RESUMEN
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 ß-mercaptoethanol commonly used in biochemical studies. Heating cold water extract contained 580 µg/mL FIP-fve and 452 µg/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.
Asunto(s)
Flammulina/química , Manipulación de Alimentos/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Micotoxinas/análisis , Proteínas Fúngicas/análisis , Calor , Estabilidad ProteicaRESUMEN
A rapid analytical approach, on-line desalting HPLC-UV-ESI-MS method, for the analysis of FIP-fve and flammutoxin (FTX), two important bioactive proteins in the fruiting bodies of Flammulina velutipes, was developed. In this study, a highly efficient desalting method is provided using molecular weight cut-off centrifugal filtration and on-line desalting. Sample preparation followed by an on-line desalting HPLC-UV-ESI-MS system was employed for simultaneous desalting and detection and identification of FIP-fve and FTX. Results indicated that using trifluoroacetic acid as a modifier on a C18 reversed-phase column renders effective separation. ESI-MS revealed that the apparent molecular masses of FIP-fve and FTX were 12,749.1 Da and 21,912.5 Da, respectively. Eleven milligrams of FIP-fve was obtained from 100 g of fresh fruiting bodies, and UV detection was performed at 280 nm using bovine serum albumin as the standard protein. The calibration curve was linear in the concentration range of 0.29-4.69 mg/mL (r2 = 0.9999). FTX and a series of degradation products were isolated from F. velutipes using 35% saturated ammonium sulfate on a DEAE cellulose column. The complete identification of FTX and a series of degradation products were carried out by precipitation of various ammonium sulfate concentrations (0-45%, 45-65% and 65-90%), in-gel trypsin digestion, and MS analysis with combined database search. The molecular weights of FTX and a series of degradation products were 29,957.2 Da, 27,480.2 Da, 26,512.5 Da, and 21,912.5 Da.