Violet red bile agar

Escherichia coli BCRC 11,634 and Staphylococcus aureus BCRC 10,451 were purchased from the Biosources Collection and Research Center (Hsinchu, Taiwan). EDTA, sodium bicarbonate (NaHCO₃), and nisin (10⁶ IU) were obtained from Sigma Chemical Co. (Gillingham, UK). Chitosan powder was obtained from Applied Chemical Co., Ltd. (Kaohsiung, Taiwan). Bacto agar, nutrient broth (NB), nutrient agar, plate count agar (PCA), Pseudomonas isolation agar, starch ampicillin agar (SAI), thiosulphate-citrate-bile salts-sucrose, tryptic soy broth, and violet red bile agar were supplied by Becton Dickinson (Sparks, MD, USA). Polylactic acid (PLA, manufactured by Natureworks@ (4032D), Minnetonka, MA, USA) had a weight-average molecular weight (Mw) of 1.96 × 10⁴ Da, as determined by gel permeation chromatography.

Aeromonas hydrophila BCRC 13881, Escherichia coli BCRC 11634, Pseudomonas fluorescens BCRC 11028, Staphylococcus aureus BCRC 10451, and Vibrio parahaemolyticus BCRC 12863 were purchased from the Biosources Collection and Research Center (Hsinchu, Taiwan). Acetic acid, acetonitrile, dimethyl sulfoxide (DMSO), glycerol, methanol, and sodium hydroxide (NaOH) were purchased from Fluka (Garage Gmbh, Buchs, Switzerland). Sodium bicarbonate (NaHCO₃) was obtained from Sigma Chemical Co. (Gillingham, UK). Chitosan powder was obtained from Applied Chemical Co., Ltd. (Kaohsiung, Taiwan). Bacto agar, nutrient broth (NB), nutrient agar (NA), plate count agar (PCA), Pseudomonas isolation agar (PIA), starch ampicillin agar (SAI), thiosulphate-citrate -bile salts-sucrose (TCBS), tryptic soy broth (TSB), and violet red bile agar (VRBA) were supplied by Becton Dickinson (Sparks, MD, USA). Polylactic acid (PLA, manufactured by Natureworks@ (4032D), Minnetonka, MA, USA) exhibited a weight-average molecular weight (Mw) of 1.96 × 10⁴ Da, as determined by gel permeation chromatography. Poly(butyleneadipate-co-terephthalate) (PBAT, Ecoflex F blend C1200 with Mn of 24,400 g/mol) and ADR (ADR-4468, a chain extender) were purchased from BASF (Ludwigshafen, Germany). Talc powder (mean particle size: 8.8 μm, surface area: 5500 cm²/g) was purchased from Chu Shin Chemical Corp. (HsinChu, Taiwan)

Standard method [15 ] was used for bacterial growth measurement, 1 g of sample taken aseptically from the surface of the meat was put in a sterile bag and prepared in triplicate (Nasco Whirl-Pak, Fort Atkinson, WI, USA). Sample was homogenized with 9 mL sterilized 0.1% (w/v) peptone saline for 2 min using a stomacher (400, Seward Laboratory, Worthing, UK). Decimal dilutions were prepared using sterilized 0.1% (w/v) peptone saline. Total aerobic plate count, lactic acid bacteria and total coliform were enumerated using plate count agar, de Man-Rogosa-Sharpe agar and violet red bile agar, respectively (Difco Laboratories Inc., Livonia, MI, USA). The plates for the enumeration of total aerobic plate count and total coliform were incubated at 37°C for 24 to 48 h, while those for the enumeration of lactic acid bacteria were incubated at 35°C for 48 h. Microbial population was expressed as log colony-forming unit (CFU)/g.

Experimental diets and excreta samples were analyzed in triplicate for dry matter (DM, method 930.15;
[16 ]), crude protein (CP, method 990.03;
[16 ]), ash (method 942.05;
[16 ]), calcium, and phosphorus (method 985.01;
[16 ]). Gross energy was measured by a bomb calorimeter (Model 1261, Parr Instrument Co., Moline, IL), and chromium concentrations of experimental diets and excreta samples were determined with an automated spectrophotometer (Jasco V-650, Jasco Corp., Tokyo, Japan) according to the procedure of Fenton and Fenton
[17 (link)]. The ATTD (%) of nutrients was calculated by the following formula:

Where
N_f = nutrient concentration in feces (%)
N_d = nutrient concentration in diet (%)
C_f = chromium concentration in feces (%)
C_d = chromium concentration in diet (%)
The microbiological assay of excreta was carried out by the procedure suggested by Choi et al.
[18 (link)]. The microbial groups enumerated were total anaerobic bacteria (TAB; plate count agar, Difco Laboratories, Detroit, MI, USA), Bifidobacterium spp. (MRS agar), coliforms (violet red bile agar, Difco Laboratories, Detroit, MI, USA), and Clostridium spp. (Tryptose sulphite cycloserine agar, Oxoid, Hampshire, UK). The anaerobic conditions during the TAB and Clostridium spp. assays were created by using the gas-pak anaerobic system (BBL, No. 260678, Difco, Detroit, MI, USA).