Identification of 4-hydroxyphenethyl alcohol and 6-hydroxynicotinic acid as novel spoilage biomarkers in refrigerated beef

Traditional freshness indices, including total viable count (TVC) and total volatile basic nitrogen (TVB-N), have limitations in elucidating the underlying molecular mechanisms of meat spoilage. This study investigated changes in beef freshness indices during refrigerated storage at 4 °C and determined that beef entered the spoilage stage on day 7. Untargeted metabolomics characterized metabolic changes and revealed two candidate biomarkers, 4-hydroxyphenethyl alcohol and 6-hydroxynicotinic acid. On days 7 and 11, the relative abundance of 4-hydroxyphenethyl alcohol was 28.88 and 115.20 times that on day 0, respectively, whereas that of 6-hydroxynicotinic acid was 33.47 and 66.66 times. Targeted quantification showed that 4-hydroxyphenethyl alcohol reached 1025.69 μg/kg on day 7 and 5081.85 μg/kg on day 11, while 6-hydroxynicotinic acid reached 685.18 μg/kg on day 7 and 1368.12 μg/kg on day 11. These biomarkers provide promising targets for spoilage warning, rapid freshness assessment, and shelf-life prediction of refrigerated beef.