To investigate the factors that contribute to biofilm degradation, we performed an enzyme treatment on the formed biofilm. Enzymatic solutions with a final concentration of 5 μg/mL of proteinase K (Nacalai Tesque), α-amylase (Wako Pure Chemical Industries Co. Ltd.), or lipase (Nacalai Tesque) in Tris-HCI buffer pH 7.0 were prepared. Each enzymatic solution was then added to wells containing biofilm, as a single or combination enzyme mixture and incubated under static anaerobic conditions at 37°C for 24 h. Biofilm measurements were performed as previously described. Biofilm without this treatment was used as a control.
α amylase
Manufactured by Fujifilm
Sourced in Japan
α-amylase is an enzyme that catalyzes the hydrolysis of starch into simpler sugars. It is a widely used laboratory reagent for the analysis and processing of carbohydrates.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Nacalai Tesque (1 mentions)
Sodium hydroxide (naoh), by Nacalai Tesque (1 mentions)
Disodium hydrogen phosphate, by Nacalai Tesque (1 mentions)
Soluble starch, by Fujifilm (1 mentions)
3 5 dinitrosalicylic acid, by Merck Group (1 mentions)
Potassium dihydrogen phosphate, by Nacalai Tesque (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Lysozyme, by Fujifilm (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Catalase, by Fujifilm (1 mentions)
Mucin, by Fujifilm (1 mentions)
Acetaldehyde, by Fujifilm (1 mentions)
4 hydroxy 2 2 6 6 tetramethylpiperidine n oxyl tempol, by Merck Group (1 mentions)
Collagenase, by Fujifilm (1 mentions)
4 protocols using α amylase
1
Enzymatic Biofilm Degradation Assay
2
Starch Hydrolysis by α-Amylase
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Sodium hydroxide, potassium sodium tartrate, disodium hydrogenphosphate, and potassium dihydrogenphosphate were obtained from Nacalai Tesque, Kyoto, Japan. The 3,5-dinitrosalicylic acid was acquired from Sigma-Aldrich, St. Louis, MO, USA. α-amylase (EC 3.2.1.1, 20 U mg−1, from Bacillus subtilis) and soluble starch (solubility 20 mg mL−1 at 25 °C, pH 4.0–6.0, from potato) were purchased from Wako, Osaka, Japan. All chemicals were of analytical-reagent grade, and were used as received
Starch stock solution (10 mg mL−1) and α-amylase stock solution (1 mg mL−1) were prepared by dissolving starch and α-amylase powders in 10 mM phosphate buffer (pH 6.90), respectively, and stored at 4 °C. The DNS reagent was prepared by dissolving 0.250 g 3,5-dinitrosalicylic acid and 75.0 g potassium sodium tartrate in 50 mL, 2 M NaOH solution, and diluted with de-ionized (DI) water to total volume 250 mL. Buffers and solutions were prepared with deionized water with conductivity < 1 μS cm−1.
Starch stock solution (10 mg mL−1) and α-amylase stock solution (1 mg mL−1) were prepared by dissolving starch and α-amylase powders in 10 mM phosphate buffer (pH 6.90), respectively, and stored at 4 °C. The DNS reagent was prepared by dissolving 0.250 g 3,5-dinitrosalicylic acid and 75.0 g potassium sodium tartrate in 50 mL, 2 M NaOH solution, and diluted with de-ionized (DI) water to total volume 250 mL. Buffers and solutions were prepared with deionized water with conductivity < 1 μS cm−1.
3
Antioxidant Activity Assay Protocol
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Reagents were purchased as follows: CA was from Tokyo Chemical Industries (Tokyo, Japan). 4-hydroxy-2,2,6,6,-tetramethylpiperidine N-oxyl (TEMPOL) and HPX were from Sigma-Aldrich (St. Louis, MO, USA). DMPO and XOD were from Labotec (Tokyo, Japan). Bovine serum albumin, α-amylase, lysozyme, mucin, catalase, DMSO, and acetaldehyde were from Wako Pure Chemicals Industries (Osaka, Japan). All other reagents used were analytical grade.
4
Evaluating Inhibitor Effects on Meat Samples
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To evaluate the effects of inhibitors of the MiVET present in meat samples, three different homogenate preparation conditions were compared as follows: (i) 60-s gentle manual homogenization (recovery of rinse solution from chicken meat surface), (ii) 30-s Stomacher homogenization, and (iii) and 60-s Stomacher homogenization. An Exnizer 400 homogenizer (Organo Co. Ltd., Tokyo, Japan) was used at 230 rpm/min. Approximately 50 mL of each homogenate was transferred to a 50-mL screw-capped tube, which was centrifuged at 20,000×g for 5 min. Approximately 40 mL of each supernatant was collected into new 50-mL screw-capped tubes, and 200 µL of the supernatant was used for conventional RNA extraction. The remaining supernatant was added to 50 μL of the IMBs to bind virions, to which 100 mg of α-amylase (Fujifilm Wako Pure Chemical Co. Ltd., Osaka, Japan) and 1 mL of 0.1% collagenase (Fujifilm Wako Pure Chemical) were added. The mixture was incubated at 35 °C for 30 min at 15-20 rpm/min in a rotator. All spike tests for the comparison of the three different homogenate preparation conditions were performed in duplicate each by using the H3N2 and H4N2 AIV subtypes.
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