The Effect And Mechanism Of Ultrasound-assisted Starch Saccharification

The hydrolysis of starch based material into monosaccharides or oligosaccharides,called saccharification,plays a significant role in starch further-processing.Glucoamylase is the most commonly used enzyme to catalyze the saccharification reaction in industry.It meets the basic need in production with no pollution,but still has the limitations of time and energy consuming.In order to overcome this disadvantage,shorten processing time,reduce energy consumption and save cost,people try their best to find an effective way to improve the conversion efficiency of starch materials.Ultrasound is a non-thermal processing technology,which is safe and environmental-friendly.It meets the requirements of the food industry and has been applied in various food processing fields.It has been reported that ultrasound could degrade biological macromolecules,including starch.Meanwhile,ultrasound is certified to improve many reactions via the ultrasonic cavitation effect.However,there are few research studied the application of ultrasound in starch saccharification.In this study,glucoamylase catalyzed saccharification was studied with and without ultrasound.Based on enzymatic kinetics,degradation kinetics and thermodynamic model,the action law and mechanism of ultrasonic assisted enzymatic saccharification was systematically studied from the aspects of protein structure,enzyme-substrate characteristics,and starch fine structures.The effect of ultrasound on saccharification was studied from different objects:enzyme,the enzymatic reaction system,and substrate.Through the comparison of different reaction systems,the action pattern of ultrasound in promoting starch saccharification were explored.The main results are listed below:(1)Effect of ultrasound on glucoamylaseThe enzyme activity of glucoamylase directly treated by ultrasound with different intensity(1.8-14.4 W/m L)at 35-75?for 10-50 min was measured by DNS method.Carrying the ultrasonic treatment at 35?for 20 minutes below 10.8 W/m L,the ultrasonic intensity showed no significant effect on the activity of glucoamylase.When the ultrasonic intensity exceeded10.8 W/m L,the activity of glucoamylase decreased slightly.The effect of ultrasound on glucoamylase was greatly influenced by the temperature.The loss of enzyme activity increased with the increase of temperature.At 75?,the enzyme activity was seriously decreased.The treatment time also affected enzyme activity.Enzyme activity began to decline slightly at 40minutes.Therefore,increasing ultrasonic intensity,prolonging treatment time and increasing treatment temperature will increase the activity loss of glucoamylase.Temperature is the most important factor.Ultrasound did not change the enzyme activity in the p H range of 3.0-6.0,containing the appropriate working p H of glucoamylase.However,it reduced the thermostability of enzyme.At 35 and 45?,ultrasound had little effect on the thermostability of glucoamylase.With the increase of temperature,the thermostability of glucoamylse decreased significantly,and the adverse effects of ultrasound were aggravated.Gel electrophoresis,circular two chromatography,and fluorescence spectroscopy were used to evaluate the enzyme structure before and after ultrasound treatment.Ultrasound did not change the molecular weight of glucoamylase.No degradation or aggregate was found.There were some changes in the secondary and tertiary structures after ultrasonic treatment.After ultrasonic treatment,the content of?-helix,?-sheet and?-turn decreased while the content of random coil increased in the secondary structure of glucoamylase.A more disorder structure was obtained,which reflects the effect of ultrasound on protein conformation.The results of the tertiary structure showed that the tryptophan residues on the enzyme surface decreased after ultrasonic treatment,which also demonstrated the changed of protein conformation caused by ultrasound.(2)Ultrasound assisted starch saccharification catalyzed by glucoamylase:the action pattern and kineticsThrough the simultaneous action of ultrasound and glucoamylase on starch,the ultrasound-assisted saccharification system was established to study the improvement of enzymatic efficiency under different ultrasonic conditions.Different ultrasonic intensity(1.8-14.4 W/m L)was used at different temperature(25-75?)for different time(10-50 min).It is found that ultrasound can effectively promote the saccharification of starch.The promoting effect increased with the increase of ultrasound intensity until reaching the optimal level at 10.8W/m L.However,the promoting effect decreased when intensity was further raised.The duration of saccharification can be shortened from 50 minutes to about 30 minutes when using ultrasound assisted saccharification(10.8 W/m L,35?).However,at