TY - BOOK

T1 - Evaluation of pressure tuning of enzymes

AU - Naghshineh, Mahsa

PY - 2013

Y1 - 2013

N2 - The current industrial technique of pectin production is based on relatively harsh chemical process,which does not allow pectin to be extracted entirely with no damage to its structure. It is also not an environmentally friendly method due to acid usage, production of large amounts of waste and high energy consumption. Therefore, searching for an environmentally friendly method of pectin extraction is a task for science and industry. Employment of hydrolytic enzymes may represent a green approach to obtain intact pectin polymer. However, the low stability/activity of enzymes, and low polymer yield of enzymatic extraction limits the application of enzyme in pectin production.There is evidence that emerging technology of high hydrostatic pressure processing can result in stabilization and activation of some enzymes. Therefore, the use of high hydrostatic pressure in combination with enzyme treatment can induce significant improvement in yield of enzymeassisted extracted pectin. The application of high hydrostatic pressure technology for enzymatic extraction of pectin was investigated. Cellulase from Trichoderma reesei and xylanase from Thermomyces lanuginosusunder five different combinations (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel waste.It was found that pressure level, type and concentration of enzyme significantly influenced pectin yield and degree of esterification (DE). Enzymatic extraction of pressure treated lime peel at 200 MPa for 30 min at 50 °C using cellulase or xylanase added individually or in combination resulted in pectin yields, which were considerably higher than acid and aqueous extraction. Enzyme-assisted extraction of pectin in combination with the high pressure treatment at 100 or 200 MPa for 30 minat 50 °C improved the enzymatic release of pectin providing higher polymer yield compared to enzymatic extractions at ambient pressure. The combined use of high pressure and enzyme adds a novel dimension to biocatalysis reactio ns as being environmentally friendly and sustainable. Cellulase was also found to be less pressure sensitive than xylanase at pressure levels up to 400 MPa. Therefore, cellulase was used for subsequent multifactorial analysis. The multifactorial effect of high pressure processing (0.1, 100, 200, and 300 MPa), pressurisation time (0, 10, 20, and 30 min), and cellulase concentration (0, 50, 100, and 150 U/g lime peel) on yield and intrinsic viscosity [η]w of pectin polymers extracted from lime peel were studied. All factors had significant effects on pectin polymer yield. The results showed that [η]w of pectin polymers was significantly influenced by high pressure and cellulase concentration. However, pressurisation time did not have any significant effect on [η]w. Moreover, the combined effects ofcellulase concentration and high pressure or pressurisation time significantly affect pectin polymer yield and [η]w. The emerging technology of high pressure processing had considerably improved the yield of enzymatically extracted pectin polymers by stimulating the rate of cellulase-catalysed reactions. The increase of pectin polymer yield was significantly correlated (r = –0.717) with reduction of [η]w.Lastly, knowledge of DE of pectin is of great importance for better understanding of pectin functionality and application. A novel method for automated determination of DE of pectin using a sequential injection analysis (SIA) system was developed. The optimisation of the SIA system parameters was carried out using face-centered central composite response surface methodology (RSM). A calibration graph for determination of non-esterified galacturonic acid (GalA) content in pectin solutions with linear range of 0.08 – 0.34% (w/v) and the limit of detection (LOD) of 0.057%(w/v) under optimal condition was achieved. The difference between concentrations (w/v, %) of total GalA and non-esterified GalA was applied to estimate DE (%) of pectin samples. The proposed automated method was used to determine the DE (%) of seven commercial pectin samples. The comparison between the results obtained by the proposed automated method using SIA and a reference method (FCC, 1981) was performed. Results indicated a good agreement (tstat < tcrit) and correlation (R2 = 0.998) between the proposed automated method and the manual reference method. The suggested method provided precision of less than 6% RSD, (n = 10) with a samplethroughput of 15 samples h–1. Determination of DE of pectin using SIA requires remarkably low consumption of sample and reagents, which thereby makes this method environmental friendly.

