Plate count agar pca

Thymol was purchased from Sigma Chemical Co. (St. Louis, MO) and resveratrol (with approximately 98% purity) was provided by Glory Biotech Co. (Chiayi, Taiwan). Alfalfa sprouts and fresh mushrooms were purchased from a local supermarket (Chiayi, Taiwan).
Two G(−) bacteria, E. coli BCRC 10675 and E. coli O157:H7 BCRC 15374, and two G(+) bacteria, S. aureus BCRC 12655 and S. aureus 10780, were ordered from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Tryptic soy broth (TSB), tryptic soy agar (TSA), and plate count agar (PCA) used for antibacterial activity assessment and total viable count (TVC) enumeration were purchased from Merck (Darmstadt, Germany).

Experimental systems were designed to analyze the inhibitory effect of bioprotective LAB strains and lactic acid (0.6%) as an adjuvant against EHEC using a storage temperature of 8 °C to approximate real meat processing conditions.
For this purpose, bovine semimembranosus muscle was obtained from a local abattoir. After removing fat and connective tissue in aseptic conditions, it was processed to prepare the experimental systems. Briefly, the surface of the meat pieces as well as the working tools (knives, forceps) were sprayed with 96° alcohol and dried in laminar flow and subjected to UV radiation for 30 min. The top layer of meat (approximately 2 cm) was discarded using a knife (regularly flamed). The remaining meat was aseptically divided in two portions: the first one was cut into small pieces and processed to obtain ground meat (GM); the other portion was sliced into approximately 0.5 cm wide steaks to obtain meat discs by using a stainless steel circle tool (3 cm diameter, 1 cm thick). The ground meat and meat discs were placed separately in bags and sealed at a final vacuum of 99% using a Turbovac 320 ST vacuum packaging machine (Howden Food Equipment, Holland) and stored at −20 °C until required for the experiments (Figure 1). Control of microbial loads of the meat systems was carried out by plating on plate count agar (PCA) (Merck, Buenos Aires, Argentina).

Microbiological analysis of the sausages was performed after 1, 4, 8, 11, 15, 22, and 29 days of refrigerated storage at 4 °C. The samples were prepared according to ISO 6887-2-2017. The total psychrotrophic count (TPC) was determined on Plate Count Agar (PCA) (Merck, Darmstadt, Germany) following incubation at 6 °C for 6 days. The lactic acid bacteria (LAB) were counted on Man Rogosa Sharpe Agar (MRS) (Merck, Darmstadt, Germany) following incubation for 72 h at 30 °C. The quantity of Enterobacteriaceae was determined on Violet Red Bile Dextrose Agar (VRBD) (Merck, Darmstadt, Germany) following incubation at 30 °C for 24 h. The lowest detection limit of the applied enumeration techniques is 10 CFU/g.

Total viable count, psychrotrophic bacteria, lactic acid bacteria (LAB), and Enterobacteriaceae populations of the samples were evaluated during storage days.
Fish fillet (10 g) was mixed with 90 mL of 0.1% sterile peptone water (Merck) in a sterile stomacher bag and stomached for 1 min. For microbial enumeration, 0.1 mL of serial dilutions of fillet homogenates was spread on the surface of agar plates. Total viable counts (TVC) were determined using Plate Count Agar (PCA, Merck) after incubation at 37°C for 24 h. Psychrotrophic bacteria were determined on Plate Count Agar and the plates were incubated at 7°C for 10 days. Lactic acid bacteria (LAB) were counted on de Man Rogosa Sharpe agar (MRS, QUELAB) incubated at 35°C for 2 days. Enterobacteriaceae were enumerated by the pour‐overlay method using Violet Red Bile Glucose (VRBG) agar (Merck). The plates were incubated at 37°C for 24 h. Three replicates of at least three appropriate dilutions depending on the sampling day were enumerated. The number of colony‐forming units was reported as Log₁₀ CFU/g samples (Yousef et al., 2022 ).

For microbiological enumeration, a 10 g sample was transferred to a sterile stomacher bag with 90 mL of sterilized Ringer solution (Merck, Darmstadt, Germany) and was homogenized for 60 s. Samples (0.1 mL) of tenfold serial dilutions of fish homogenates were spread onto the surface of the appropriate media in Petri dishes for enumeration of different spoilage bacteria^{27 (link)}. Total aerobic count was enumerated on Plate Count Agar (PCA, Merck, Darmstadt, Germany) after incubation at 25 °C for 72 h. Pseudomonas spp. were enumerated on Cetrimide Agar (CFC, Merck, Darmstadt, Germany) after incubation at 25 °C for 48 h. For Enterobacteriaceae spp. enumeration the pour-plate method was used, using the Violet Red Bile Dextrose Agar (VRBD, Merck, Darmstadt, Germany), incubated at 25 °C for 48 h. Two replicates of at least three appropriate dilutions were enumerated. The microbial growth was modelled using the Baranyi Growth Model^{28 (link)}. For curve fitting the in-house program DMfit (Inst. Food Research, Reading, U.K.) was used, and kinetic parameters such as the rate (k) of the microbial growth were estimated. Total volatile basic nitrogen analysis was conducted on a single TCA extraction by distillation in a Kjeldhal rapid distillation unit and titration with hydrochloric acid²⁹ .