high temperature of 75?,the efficiency of ultrasonic assisted saccharification decreased significantly,indicating that high temperature is not conducive to the process of ultrasonic assisted saccharification.Enzymatic kinetics investigated by Michaelis-Menten model showed a reduction of K_mvalue and an increase of V_m value.The value of K_cat/K_m,indicating the enzymatic efficiency,was increased.A better affinity of glucoamylase to starch substrate and also a higher enzymatic efficiency were obtained with the assistant of ultrasound.The degradation kinetics of starch in ultrasonic assisted saccharification and enzymatic saccharification were studied by the first-order kinetic model at 25-65?.The results showed that the degradation rate constants of ultrasonic assisted saccharification increased by 35.59%,33.16%,10.91%and 13.74%respectively at 25-55?.When the temperature raised to 65?,the rate constant decreased by14.78%.It can be inferred that the activity of glucoamylase was affected by ultrasound at 65?.The activation energy(E_a),enthalpy change(?H)and entropy change(?S)are lowered with the assistant of ultrasound.It shows that ultrasound improves the efficiency of the reaction,reduces the lowest energy requirement for the reaction,and makes the reaction easier to start.(3)Effect of ultrasonic assisted saccharification on starch propertiesThe changes of physicochemical and structural properties of starch caused by the reaction of enzymatic saccharification,ultrasonic treatment and ultrasound assisted enzymatic saccharification were studied through chromatography,nuclear magnetic resonance and in vitro digestion model.Through the structural analysis,the mechanism of ultrasonic assisted saccharification was further clarified.The molecular weight of starch decreased after each reaction,in which the effect of ultrasonic assisted saccharification was the largest(decreased to1.00×10⁵g/mol at 50 min),followed by ultrasonic treatment.The M_w change caused by enzymatic saccharification was much less than the former two.Compared with glucoamylase,ultrasound led to more severe decrease in starch molecular weight.The decrease of M_w results in the increase of starch solubility and the decrease of viscosity.Ultrasonic treatment reduced the chain length of amylose significantly,but showed no change on the chain length distribution of amylopectin.Glucoamylase can effectively shorten the chain length of amylose,as well as amylopectin.The average chain length of amylopectin length decreased owing to the shortening of the external chain.Ultrasonic assisted saccharification can further promote the shortening of starch chain by glucoamylase.Enzymatic saccharification and ultrasonic assisted saccharification increased the branching density of starch by 76.0%and 84.4%,respectively.Since the breakage of glycoside bonds induced by ultrasound has no selectivity,thus showing no influence on the branching density of starch.The digestibility of starch changed little after different treatments,while enzymatic saccharification led to a slight improvement of starch digestibility.(4)Study on the mechanism involved in ultrasound assisted saccharificationBy studying the different effect of ultrasound on substrate,enzyme and mixed reaction system using DNS method,the main origin of ultrasonic facilitation on saccharification was determined.Ultrasonic pretreatment of starch was found to improve the saccharification slightly,while the facilitation was far lower than that of ultrasound assisted saccharification.It can be concluded that the main reason leading to the ultrasonic facilitation on saccharification is the ultrasonic effect on reaction system,followed by the ultrasonic effect on starch molecules.The main mechanism of ultrasound-assisted starch saccharification is that ultrasound promotes the mass transfer of the mixed reaction system.Ultrasound lowers the mass transfer barrier around enzyme and substrate,accelerating substrate-enzyme binding and product-enzyme separation.The changes of amylose and amylopectin was analyzed by iodine binding method,showing that the decrease of amylose content caused by ultrasonic assisted saccharification is smaller than amylopectin.It can be explained that branched amylopectin contains more binding sites(non-reducing ends)for glucoamylase,leading to a more effective ultrasonic enhancement on the saccharification of amylopectin.The free radical amount under the experimental conditions,detected by methylene blue test,was not affected by ultrasonic intensity,but decreased with the increasing temperature and accumulated with time.There was no correlation between the free radical amount and the changes of enzyme activity and efficiency caused by ultrasound.Therefore the free radical effect was not the main cause of glucoamylase inactivation or saccharification acceleration in this study.