2.7. Production of extracellular enzymes
Lemon peel, passion fruit peel, and grapefruit peel were used for the induction
of pectinases because of their high content of pectin (35.0%, 18.4%, and 21.2%,
respectively) [17–19], and also because they are produced in large quantities as
waste from the fruit processing industry. The peels were ground using a food processor,
and then washed three times with double-distilled water. Peels were dried
at 50 ◦C for 24 h before being utilized as substrate for the production of extracellular
enzymes. Parental and hybrid fungi were grown in 500mL Erlenmeyer flasks containing
200mL of liquid medium [0.2% (w/v) (NH4)2SO4, 0.2% (w/v) KH2PO4, 0.2%
(w/v) K2HPO4, pH 3] supplemented with either citric pectin, lemon peel, passion
fruit peel, or grapefruit peel at a concentration of 1% (w/v). The medium was autoclaved
at 121◦C for 20 min. The flasks containing medium were inoculated with
2×106 sporesmL−1. Cultivation was carried out at 37 ◦C, on a rotary shaker (New
Brunswick Scientific Co., NJ, USA) at 200 rpm, for 120 h. Samples were collected at
24-h intervals during 120 h. Samples were centrifuged for 20 min at 2500rpm at
room temperature to remove spores. Supernatants were filtered through Millipore
0.45mmembranes, and kept frozen at −20 ◦C until used. These preparations were
used as enzyme-containing filtrates during the course of the study.
2.7. Production of extracellular enzymesLemon peel, passion fruit peel, and grapefruit peel were used for the inductionof pectinases because of their high content of pectin (35.0%, 18.4%, and 21.2%,respectively) [17–19], and also because they are produced in large quantities aswaste from the fruit processing industry. The peels were ground using a food processor,and then washed three times with double-distilled water. Peels were driedat 50 ◦C for 24 h before being utilized as substrate for the production of extracellularenzymes. Parental and hybrid fungi were grown in 500mL Erlenmeyer flasks containing200mL of liquid medium [0.2% (w/v) (NH4)2SO4, 0.2% (w/v) KH2PO4, 0.2%(w/v) K2HPO4, pH 3] supplemented with either citric pectin, lemon peel, passionfruit peel, or grapefruit peel at a concentration of 1% (w/v). The medium was autoclavedat 121◦C for 20 min. The flasks containing medium were inoculated with2×106 sporesmL−1. Cultivation was carried out at 37 ◦C, on a rotary shaker (NewBrunswick Scientific Co., NJ, USA) at 200 rpm, for 120 h. Samples were collected at24-h intervals during 120 h. Samples were centrifuged for 20 min at 2500rpm atroom temperature to remove spores. Supernatants were filtered through Millipore0.45mmembranes, and kept frozen at −20 ◦C until used. These preparations wereused as enzyme-containing filtrates during the course of the study.
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