Safety analysis of D. hansenii inoculation
Hemolysis: Hemolytic properties were examined, following the guidelines of the American Society for Microbiology [
21]. The cultured
D. hansenii isolates (SMFM201812-1, SMFM201812-3, SMFM201905-4, SMFM201905-5, and SMFM201905-15) were streaked onto Columbia agar plates containing 5% sheep blood (bioMerieux, Marcy l’Etoile, France) and incubated at 20°C for 48 h to evaluate their hemolytic activities. Strains without a clear zone around the colonies were considered non-hemolytic. Meanwhile, strains with partially clear zones around the colonies with green color were considered to exhibit
α-hemolysis, whereas those with clear transparent zones around the colonies were considered to exhibit
β-hemolysis.
Cytotoxicity: The cytotoxicity was measured by modifiying the method of Sadeghi-Aliabadi et al [
22]. HT-29 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 1% penicillin-streptomycin (Welgene, Korea) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C and 5% CO
2. The cells were subcultured when more than 80% were attached to the T-75 flask. For trypsinization, cells were incubated with 3 mL trypsin (Gendepot, Barker, TX, USA) for 3 min. The trypsinized cells were centrifuged at 217
g and 25°C for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 mL of DMEM. The cell suspension (1 mL) was transferred to a new T-75 flask and cultured in 19 mL of fresh DMEM. When more than 80% of cells were attached to the 75-T flask, the subcultured cells were treated with 3 mL of trypsin for 3 min. The cell suspension was centrifuged at 217
g and 25°C for 5 min. Next, the cell pellet was washed with the medium and resuspended in DMEM. The cells were then seeded in a 96-microtiter plate at a density of 5×10
4 cells/mL and incubated at 35°C with 5% CO
2 for 24 h. In individual wells of the microtiter plate, 20 μL of the
D. hansenii isolates (5 log CFU/mL) were incubated with 180 μL of DMEM without antibiotics at 37°C and 5% CO
2 for 24 h. The culture supernatant was carefully removed from each well using a micropipette. The samples were then incubated with 5 mg/mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, St Louis, MO, USA) reagent at 37°C and 5% CO
2 for 4 h to allow the metabolism of MTT by HT-29 cells. The supernatant was removed using a micropipette, and the samples were solubilized in dimethyl sulfoxide (Samchun, Korea). The absorbance of the mixture at 540 nm (OD
540) was measured using a microplate spectrophotometer (BioTek, Winooski, VT, USA). Cytotoxicity was evaluated by measuring cell viability, and cell viability was calculated using the following equation:
Analysis of D. hansenii-inoculated beef quality during dry aging production of dry-aged beef: D. hansenii isolates (SMFM 201812-1, SMFM201812-3, SMFM201905-4, SMFM201905-5, and SMFM201905-15) (100 μL) stored at −70°C were inoculated into 10 mL PDB and cultured at 20°C for 72 h. For subculturing, 0.1 mL aliquots of the cultures were inoculated into 10 mL fresh PDB and cultured at 20°C for 72 h. The subculture was transferred to a 15-mL conical tube and centrifuged at 1,912 g and 4°C for 15 min. The cells were washed twice with phosphate-buffered saline (PBS, pH 7.4; 8.0 g NaCl, 1.5 g NaHPO4, 0.2 g KH2PO4, 0.2 g KCl in 1 L distilled water). The cell density of the suspension was adjusted to 6 log CFU/mL before inoculation. The cattle (22 month old Holstein steer) was slaughtered in Anseong, Korea on August 6, 2019. The grade of the beef was grade 2 which is the fourth grade out of five grades (1++, 1+, 1, 2, and 3) in South Korea. The rump beef within 1 week after slaughtering was subjected to UV irradiation for 10 min in a bio cabinet. Next, rump beef (600 g) was evenly sprayed with 3 mL of five D. hansenii isolates using a sprayer. The control samples were sprayed with PBS. The inoculated beef sample was incubated at room temperature for 15 min to allow D. hansenii attachment and subjected to dry aging at 5°C and 75% relative humidity in a thermo-hygrostat (KCL-2000; EYELA, Tokyo, Japan) for 4 weeks.
