Study Of Ultrasound Assisted Walnut Protein Extraction And Enzymolysis And The Effect Of Ultrasound

The walnut meal is a by-product of walnut oil after being squeezed,and the quantity is huge.Most walnut meals are used to feed animals or discarded.Walnut meal is still a potential source of protein.However,the studies of walnut protein have mainly concentrated on the walnut protein preparation by alkali extraction.This traditional method is not only time consuming and inefficient,but also the process is not conducive to the future industrial production.Thus,efficient preparation and utilization methods are important to study in order to overcome these shortcomings.The objectives of this study were?1?to optimize the sweep frequency ultrasound assisted alkali extraction of protein from walnut meal and evaluate the changes of protein properties caused by ultrasonic treatment compared to the traditional alkali extraction,?2?to establish a suitable kinetic model of sweep frequency ultrasound assisted alkali extraction,and reveal the effect of ultrasound,and?3?to optimize the ultrasound assisted enzymolysis of walnut protein and evaluate the properties of walnut peptides compared to the traditional enzymolysis.The main conclusions are as follows:?1?The results of sweep frequency ultrasound assisted alkali extraction of walnut protein showed that all ultrasonic factors had significant effects on the yield and content of protein.The optimum conditions were obtained as follows:solid-liquid ratio of 1:12,pH value of 9.0,initial temperature of 15^oC,ultrasonic frequency of 28 kHz,sweep frequency amplitude of 1.5 kHz,sweep frequency cycle of 100 ms,duty ratio of 77%,and ultrasonic time of 90 min.Under these conditions,the yield and content of walnut protein were 14.7%and 34.7%,respectively.Compared to the traditional alkali extraction?10.9%and 31.6%?,ultrasonic treatment significantly increased the yield and content by 34.9%and 9.8%,respectively.It was concluded that ultrasound can significantly increase the yield and content of walnut protein.The results of FT-IR,fluorescence,CD and SEM showed that ultrasonic treatment increased hydrogen bond force within the protein and the formation of cyclic amides,destroyed the tertiary structure,and made the secondary structure became more order and the microstructure became more dispersive,which contributed to the improvement of the thermal stability of walnut protein.?2?The kinetics results of sweep frequency ultrasound assisted alkali extraction showed that both ultrasonic power intensity?UPI?and ultrasonic temperature increased the concentration and yield of soluble protein,and with a synergetic effect.It was concluded that ultrasound significantly increased the extraction efficiency of walnut protein.Without considering the effect of protein aggregation,the real kinetic model was obtained as follows:y?28?8.588E-6×P^0.015T^2.801[1-exp(-7.896E-15P^0.957T^2.702×t)]?10?0.1909 exp(-7.896E-15×P^0.957T^2.702×t).In this model,the yield of soluble protein y was related to the ultrasonic power intensity,ultrasonic temperature,and ultrasonic time.The result of FT-IR showed that within a short ultrasonic treatment time,ultrasonic treatment caused the C=O groups to form cyclic amides.The result of fluorescence indicated that the tertiary structure of walnut protein was destroyed by ultrasonic treatment.The result of CD showed that the secondary structure of walnut protein was obviously changed by ultrasonic treatment and became more order.The result of SEM suggested that the microstructures of soluble protein and left residue were obviously changed by ultrasonic treatment and the microstructure became more dispersive.?3?Ultrasound working mode screening test results showed that probe type ultrasound?F,G and H groups?significantly improved the activity of walnut polypeptides than flat plate ultrasound?A,B,C,D and E?.In which the probe type ultrasound mode of 20/40 kHz?H group?had the highest ACE inhibitory activity.Therefore the optimum conditions of ultrasound assisted enzymolysis of walnut protein for walnut polypeptides were obtained as follows:substrate concentration of 20 g/L,enzyme amount of 16500 U/g,temperature of 50^oC,pH of 9.0,ultrasonic power intensity of 100 W/L,frequency combination of 20/40 kHz,duty ratio of 10s/5s,ultrasonic time of 30 min,and left enzymolysis time of 120 min.Under these conditions,the DH value and ACE inhibitory activity of peptides were 24.5%and 59.3%,respectively.Compared to the traditional enzymolysis,ultrasonic treatment significantly increased the ACE inhibitory activity by 23.8%.During the enzymolysis process,ultrasonic treatment made the molecular weight distribution of polypeptides more concentrated in 200-1000 Da and the content in 200-1000 Da was increased by 30.3%.In addition,ultrasonic treatment significantly increased the hydrophobic amino acid content by 4.0%.The increases of the content of polypeptides in 200-1000 Da and the content of hydrophobic amino acids were beneficial to produce highly active polypeptide component,which might account for the increased ACE inhibitory activity caused by ultrasonic treatment.