The present investigation explored the potential of various physical, chemical, thermal, non-thermal and biological interventions, individually and in different combinations, to reduce bacterial populations on meat surfaces, thereby improving the quality and shelf stability of raw beef during refrigerate storage. Clove essential oil, hot water washing, UV light treatment and bacteriophage therapy were employed individually on meat fillets (beef) to evaluate their potential to improve safety and quality of meat. Subsequently, different combinations of these antimicrobial interventions were used as multiple hurdle technology to inactivate surface microflora of beef. Prior to any applications, clove powder was nutritionally characterized, and following optimization of an extraction protocol, antioxidant potential and antimicrobial efficacy of clove extracts was measured. For the extraction of polyphenols, four extraction methods were used: ethanol extraction (EE); n-hexane extraction (HE); petroleum ether extraction (PE); and steam distillation extraction (SE). Among these extraction methods, clove oil obtained through HE demonstrated highest extraction yield (48.84±0.13%), total phenolic contents (54.05±0.93 mg GAE/g), total flavonoid contents (15.54±0.46 mg quercetin/g), FRAP value (0.69±0.03 mg/mL) and DPPH activity (0.29±0.01 mg/mL). Additionally, HE clove extract showed the highest antimicrobial activity against all experimental pathogens, producing zones of inhibition of 24.27±0.32 mm for S. Typhimurium, 25.8±0.20 mm for E. coli and 29.67±0.35 mm for L. monocytogenes. With highest susceptibility shown by L. monocytogenes. Moreover, response surface methodology was applied to optimize the treatment combinations for all antimicrobial interventions before their final application on meat. For clove oil treatment, meat samples were dipped in solutions of three concentrations (0.5%, 1.0%, 1.5%) for three different time periods (1 min, 2 min, 3 min) and lowest total plate count (3.27 log CFU/g) and Listeria monocytogenes concentrations (2.29 log CFU/g) were recorded in samples immersed in 1.5% clove solution for 3 min. Additionally, immersion in clove solutions was helpful in improving meat qualityand oxidative stability during storage of 15 days. For hot water treatment, total plate count and L. monocytogenes concentrations were lowest on meat samples washed with hot water at 90°C for 90 s i.e., 2.62 log CFU/g and 2.27 log CFU/g respectively. However, drip loss and purge loss percentages were slightly higher and slight discoloration was seen with increase in heat. However, pH, TVBN, and texture values remained in acceptable ranges for all the hot water-treated samples. UV application also reduced total bacteria concentrations with lowest concentrations of total plate counts (2.86 log CFU/g) and L. monocytogenes numbers (2.71 log CFU/g) recovered on meat samples treated with UV light at 8 cm distance for 180 s. Likewise, treat with bacteriophages reduced growth of L. monocytogenes during storage from 5.2 log CFU/g on Day 1 to 3.9 log CFU/g on Day 5, 3.5 log CFU/g on Day 10 and 2.9 log CFU/g on Day 15. Among multiple hurdles, obtained log values of total plate count and L. monocytogenes numbers were 4.35 and 3.92 log CFU/g for dipping in clove oil solution (0.5%) at 60°C for 1 min., 3.31 and 3.26 log CFU/g for hot water wash (60°C) + UV treatment, 3.43 and 3.25 log CFU/g for clove solution dipping (0.5%) + UV treatment and 2.52 & 2.30 log CFU/g for hot water wash (60°C) + clove solution dipping (0.5%) + UV treatment. For phage combinations, L. monocytogenes concentrations were 2.91 log CFU/g for Phage + 0.5% clove essential oil, 2.39 log CFU/g for phage + UV treatment and 1.8 log CFU/g for phage + clove extract + UV treatment. Taken together, these data indicate that individual mitigation treatments each reduced bacterial concentrations on meat surfaces, but multi-hurdle approaches that employed the different treatments on sequence were most effective.Importantly, most mitigation treatments reduced bacterial concentrations without a concomitant decreased on meat quality as measured here.
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