Study Of Walnut Protein Hydrolysate On Memory Improvement And Its Tabletting Properties

Learning and memory is not only one of the important functions of brain,but also the key ability for human beings and animals to survive and evolve.In modern society,high pressure and aging population lead to memory impairment and decline of people of different ages.It has become a major health problem threatening public health safety.The aim of this study is to investigate the memory improving peptides（MIP）prepared by enzymatic hydrolysis.Firstly,memory improving effects of different protein hydrolysates were compared in order to screen the optimal protein material.Walnut protein hydrolysates which are rich in MIPs were prepared by controlled enzymatic hydrolysis.Then the memory improving effects on memory impaired mice were investigated through the behavioral tests,detection of cholinergic system function and neurons in the CA3 region of brain.Finally,the tabletting properties of MIP were studied to develop new product of memory improving peptides and provide theoretical and methodological guidance for producing new nutritional and healthy food.The comparison of compositions,in vitro memory improving effects were conducted of soybean protein hydrolysate（SPH）,walnut protein hydrolysate（WPH）,peanut protein hydrolysate（PPH）and cod protein hydrolysate（CPH）.Results showed the molecular weight distribution of peptides in plant protein hydrolysates concentrated in the 1-3 kDa fractions.However,the fraction below 1 kDa of CPH was the highest（39.29%）.Amino acids analysis presented that WPH was rich in memory improving amino acids（such as arginine,tyrosine and phenylalanine）.SPH（0.10-0.20 mg/ml）in a low concentration、WPH（0.50 mg/ml）in a medium concentration,but PPH（1.00 mg/ml）and CPH（1.00 mg/ml）in higher concentration could significantly increase PC12 cells viability.Pretreatment with protein hydrolysate（2mg/ml）could significantly protect PC12 cells from H₂O₂ impairment.As for this,SHP and WPH presented higher activity.In addition,the mice inhibitory avoidance test showed that WPH was the best one to significantly improve the ability of memory acquisition,consolidation and reproduction in normal mice.All the four protein hydrolysates presented differential ability of ameliorating the acquired memory of the anisodine-induced memory impairment mice.Among them,SPH was the best one in memory acquisition.All the four protein hydrolysates could ameliorate the consolidated memory of the anisodine-induced memory impairment mice,but SPH,WPH and CPH showed better.Among the four protein hydrolysates,only SPH and PPH could improve the memory retrieval ability of the anisodine-induced memory impairment mice.The protection effect of the four hydrolysates is as follows:SPH>WPH>HHP>CPH in the in vitro acetylcholinesterase inhibitory activities,which indicates that the in vitro acetylcholinesterase inhibitory activities of hydrolysates were related to the improvement of memory function in PC12 cells and mice.Then,defatted walnut meal was chosen as the experimental material.The extraction process of walnut protein was developed by analyzing the influence of extraction parameters of alkaline dessolving and acid precipitation on protein leaching rate.The final extraction process was as followed:the ratio of p H 9 alkali solution and walnut meal was 16:1,the extraction time was 2 h at 55 ^oC,the pH of acid precipitation was 4.0.By this process,the protein leaching rate was reaching to 47.15%.The enzymatic hydrolysis efficiency（protein recovery and hydrolysis degree）and the inhibition rate of acetylcholinesterase were taken as the guide for optimization of MIP preparation process.The addition amount of alcalase enzyme and flavor protease was 8‰（E/S）and 2‰（E/S）,respectively.The hydrolysis time was 12 h and the substrate protein concentration was 8%.The protein recovery and peptide yield were 85.23%and 68.94%,respectively,and the inhibitory activity of acetylcholinesterase was 54.37%.The prob tests in Morris water maze showed that WPH exhibited a similar effect with piracetam in improving the spatial exploration ability of mice.It could significantly reduce the escape latency and increase the crossing times（p<0.05）.These results indicated that WPH could effectively ameliorate the spatial memory deficits induced by scopolamine in mice.Further,the effects of WPH with different hydrolysis time（8h,12h,16 h）on memory improving were evaluated by testing the learning and memory ability of mice.Results showed that WPH-16 exhibited better memory improving effects which indicated that different hydrolysis time could release or assemble new memory peptides.WPH-16 could improve scopolamine induced mice memory impairment through increasing Ach R amount.It indicates that peptides in protein hydrolysates act the key role.WPH-16 at a medium and high dose could significantly inhibit or ameliorate the deformation and swelling of capillaries,maintain the basic morphology of neurons,increase the number and length of synapses and the secretion of granular material in presynaptic membranes.But WPH-16 at a high dose could exhibit better improvement effects.Finally,the process of a memory improving product was studied.The powder property tests of WPH showed that WPH was irregular polygon in shape and its material belongs to plastic material.It has good compressible,but poor fluidity and easy to absorb moisture.The optimum formula of direct compression tablet was determined through an orthogonal experiment analysis.The formula was composed of WPH powder,erythritol,microcrystalline cellulose,and magnesium stearate and silica（2:1）.The ratio was 40:37.5:20:1.5:1.The optimized process parameters of WPH compounds were as follows:3-D mixing for 10 min with a rotation speed of 6 rmp/min,press with a pressure of 20 kN,and tabletting with a speed of 30 rmp/min.The accelerated test and long-term stability test showed that WPH tablets met the standard requirements and exhibited broad market prospects.