Probiotic Spores of Shouchella clausii SF174 and Displayed Bromelain Show Beneficial Additive Potential

Abstract

Probiotics have health-beneficial properties mainly due to either a direct action on the host or the modulation of the host microbiota. Health-beneficial properties have also been associated with a variety of plant-derived molecules, widely used as dietary supplements. This study explores the possibility of combining the actions of probiotics and of plant-derived molecules by developing beneficial, probiotic-carrying, heterologous molecules. To this extent, spores of SF174, a well-characterized probiotic strain of Shouchella clausii (formerly Bacillus clausii), were used to bind bromelain, a plant-derived mixture of endopeptidases with beneficial effects. Probiotic spores displaying bromelain maintained their antioxidant activity and acquired the endopeptidase activity of the heterologous molecule. The endopeptidase activity was stabilized by the interaction with the spore and largely preserved from degradation at simulated gastric conditions. Under conditions mimicking those encountered in the intestine, as well as upon spore germination, active bromelain was released from the spore surface. The in vitro results reported in this study support the idea that probiotics carrying beneficial heterologous molecules combine the health properties of the probiotic with those of the delivered molecule and pave the way for the development of a novel class of functional probiotics.

Keywords: Bacillus clausii; antioxidants; beneficial bacteria; functional probiotics; surface display.

Figure 1

Bromelain endopeptidase activity before fractionation (total units, grey bars) and after fractionation by centrifugation, with pellet (adsorbed units, orange bars) and supernatant (Free units, blue bars). (A) Adsorption reaction performed in citrate buffer at pH 4.5. (B) Adsorption reaction performed in phosphate or acetate buffer at pH 4.5. (C) Adsorption reaction performed in acetate buffer at various pH values. The data represent the mean of three independent experiments, and the error bars are the standard error of the mean. p-values were calculated by using two-tailed T-test with ns indicating p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, respectively.

Figure 1

Bromelain endopeptidase activity before fractionation (total units, grey bars) and after fractionation by centrifugation, with pellet (adsorbed units, orange bars) and supernatant (Free units, blue bars). (A) Adsorption reaction performed in citrate buffer at pH 4.5. (B) Adsorption reaction performed in phosphate or acetate buffer at pH 4.5. (C) Adsorption reaction performed in acetate buffer at various pH values. The data represent the mean of three independent experiments, and the error bars are the standard error of the mean. p-values were calculated by using two-tailed T-test with ns indicating p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, respectively.

Figure 2

Endopeptidase activity of free (blue line) and spore-adsorbed (orange line) bromelain after various times of exposure at SGF at pH 2.5. The data are reported as a percentage of the bromelain activity, considering as 100% the activity before incubation at SGF conditions. The data represent the mean of three independent experiments, and the error bars are the standard error of the mean. p-values were calculated by using a two-tailed T-test with *** and **** indicating p < 0.001 and p < 0.0001, respectively.

Figure 3

Endopeptidase activity of bromelain before fractionation (total units, grey bar) and after SIF treatments and fractionation leading to the pellet (adsorbed enzyme, orange bars) and the supernatant (free enzyme, blue bars). The data represent the mean of three independent experiments, and the error bars are the standard error of the mean. p-values were calculated by using a two-tailed T-test with ** and *** indicating p < 0.01 and p < 0.001, respectively.

Figure 4

(A) Efficiency of germination monitored by OD loss of B. subtilis SF106 (orange line) and S. clausii (blue line) spores (1 × 10⁷). (B) Efficiency of germination monitored by OD loss of S. clausii spores (1 × 10⁹ spores) adsorbed (orange line) or not (blue line) with bromelain. The arrows indicate the time of collection of samples for panel C. (C) Endopeptidase activity of bromelain at the induction of germination (T0) and at the indicated times after the induction of germination. Samples of each time point were fractionated, and fractions were independently assayed: pellet (adsorbed enzyme, orange bars) and supernatant (free enzyme, blue bars). The data represent the mean of three independent experiments, and the error bars are the standard error of the mean. p-values were calculated by using a two-tailed T-test with ns indicating p > 0.05, * p < 0.05, ** p < 0.01, and **** p < 0.0001, respectively.

Figure 5

Antioxidant activity of spores (dark green bars), bromelain (dark blue bars) and spore adsorbed bromelain (dark orange bars). Light bars report samples analyzed after SGF treatment. (A) Hydrogen peroxide and (B) free radicals scavenging activities. The data represent the mean of three independent experiments, and the error bars are the standard error of the mean. p-values were calculated by using a two-tailed T-test with ns indicating p > 0.05, * p < 0.05, *** p < 0.001, and **** p < 0.0001, respectively.

Figure 6

Endopeptidase activity of food-grade bromelain before fractionation (grey bars) or after fractionation by centrifugation, with pellet (adsorbed enzyme, orange bars) and supernatant (free enzyme, blue bars). p-values were calculated by using a two-tailed T-test with ns indicating p > 0.05 and **** p < 0.0001, respectively.