Pepsin

Fresh livers of sheep naturally infected with E. granulosus were collected from a slaughterhouse in Urumqi, Xinjiang, China. The contents of the noncalcified, noninfected, intact unicystic E. granulosus vesicles were aseptically extracted from the livers of sheep infected. The contents were transferred to sterile centrifuge tubes (CORNING), centrifuged at 1500g for 10 min to extract the supernatant, which was then treated with endotoxin removal, and filtered through a 0.22 μm membrane (Millipore) and quantified for protein concentration before being frozen at −80°C. To naturally eliminate fragments of the brood capsule from the protoscoleces sediment, it was digested with 1% pepsin (BioFroxx) and then rinsed three times with sterile PBS containing 100 U/mL penicillin‐streptomycin solution (Gibco). The sediment was stained with 0.5% eosin (BKMAMLAB) for 5 min, and the number of active protoscoleces (>90%) was counted microscopically and diluted to 10,000 protoplasts/mL in suspension with sterile PBS containing penicillin‐streptomycin solution and kept on standby.¹¹

2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTZ), 1,1-Diphenyl-2-picrylhydrazyl Free (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Nutrient Broth, Agar Powder, and Trypsin (1:250) are from Beijing Solarbio Science & Technology Co., Ltd. Pepsin (1:3000) from BioFroxx, Germany. Escherichia coli (E. coli) is used as standard quality control strain ATCC 25922 and Staphylococcus aureus (S. aureus) is used as standard quality control strain ATCC 25923. Penicillin sodium is from Shanghai yuanye Bio-Technology Co., Ltd, Lot: S15J7Y9073, mass fraction 97%. Gallic acid is from Chengdu Kelong Chemical Reagent Factory, mass fraction 99%. Gallic acid ethyl ester is from Chengdu Prefa Technology Development Co. Lot: PRF20090442, mass fraction 98%. Folin–Ciocalteu’s phenol reagent is a homemade laboratory product. Other chemical reagents are all of analytical purity grade. Methanol is chromatographically pure grade. Water is purified water.

The stability of the antifungal effect of metabolites from Lpb. plantarum LPP703 was investigated. To determine the heat sensitivity of the antifungal effect, aliquots of Lpb. plantarum LPP703 CFS were separately subjected to 60, 80, 100, and 121 °C for 2 h. The sensitivity of Lpb. plantarum LPP703 CFS to various kinds of proteases was investigated by incubating aliquots of the CFS separately with 1 mg/mL of pepsin, trypsin, protease K, and papain (BioFroxx, Einhausen, Germany). The hydrolysis reaction was performed under the optimal conditions for each protease. After 2 h, the hydrolysis was terminated by boiling at 100 °C for 5 min. The pH sensitivity was assessed by adjusting the pH value of aliquots of Lpb. plantarum LPP703 CFS to 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. The antifungal effect of all the treated Lpb. plantarum LPP703 CFS was assayed by microplate inhibition analysis, as described in Section 2.4 above.