISOBAX^TM) is an inhibitor of growth and virulence of pathogenic bacteria. To test for its effect on probiotic bacteria, WH was tested on the growth and biofilm formation of a commercially available probiotic Lactobacillus plantarum PS128. As these bacteria are aerotolerant, the experiments were carried out aerobically and in nutritionally inadequate/poor (nutrient broth) or adequate/rich (MRS broth) conditions. Interestingly, despite its negative effect on the growth and biofilm formation of pathogenic bacteria such as Staphylococcus epidermidis, WH promotes the growth of the probiotic bacteria in a nutritionally inadequate environment while maintaining their growth under a nutritionally rich environment. In the absence of WH, no significant biofilm is formed on the surfaces tested (polystyrene and alginate), but in the presence of WH, biofilm formation was significantly enhanced. These results indicate that WH may thus be used to enhance the growth and survival of probiotics.

Keywords: Lactobacillus plantarum; biofilm; prebiotic; probiotic; witch hazel (Hamamelis).

Figure 1

The effect of witch hazel extracts on the growth of S. epidermidis. Bacteria were grown overnight in TSB in the presence of increasing amounts of witch hazel extracts, ethanol, or culture broth alone (control). OD₆₃₀ was determined after 24 h. Witch hazel extracts tested: WH (whISOBAX, StaphOff Biotech), CareOne extract (CareOne), and NFH (Nutritional Fundamentals for Health).

Figure 2

The effect of witch hazel extracts on biofilm formation by S. epidermidis. Bacteria were grown overnight in TSB in the presence of increasing amounts of witch hazel extracts, ethanol, or culture broth alone (control). After 24 h, unbound bacteria were removed, biofilm bacteria stained in crystal violet, stain dissolved in SDS and OD₆₃₀ were determined. Witch hazel extracts tested: WH (whISOBAX, StaphOff Biotech), CareOne extract (CareOne), and NFH (Nutritional Fundamentals for Health).

Figure 3

The effect of witch hazel on the growth of L. plantarum. (a) Bacteria were grown overnight in MRS both in the presence of increasing amounts of whISOBAX, ethanol (50%, starting solution) or culture broth alone (control). OD₆₃₀ was determined after 48 h. (b) Bacteria were grown overnight in nutrient both (Nut) in the presence of increasing amounts of whISOBAX, ethanol (50%, starting solution) or culture broth alone (control). OD₆₃₀ was determined after 48 h.

Figure 4

The effect of witch hazel on biofilm formation by L. plantarum. Bacteria were grown overnight in MRS (a) or nutrient broth (Nut) (b) in the presence of increasing amounts of whISOBAX (WH), ethanol (Eth), or medium alone (control). After 48 h, unbound bacteria were removed, bound (biofilm) bacteria were stained in crystal violet, stain was dissolved in SDS, and OD was determined. (c) Bacteria were grown with a final 1:20 dilution of WH or Eth control (see (b) above). Bound cells were resuspended in nutrient broth, and undiluted samples were streaked on MRS agar plates, which were placed in anaerobic chambers for 24 h.

Figure 4

The effect of witch hazel on biofilm formation by L. plantarum. Bacteria were grown overnight in MRS (a) or nutrient broth (Nut) (b) in the presence of increasing amounts of whISOBAX (WH), ethanol (Eth), or medium alone (control). After 48 h, unbound bacteria were removed, bound (biofilm) bacteria were stained in crystal violet, stain was dissolved in SDS, and OD was determined. (c) Bacteria were grown with a final 1:20 dilution of WH or Eth control (see (b) above). Bound cells were resuspended in nutrient broth, and undiluted samples were streaked on MRS agar plates, which were placed in anaerobic chambers for 24 h.

Figure 5

The effect of witch hazel (WH) on biofilm formation by L. plantarum on alginate beads. Bacteria were incubated with alginate beads in the presence of WH diluted 1:80 (WH 2.5) or ethanol control and the OD of adherent bacteria was determined.

Figure 6

The effect of witch hazel on anaerobic vs. aerobic growth of L. plantarum in nutrient broth. Bacteria were grown overnight in nutrient broth in the presence of increasing amounts of whISOBAX (WH) or ethanol (Eth) control. After 48 h, OD was determined.