Low-grade green coffee with Bacillus amyloliquefaciens NY124 protease: extraction, purification, and functional synergy of chlorogenic acid and trigonelline

Defective green coffee beans contain bioactive compounds like chlorogenic acid (CGA) and trigonelline, but high caffeine limits applications. For cost-effective, high-purity industrial use of functional ingredients, this study extracted and purified CGA and trigonelline from low-grade green coffee beans using bacterial enzyme and open column chromatography. Steam pretreatment enhanced the CGA content reaching 35 g/kg of dry weight. Among six strains, Bacillus amyloliquefaciens NY124 was selected for its superior CGA and trigonelline extraction. Its enzyme, a 42 kDa protease, was purified and confirmed by SDS-PAGE and zymography. CGA production doubled with 1.88 U/mL NY124 protease, 7.87% (w/v) green coffee, and 6.85 h via response surface methodology. Diaion HP-20 resins removed 89% of caffeine while silica column chromatography yielded caffeine-free CGA, and trigonelline both achieving high (> 95%) purity. The decaffeinated isolate (Decaf), comprising CGA and trigonelline, exhibited greater functional activity than either fraction alone, highlighting their synergistic effect. Decaf exhibited tenfold higher antioxidant activity than control, along with 52.97% α-glucosidase inhibition (five-fold over control). Decaf isolate demonstrated anti-obesity effects by significantly reducing lipid accumulation by 37% and enhancing lipolysis, evidenced by an 89% increase in free glycerol release compared to the non-treated 3T3-L1 adipocytes.