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8 protocols using violet red bile agar

1

Characterizing NSLAB Dynamics in Ripening Cheese

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For bacteriological analysis, cheeses were aseptically sampled at Day 1, 14, 28, and Month 3 of ripening. The samples were placed in a sterile stomacher bag, diluted 1:10 with sterile 2% trisodium citrate and homogenized using a stomacher (Iul Instruments, Barcelona, Spain) for 5 min. Independent duplicate samples were taken at each time point and serial dilutions were prepared as required. Starter cells were enumerated on LM17 agar (Merck, Darmstadt, Germany) after incubation at 30°C for 3 days. Total NSLAB (lactobacilli) were enumerated on Lactobacillus selective (LBS) agar (BD, Oxford, UK) after 5 days incubation at 30°C. Coliforms and enterococci were enumerated on violet red bile agar (BD) and Kanamycin aesculin azide agar (Merck) after 24 h incubation at 30°C.
To confirm that the majority of NSLAB belonged to the inoculated adjuncts, Pulsed-Field Gel Electrophoresis (PFGE) was performed as described by Stefanovic et al. (2017a (link)). Isolates from Month 3 samples were analyzed, and the PFGE patterns were compared with the patterns of the three adjuncts (Figure 2B).
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2

Enumeration of Food Microorganisms

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Enumeration of total coliforms was done using Violet Red Bile Agar (BD, Bioxon, México City, México) incubated on aerobic conditions during 24 h at 37 °C; molds and yeast were determined aerobically on Potato Dextrose Agar (BD, Bioxon, México City, México) acidified with 10% (w/v) (1.5 mL/100 mL) tartaric acid and incubated for 5 days at 25 °C. Presence of coagulase-positive staphylococci was determined spreading 100 µL of each dilution on Baird Parker Agar plates (BD, Bioxon, México City, México) enriched with egg yolk tellurite emulsion incubated at 37 °C for 2 days. Presumptive colonies were tested for catalase, coagulase and Gram strain, in order to identify them as coagulase positive staphylococci [12 (link)].
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3

Enumeration of Cheese Microbiota

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Cheese was sampled aseptically using a cheese trier at 1, 10, 35, 45, and 95 d of ripening. The samples were placed in a sterile stomacher bag, diluted 1:10 with sterile 2% trisodium citrate buffer (VWR) and homogenized using a stomacher (Iul Instruments, Barcelona, Spain) for 10 min. Independent duplicate samples were taken at each time point and dilutions were prepared as required. Viable S. thermophilus cells were enumer-ated aerobically on Ellikers (BD) agar supplemented with 0.5% beef extract (BD) after 3 d of incubation at 42°C. Lactobacillus helveticus cells were enumerated anaerobically on MRS agar (BD) at pH 5.4 after 3 d at 45°C. Lactobacillus casei cells were plated on MRS medium supplemented with vancomycin (Sigma, Arklow, Ireland) as per Ong et al. (2006) . Total NSLAB were enumerated anaerobically on Lactobacillus selective agar (BD) for 5 d at 30°C. Coliforms were plated on violet red bile agar (BD) at 30°C for 1 d. Propionic acid bacteria were enumerated on sodium lactate agar after 7 d incubation at 30°C (Drinan and Cogan, 1992) .
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4

Evaluating Colostrum Bacterial Contamination

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To evaluate the level of contamination in colostrum samples, we measured total aerobic bacterial count (TBC) and total coliform count (TCC). We prepared 10-fold serial dilutions of each sample in 9 mL of sterilized PBS (Sigma Aldrich). For TBC, plates were prepared by placing 100 µL of each relevant dilution in sterile Petri dishes and adding sterilized plate count agar (Becton Dickinson), previously cooled to 50°C, into each plate before mixing and allowing to solidify. The TCC was prepared as described above, apart from the use of violet red bile agar (Becton Dickinson) instead of plate count agar. Plating was carried out in duplicate for each sample. The TCC and TBC plates were incubated at 30°C and 37°C for 72 h and 24 h, respectively. Duplicate plates containing colonies within the range of 30 to 300 were used to calculate mean colony forming units per milliliter in the original samples. Cut-off points of 100,000 and 10,000 cfu/mL for TBC and TCC, respectively, were used to classify samples as satisfactory or unsatisfactory (Godden, 2008 (link)).
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5

Antimicrobial Efficacy of PLA-Nisin Composites

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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.
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6

Microbial Strain Cultivation and Polymer Characterization

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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)
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7

Bacterial Growth Quantification in Meat

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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.
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8

Nutrient Digestibility and Gut Microbiome

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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
Nf = nutrient concentration in feces (%)
Nd = nutrient concentration in diet (%)
Cf = chromium concentration in feces (%)
Cd = 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).
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