Adsorption of Shiga Toxin to Poly‐γ‐Glutamate Precipitated

Abstract

We screened foods containing indigestible ingredients in the ability to adsorb Shiga toxin (Stx). When 5 mg of foods and dietary fibers such as dry vegetables and inulin were mixed and incubated with 0.5 mL of Stx solution (100 ng/mL) containing 0.5% bovine serum albumin, both Stx1 and Stx2 seemed to be adsorbed by only a fermented food, natto (a traditional Japanese food prepared from steamed soybeans by the biological action of Bacillus subtilis). We purified the Stx-adsorbing substance from natto by extraction with H O, acid treatment, Proteinase K treatment, and an ion exchange chromatography. The purified substance showed an average molecular mass of about 600 kDa. We identified it as poly-γ-glutamate (PGA) by amino acid analysis of its hydrolysate and peptide analysis after its treatment with Proteinase K. Purified PGA (MW: molecular weight = about 600 kDa) was considered to adsorb both Stx1 and Stx2 when we separated adsorbed and unadsorbed Stxs (MW = about 72 kDa) by an ultrafiltration method with a centrifugal filter unit (MWCO: molecular weight cut-off = 100 K). However, PGA with the ability to adsorb Stx was an insoluble form precipitated in the filter unit during centrifugation. PGA precipitated beyond the saturated density was also confirmed to well adsorb both Stx1 and Stx2 by an equilibrated dialysis method. To the best of our knowledge, this is the 1st report on food-adsorbing Stx.

Determination of binding affinity of poly-γ-glutamate to Shiga toxin

“…Although several polymers including the globotriose moiety of Gb3 have been reported to adsorb Stx (Li, Wu, Cheng, & Lv, ; Miyagawa et al, ; Miyake et al, ; Nishikawa et al, ; Watanabe et al, ), there are few reports on foods with an ability to adsorb Stx. Previously, we reported that Stx bound to PGA precipitated beyond the saturated density, but not to soluble PGA (Goto, Tsuji, Kanemaru, & Yokoigawa, ). If chemically insolubilized PGA adsorbs Stx, the toxin in gut lumen may be excreted with the PGA into feces.…”

“…Previously, we reported that Stx bound to PGA precipitated beyond the saturated density, but not to soluble PGA (Goto, Tsuji, Kanemaru, & Yokoigawa, 2016). If chemically insolubilized PGA adsorbs Stx, the toxin in gut lumen may be excreted with the PGA into feces.…”

“…PGA was purified from natto as described previously (Goto et al, ). EAH–Sepharose TM 4B was obtained from GE healthcare, Uppsala, Sweden.…”