Skim Milk Culture of Lactobacillus johnsonii SBT0309 Increases Intestinal Alkaline Phosphatase Activity and Inhibits Lipopolysaccharide-Induced Interleukin-8 Production in Intestinal Epithelial Cells

Abstract

Background/objectives: Intestinal alkaline phosphatase (IAP) is an enzyme expressed in the intestinal brush border, which may exert anti-inflammatory effects by detoxifying lipopolysaccharides (LPSs), thereby preventing metabolic disorders. Various food components have been reported to influence IAP activity. However, few studies have evaluated the effects of fermented milk on IAP activity. In this study, we aimed to investigate fermented milk with high IAP-activating capacity and investigate its effect.

Methods: We screened a skim milk culture (SC), a fermented milk model, using differentiated Caco-2 cells. We investigated the effect of SC on IAP activity and gene expression in the Drosophila midgut. Quantitative PCR and immunoblot assays were conducted to examine gene and protein levels.

Results: Among the SC samples from different lactic acid bacteria or bifidobacteria, the SC of Lactobacillus johnsonii SBT0309 (LJ0309 SC) demonstrated a particularly strong capacity to activate IAP in Caco-2 cells, demonstrated by significantly increased IAP gene expression and protein levels in Caco-2 cells. Additionally, LJ0309 SC inhibited increased secretion of IL-8 in LPS-stimulated Caco-2 cells. Finally, in Drosophila melanogaster fed LJ0309 SC, we observed an increase in both IAP activity and gene expression in the midgut.

Conclusions: LJ0309 SC increased IAP activity and gene expression in both Caco-2 cells and the Drosophila midgut, and inhibited the inflammatory response in LPS-stimulated Caco-2 cells. Although further in vivo studies are required, LJ0309 SC might help to ameliorate LPS-induced inflammation and disease via IAP activation.

Keywords: Caco-2 cells; Drosophila melanogaster; Lactobacillus johnsonii SBT0309; fermented milk; intestinal alkaline phosphatase; lipopolysaccharide; skim milk culture.

Figure 1

Screening of SC samples to increase IAP activity of Caco-2 cells. IAP activity in differentiated Caco-2 cells treated with butyrate (2 mM), lyophilized SM (5 mg/mL), or lyophilized SCs (5 mg/mL) for 7 days. SC samples were prepared with Lactobacillus (L.) delbrueckii subsp. jacobsenii, L. johnsonii, or Streptococcus thermophilus as listed in Table S2. Data are shown as mean + SD (n = 3) and analyzed using one-way ANOVA followed by Dunnett’s test (** p < 0.01, *** p < 0.001), which compared the control group with the other groups. IAP, intestinal alkaline phosphatase; SC, skim milk culture; SM, skim milk medium.

Figure 2

LJ0309 SC increases IAP gene expression and protein levels in Caco-2 cells. IAP (a) mRNA and (b) protein levels in differentiated Caco-2 cells treated with butyrate (2 mM) or lyophilized LJ0309 SC (5 mg/mL) for 7 days. IAP mRNA was analyzed 3 and 5 days after sample addition. The mRNA expression was determined relative to the mean value of the control group at the same time point. IAP and β-actin were detected using immunoblotting and the IAP/β-actin ratio was quantified using densitometry. Data are shown as mean + SD (n = 3) and analyzed using one-way ANOVA followed by Dunnett’s test (* p < 0.05, ** p < 0.01), which compared the control group with the other groups at the same time point. IAP, intestinal alkaline phosphatase; SC, skim milk culture.

Figure 3

LJ0309 SC inhibited the IL-8 secretion in LPS-stimulated Caco-2 cells. (a) IAP mRNA and (b) IL-8 secretion in differentiated Caco-2 cells treated with LPS (10 µg/mL) and lyophilized LJ0309 SC (5 mg/mL) for 7 days. (a) IAP mRNA was quantified using qPCR and (b) IL-8 levels were determined in the culture supernatant. Data are shown as mean + SD (n = 3) and analyzed using two-way ANOVA followed by Tukey–Kramer’s test (^{a, b, c} p < 0.05). LPS, lipopolysaccharide; SC, skim milk culture.

Figure 4

LJ0309 SC increased IAP gene expression and activity in the Drosophila midgut. (a) IAP activity and (b) CG5150 and CG10827 mRNA expression levels in the midgut of flies fed experimental diets containing 2.5% lyophilized SM or 2.5% lyophilized LJ0309 SC for 5 weeks. Data are shown as mean + SD [(a) n = 24, 25 (72, 75 flies), (b) n = 12, 13 (60, 65 flies)] and analyzed using Student’s t-test (** p < 0.01, *** p < 0.001). SC, skim milk culture; SM, skim milk medium.