Microbial analysis: Microbial analysis of dry-aged beef crusts was performed after 4 weeks of dry aging. After trimming the crust, 10 g of dry-aged meat crust and 20 mL of 0.1% BPW were transferred to a filter bag (3M, St. Paul, MN, USA). The samples were homogenized for 60 s using a pummeller (BagMixer 400 W; Interscience, France). The homogenate was serially diluted with 0.1% BPW. Each dilution (0.1 mL) was plated onto tryptic soy agar (TSA; Becton Dickinson, USA) or PDA supplemented with 10% tartaric acid. The samples plated onto TSA plates were incubated at 37°C for 24 h to determine the total aerobic bacterial counts. To enumerate yeast, each dilution of the sample was plated onto PDA supplemented with 10% tartaric acid and cultured at 20°C for 48 h.
pH: Before measurement, the pH meter was calibrated using pH 4.01, pH 7.00, and pH 10.01 buffered solution (Thermo Fisher Scientific Inc., San Jose, CA, USA), and the pH was measured by modifying the method of Calicioglu et al [
23]. The dry-aged beef sample (10 g) along with 0.1% BPW (20 mL) was transferred to sterile sample bags and homogenized for 60 s using a pummeller. The pH value of the homogenate was measured using a pH meter (Thermo Fisher Scientific Inc., USA).
Anlaysis of thiobarbituric acid reactive substance: Lipid rancidity was measured following the methods described by Park et al [
24] and Hwang et al [
25]. The samples (1 g) along with 3 mL distilled water were placed in sterile sample bags and homogenized for 60 s using a pumeller. Next, 1 mL aliquots of the homogenate without solid contents were incubated with 2 mL of 20 mM 2-thiobarbituric acid/20% trichloroacetic acid at 100°C for 15 min in a water bath. The reaction was stopped by cooling the reactant in flowing water. After filtering the supernatants with No. 1 filter paper (Advantec, Tokyo, Japan), the absorbance of the filtrate at 531 nm was measured using an Epoch microplate spectrophotometer (BioTek, USA).
Shear force: The beef sample (100 g) was heated upto 70°C the core temperature followed by cooling under running water. The sample was cut into pieces (1 cm×1 cm ×4 cm) perpendicular to the direction of the muscle fiber. The shear force was measured using a texture analyzer (TA-XT2; Stable Micro Systems Ltd., Haslemere, UK) equipped with a Warner-Bratzler shear blade. The test conditions were as follows: pre-test speed 2.0 mm/s, test speed 2.0 mm/s, post-test speed 8.0 mm/s, and distance 30.0 mm. Measurements were performed until the sample was completely cut perpendicular to the direction of the muscle fiber.
Free amino acid analysis: The sample (3 g) was incubated with 15 mL of 0.01 N HCl and homogenized using a pummeller for 1 min. The homogenate was filtered through a No. 1 filter paper (Advantec, Japan). The filtrate (600 μL) was incubated with 20 μL of internal standard solution (1 mg/mL L-citrulline; Sigma-Aldrich, USA) and 1,380 μL of acetonitrile. The mixture was left undisturbed for 30 min and centrifuged at 10,000 g for 15 min. The supernatant was filtered through a 0.2-μm syringe filter. Free amino acid content was analyzed using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc., USA) at the National Instrumentation Center for Environmental Management (Seoul, Korea) as follows: column, Inno C18 column (4.6 mm×150 mm, 5 μm); column temperature, 40°C; injection volume, 0.5 μL; mobile phase A, 40 mM sodium phosphate (pH 7); mobile phase B, distilled water/acetonitrile/methanol = 10%/45%/45% (v/v).
Fatty acid analysis: The sample (1 g) was mixed with 2 mL of pyrogallol, 2 mL of an internal standard solution (triundecanoin), and 10 mL of 8.3 M HCl, and the mixture was sealed with parafilm. To promote decomposition, the samples were incubated at 70°C to 80°C for 40 min in a drying oven. The decomposed product was incubated with 25 mL of ethyl ether for 5 min with shaking, followed by incubation with 25 mL of petroleum ether for 5 min with shaking. The sample was evaporated at 35°C to 40°C using a nitrogen concentrator, vortex-mixed with 2 mL of chloroform and 3 mL of ethyl ether, and concentrated at 40°C using a nitrogen microconcentrator. The concentrate was incubated with 2 mL of 7% BF3-methanol and 1 mL of toluene at 100°C for 45 min, followed by incubation at room temperature. Next, the sample was incubated with 5 mL distilled water, 1 mL isooctane, and 1 g Na2SO4; vortexed; and centrifuged at 448 g for 5 min. The supernatant was used as a test solution. Gas chromatography was performed using Agilent 7890A (Agilent, Palo Alto, CA, USA) with an SP-2560 (100 m×0.25 mm×0.2 μm) column. The chromatography conditions were as follows: sample inlet temperature, 225°C; detector temperature, 285°C; and mobile phase flow rate, 0.75 mL/min. The results of the test samples were compared with those of the standard solution (a fatty acid standard: isooctane). The analysis was performed at the Food Analysis Research Center (Suwon, Korea).