AB - The current industrial technique of pectin production is based on relatively harsh chemical process,which does not allow pectin to be extracted entirely with no damage to its structure. It is also not an environmentally friendly method due to acid usage, production of large amounts of waste and high energy consumption. Therefore, searching for an environmentally friendly method of pectin extraction is a task for science and industry. Employment of hydrolytic enzymes may represent a green approach to obtain intact pectin polymer. However, the low stability/activity of enzymes, and low polymer yield of enzymatic extraction limits the application of enzyme in pectin production.There is evidence that emerging technology of high hydrostatic pressure processing can result in stabilization and activation of some enzymes. Therefore, the use of high hydrostatic pressure in combination with enzyme treatment can induce significant improvement in yield of enzymeassisted extracted pectin. The application of high hydrostatic pressure technology for enzymatic extraction of pectin was investigated. Cellulase from Trichoderma reesei and xylanase from Thermomyces lanuginosusunder five different combinations (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel waste.It was found that pressure level, type and concentration of enzyme significantly influenced pectin yield and degree of esterification (DE). Enzymatic extraction of pressure treated lime peel at 200 MPa for 30 min at 50 °C using cellulase or xylanase added individually or in combination resulted in pectin yields, which were considerably higher than acid and aqueous extraction. Enzyme-assisted extraction of pectin in combination with the high pressure treatment at 100 or 200 MPa for 30 minat 50 °C improved the enzymatic release of pectin providing higher polymer yield compared to enzymatic extractions at ambient pressure. The combined use of high pressure and enzyme adds a novel dimension to biocatalysis reactio ns as being environmentally friendly and sustainable. Cellulase was also found to be less pressure sensitive than xylanase at pressure levels up to 400 MPa. Therefore, cellulase was used for subsequent multifactorial analysis. The multifactorial effect of high pressure processing (0.1, 100, 200, and 300 MPa), pressurisation time (0, 10, 20, and 30 min), and cellulase concentration (0, 50, 100, and 150 U/g lime peel) on yield and intrinsic viscosity [η]w of pectin polymers extracted from lime peel were studied. All factors had significant effects on pectin polymer yield. The results showed that [η]w of pectin polymers was significantly influenced by high pressure and cellulase concentration. However, pressurisation time did not have any significant effect on [η]w. Moreover, the combined effects ofcellulase concentration and high pressure or pressurisation time significantly affect pectin polymer yield and [η]w. The emerging technology of high pressure processing had considerably improved the yield of enzymatically extracted pectin polymers by stimulating the rate of cellulase-catalysed reactions. The increase of pectin polymer yield was significantly correlated (r = –0.717) with reduction of [η]w.Lastly, knowledge of DE of pectin is of great importance for better understanding of pectin functionality and application. A novel method for automated determination of DE of pectin using a sequential injection analysis (SIA) system was developed. The optimisation of the SIA system parameters was carried out using face-centered central composite response surface methodology (RSM). A calibration graph for determination of non-esterified galacturonic acid (GalA) content in pectin solutions with linear range of 0.08 – 0.34% (w/v) and the limit of detection (LOD) of 0.057%(w/v) under optimal condition was achieved. The difference between concentrations (w/v, %) of total GalA and non-esterified GalA was applied to estimate DE (%) of pectin samples. The proposed automated method was used to determine the DE (%) of seven commercial pectin samples. The comparison between the results obtained by the proposed automated method using SIA and a reference method (FCC, 1981) was performed. Results indicated a good agreement (tstat < tcrit) and correlation (R2 = 0.998) between the proposed automated method and the manual reference method. The suggested method provided precision of less than 6% RSD, (n = 10) with a samplethroughput of 15 samples h–1. Determination of DE of pectin using SIA requires remarkably low consumption of sample and reagents, which thereby makes this method environmental friendly.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122699772205763

M3 - Ph.D. thesis

BT - Evaluation of pressure tuning of enzymes

PB - Department of Food Science, Faculty of Science, University of Copenhagen

ER -