Circular bioeconomy-driven carotenoid production by Agrococcus sp. NP24 using cheese whey byproduct: process optimization and bioactivity assessment

Growing interest in the circular economy has promoted the use of agri-food wastes as fermentable and readily available substrates for microbial cultivation, offering a sustainable and cost-effective strategy for natural pigment production. In this study, cheese whey was utilized as a nutritional substrate for pigment synthesis by an isolated strain identified as <i>Agrococcus</i> sp. NP24 (PQ097720.1). The work further aimed to characterize the produced pigment and evaluate its bioactivity. The culture medium was optimized using a Box-Behnken design (BBD). The carotenoid profile of the extracted pigment was analyzed by HPLC-DAD and LC-MS. Pigment stability was assessed across a range of pH values and temperatures, and its antimicrobial, antioxidant, and anticancer activities were examined. The pigment was identified as zeaxanthin monoester (C14:0). The maximum pigment yield (0.0567 mg/mL extract) was achieved after 72 h at 20 °C using a medium containing 80% whey (v/v), 0.5% peptone (w/v), 0.97 g % casein (w/v), and supplemented with 0.5% (w/v) yeast extract and 0.5% (w/v) MgSO₄. The pigment remained fully stable up to 50 °C. Acidic conditions (pH 3-5) enhanced pigment absorbance compared to neutral and alkaline pHs. In contrast, exposure to daylight markedly reduced pigment stability, leaving only 26% residual activity after 1 h. The pigment exhibited potent antioxidant activity with an IC₅₀ of 6 μg/mL. It also showed cytotoxic and significant selectivity against the triple-negative breast cancer cell line MDA-MB-231 and the colorectal carcinoma cell line HCT-116, with IC₅₀ values of 3.3 mg/mL and 0.56 mg/mL, respectively, while no cytotoxicity was observed toward the HepG-2 hepatoblastoma cell line. The carotenoid did not display significant antimicrobial activity. In conclusion, the cost-effective production of NP24 carotenoids, combined with their favorable stability and bioactivity, supports their potential use as natural colorants in food applications.