Optimization for Ultrasound-microwave Assisted Extraction of Pectin Methylesterase from Jujube Using with Orthogonal Design Methodology

Optimization of conditions for Jujube pectin methylesterase extraction was investigated using orthogonal design methodology. Extraction parameters which are employed in this study are Liquid-Solid Ratio (LSR) (1:5-20), NaCl content (0-2.0 N), ultrasonic time (0-80 min) and they were optimized using a three-factor orthogonal Design coupled with range and variance analysis. The results showed that, NaCl content and solid-liquid ratio have significant effect on the activity of pectin methylesterase. The satisfactory conditions for Jujube pectin methylesterase extraction were obtained as follows: 1:10 of LSR, 1 N of NaCl content and 40 min of ultrasonic time. Among the studied factors, NaCl content had the greatest influence on yield. Under these conditions, the experimental activity of Jujube pectin methylesterase reach the highest, was 0.6841 U/g.


INTRODUCTION
Pectin is abundant in jujube and mainly high methoxyl pectin.Pectin is widely distributed in plants, constituting the main component of plant cell walls.Pectin is chain polymer with alpha-D-galacturonic acid as structure unit, connected with alpha-1, 4-glycosidic bond (Lacroix et al., 2005).Before pectin secreted into the cell wall, galacturonic acid in the main chain would esterification with methanol.Pectin methylesterase could catalyze pectin demethyl esterification to take off free carboxyl groups, methanol and hydrogen ions (Polydera et al., 2004).This will change the pH value around cell wall, which affects other pectic enzymes, such as polygalacturonase and the activity of pectin lyase, etc. Pectin methylesterase play an important role in plant cell wall metabolism process (Micheli et al., 1998).
At present, enzyme exaction and purification method include crude enzyme preparation, salting out with ammonium sulfate, organic solvent precipitation, chromatography, polyacrylamide gel electrophoresis, Sephadex gel filtration, isoelectric focusing, aqueous two-phase extraction (Kobayashi et al., 1999).Determination methods include viscosity reduction, titration, AJDA, ultraviolet absorption and so on.Given to obtain the larger pectin methylesterase activity and ease operation, crude enzyme preparation and sodium iodate titration are chosen as exaction and determination method (Jaramillo et al., 2013).
However, extraction technology of pectin methylesterase in jujube has not reported clearly until now.In this study, pectin methylesterase preparation technology is optimized in Liquid-Solid Ratio (LSR), NaCl content, ultrasonic time using a three-factor orthogonal design methodology, so as to provide advice for pectin methylesterase function on improving fruit and vegetable quality and its activity regulation research.
Orthogonal design: Based on the results of single factor experiment on liquid ratio, NaCl content and ultrasonic time, L 9 (3 4 ) orthogonal test designed as Table 2 to choose proper extraction parameters of pectin methylesterase.

RESULTS AND DISCUSSION
The influence of liquid ratio on pectin methylesterase activity: It can be seen from Fig. 1 that pectin methylesterase activity increased first, reach the peak of 0.67U/g at liquid ratio of 1:10, significantly higher than other treatment (p<0.05), then decreased gradually with liquid ratio adding.So it is advisable to choose liquid ratio of 1:10.
The influence of NaCl content on pectin methylesterase activity: Pectin methylesterase activity presented a increase-stable-decrease trend with NaCl content adding, peak at NaCl content of 1.0 and 1.5 N, reach maximum pectin methylesterase activity of 0.66 U/g (Fig. 2).So it is advisable to choose NaCl content of 1.0 N.
The influence of ultrasonic time on pectin methylesterase activity: From Fig. 3, pectin methylesterase activity rose first, reach the peak of 0.68U/g at 60 min, significantly higher than other treatment (p<0.05), then reduced with ultrasonic time.
That is because ultrasonic wave has the effect of splitting cells, certain ultrasonic treatment is beneficial for jujube cell to release pectin methylesterase, so enzyme activity showed a trend of increase at first.However, protein would be fractured after a long time of ultrasonic treatment, so as to enzyme activity decrease later.So it is advisable to choose ultrasonic time of 60 min.

Orthogonal test result of extraction of pectin methylesterase:
The orthogonal test result is shown in  Verification test: Three repeat tests performed according to A2B1C1 conditions, pectin methylesterase activity reach a mean of 0.6841 U/g, higher than the 9 test results in Table 3, so the A2B1C1 is appropriate extraction parameter of pectin methylesterase.
Compared with liquid-liquid extraction of pectinase produced by Aspergillus oryzae using aqueous two-phase micellar system, pectinase activity varied from 0.233 to 0.601 U mL1.The highest activity was obtained in the top phase at conditions 25 C, 8% Triton X-114 (wt/wt) and 20% concentrated crude extract (wt/wt) (Jaramillo et al., 2013).If processed for long time period, a gradual further inactivation following different, slower kinetics will be achieved.This has also been observed by other researchers (Basak and Ramaswamy, 1996;Goodner et al., 1998).

CONCLUSION
Single factor test result show proper extraction parameter is Liquid-Solid Ratio (LSR) (1:10), NaCl content (1.0N), ultrasonic time (60 min).After a threefactor orthogonal Design coupled with range and variance analysis.The results showed that, NaCl content and solid-liquid ratio have significant effect on the activity of pectin methylesterase.The satisfactory conditions for Jujube pectin methylesterase extraction were obtained as follows: 1:10 of LSR, 1 N of NaCl content and 40 min of ultrasonic time.Among the studied factors, NaCl content had the greatest influence on enzyme activity.Under these conditions, the experimental activity of Jujube pectin methylesterase reach the highest, was 0.6841 U/g.

Fig. 1 :
Fig. 1: Effect of solid-liquid ratio on the activity of PME

Table 1 :
Single factor experiment design of pectin methylesterase extraction

Table 2 :
Factors and levels for L9 (3 4 ) orthogonal array design of the preparation of crude enzyme solution

Table 3 :
Factors and levels for L9 (3 4 ) orthogonal array design and results of the preparation of crude enzyme solution