Application of thermal stability difference to remove flammutoxin in fungal immunomodulatory protein, FIP-fve, extract from Flammulina velutipes

Ching-Hsin Tung ^a,b, Chih-Chieh Lin ^a, Huei-Ju Wang ^c, Sung-Fang Chen ^d, Fuu Sheu ^e, Ting-Jang Lu ^a,*

^a Graduate Institute of Food Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan, ROC

^b Food and Drug Administration, Ministry of Health and Welfare, No. 161-2, Kunyang Street, Nangang District, Taipei, 11561, Taiwan, ROC

^c Department of Applied Science of Living, Chinese Culture University, No. 55, Hwa-Kang Road, Yang-Ming-Shan, Taipei, 11114, Taiwan, ROC

^d Department of Chemistry, National Taiwan Normal University, No. 88, Sec. 4, Ting-Chow Road, Taipei, 11677, Taiwan, ROC

^e Department of Horticulture, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan, ROC

Fungal immunomodulatory protein (FIP-fve) is a potential functional food ingredient. However, undesirable component flammutoxin (FTX) would occur in the extracted fraction of FIP-fve. In this paper, an application of heating processing instead of the intensive separation process was employed in fractionation of FIP-fve, meanwhile, exclusion of FTX was reached. Contents of FIP-fve and FTX were monitored by HPLC-UV-ESI-MS. Both FIP-fve and FTX had higher thermal stability in a lower concentration solution. Cold water could effectively extract FIP-fve and FTX from fresh mushroom without acetic acid and disulfide-bond breaking agent b-mercaptoethanol commonly used in biochemical studies. Heating cold water extract contained 580 mg/mL FIP-fve and 452 mg/mL FTX at 60 C for 5 min could effectively exclude FTX and remain 75% of FIP-fve. Adding 0.1 M trehalose or 20% ethanol did not significantly alter the stability of both proteins. The method developed is an applicable procedure for preparing FIP-fve solution free of FTX.

Keywords: FIP-fve, Flammulina velutipes, Flammutoxin, HPLC-UV-ESI-MS, Protein stability