Bacillus subtilis

PBAT (TH801T) was purchased from Xinjiang Blue Ridge Tunhe Polyester Co., Ltd (Changji, China). It has a specific gravity of 1.21 g/cm³ and a melt mass flow rate (MFR) of 5.0 g/10 min (190 °C), with a number-average molecular weight of 6.12 × 10⁴ g/mol. Reeds were collected from the shore of the lake (Baiyangdian, Baoding, China). For enzymatic degradation, the following enzymes and reagents were used: Proteinase K (≥30 units/mg) from Tritirachium album (Blirt, Gdansk, Poland), esterase (≥10 units/mg) from Bacillus subtilis (Sigma-Aldrich, Bavaria, Germany), lipase (15–25 units/mg) from Candida rugosa (Sigma-Aldrich, Bavaria, Germany), and cellulase (≥200 FPU/mL) from Trichoderma reesei (Novozymes A/S, Copenhagen, Denmark). Tris(hydroxymethyl)aminomethane-HCl (0.1 M, pH = 8.0, Sigma-Aldrich, Germany) and citric acid-sodium citrate (0.1 M, pH = 4.8, Sigma-Aldrich, Germany) were used as buffers for enzyme degradation, sodium azide (NaN₃) (Sigma Chemical Co., St. Louis, MI, USA) was used as an antifungal agent, and calcium chloride (Sigma Chemical Co., USA) was used as an activator of enzyme degradation. Other chemical reagents were obtained from Sinopharm Chemical Reagent (Shanghai, China).

Human salivary α-amylase (HSA), porcine pancreatic α-amylase (PPA), Bacillus subtilis (BSA), and Aspergillus oryzae (AOA) were purchased from Sigma Co., St. Louis. Bacillus pacificusα-amylase (BPA) was purified as recently reported by Alonazi et al. (2020), and the two larval insect α-amylases from Tribolium confusum (AOA) and Callosobruchus maculatus (CMA) were obtained, as previously described by Hivrale et al. (2011).

Folin-Ciocalteu’s phenol, potassium phosphate monobasic and dibasic, xanthine, xanthine oxidase, allopurinol, and hydrochloric acid were obtained from Sigma-Aldrich Japan K.K., Tokyo, Japan. Aluminium (III) chloride hexahydrate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium acetate, acetic acid, dibutyl hydroxuytoluene (BHT), gallic acid, and quercetin were purchased from Kanto Chemical Co. Inc., Tokyo, Japan. Acetone, potassium peroxodisulfate, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Nacalai Tesque, Inc., Kyoto, Japan. Methanol (MeOH), ethyl acetate (EtOAc), ethanol, and methanol plus were obtained from Junsei Chemical Co., Ltd., Tokyo, Japan. All of the bacteria strains, including Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Bacillus subtilis, were purchased from Sigma-Aldrich (Tokyo, Japan).

Bone allograft cubes were removed from the implanted site. After 1 and 3 days but also after 40 and 120 days the bone cubes could be manually separated from the surrounding tissue after careful preparation of the surrounding (bone) tissue by means of a (autopsy) bone saw (A1 Medical, Laurel, MS, USA). After removal of the allograft bone blocks, each allograft cube was placed centrally in Bacillus subtilis (Merck KGaA, Germany) in test agar pH 8.0 (Merck KGaA, Germany) inoculated agar plates and the test was performed as described in Section 2.4.1.

Mueller-Hinton agar (Oxoid), hydrochloric acid (HCl), sodium hydroxide Na(OH)₄, Bacillus subtilis (Merck Darmstadt, Germany, no. 64271), and Micrococcus luteus ATCC^® 10240 strain were used.

Bacillus subtilis (Merck KGaA, Germany in Test Agar pH 8.0 Merck KGaA, Germany), Staphylococcus aureus ATCC 29213 (American Type Culture Collection, LGC Standards GmbH, Wesel, Germany) and methicillin-resistant Staphylococcus aureus MRSA DSM 46320 (Leibniz Institute DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen— German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were used for antibiotic delivery and antibiotic susceptibility assays.