Ultrasound-Assisted Alkali Extraction Of Protein From Tea Residue And Its Activity Evaluation

Global tea production in 2017 stood at 5.686 million tons and China being the largest producer accounted for 2.55 million tons,which represents 44.8%of the world's total tea production.This would culminate in the generation of excessive quantities of residues from tea processing,but these residues have not been effectively exploited.It has been found that tea processing residue?referred to as tea slag?contains protein,which can be extracted for applications in animal feed and food production or they could be hydrolyzed to amino acids or peptides for other applications.However,the solubility of tea slag protein is low,and the main limiting factor for the development and utilization of tea slag protein is the low content of extractable protein,so this study intends to use ultrasonic technology to strengthen the process of protein alkali extraction of tea slag,and further the preparation of functional polypeptides through hydrolysis.The main contents and results of this paper are as follows:?1?Ultrasonic assisted alkali extraction of tea slag protein was studied.The effect of ultrasonic assisted sodium hydroxide extraction on protein extraction rate of tea slag protein was optimized and predicted by response surface method,and compared with the traditional thermo-assisted alkali extraction.The results showed that the optimum conditions of ultrasonic assisted alkali were:NaOH concentration of 0.13 M,extraction time of 13 Min,ultrasonic power of 377 W/L and solid-liquid ratio 51.5 g/L.Protein extraction yield of 138.9 mg/g was obtained under the optimum conditions,and the optimum conditions of heat-assisted alkali were:alkali concentration 0.15 M,extraction time 120min and temperature of 60 oC.Compared with the thermo-assisted alkali,the protein yield of ultrasound-assisted alkali was increased by 56.83%,the time was shortened by 89.17%,the content of total amino acids was increased by 43.08%,the content of hydrophobic amino acids was increased by 35.12%,the content of aromatic amino acids was increased by 25%,the content of positively charged amino acids was increased by 54.85%,The content of negatively charged amino acids increased by 48.05%,and the hydrolysis degree of protein decreased significantly.?2?The functional and structural characteristics of tea slag protein were studied by combining ultrasonic pretreatment with biological refining.Ethanol,Viscozyme and Ultrasound?EVU?pretreatment together with alkaline extraction resulted in the highest protein recovery yield of 80.95%with a purification factor of 8.5.Surface hydrophobicity content of 347±7.8 of EVU was significantly higher than the other treatments.Sulfhydryl content of 55.5±2.12 umol/g of EVU was also significantly higher than the other treatments.All the pretreatments had the same solubility profile but the highest was observed for EVU at pH 9-11.EVU extracted proteins had significantly improved emulsion and foam capacity.Analysis of fourier transform infrared spectroscopy?FTIR?,ultraviolet–visible spectroscopy,non-protein nitrogen content and amino acid content revealed slight modification in the protein advance structure in terms of pretreatment,however,the main peptide chain and amino acid composition were not altered.?3?The effects of sodium hydroxide concentration on the functional characteristics,structural characteristics and lysinoalanine formation of tea slag protein extraction were studied.The results showed that increasing alkali concentration from 0.0125 M to 0.150 M resulted in an increase in protein yield,solubility,foam capacity and stability up to 0.1 M,which declined further with an increase in the alkali concentration.Surface hydrophobicity,thiol and disulfide contents reached a maximum at 0.075 M and continued to decline afterward.The effect of alkaline concentration on lysinoalanine?LAL?formation showed an increase from 6.00±0.32 g/kg at 0.1 M to 29.43±1.10 g/kg at 0.125 M followed by a decline to 7.37±0.01 g/kg at 0.15 M.?4?The effect of ultrasonic pretreatment on the enzymolysis hydrolysis kinetics and thermodynamics of tea slag protein was studied.The results indicated that both ultrasound assisted and traditional enzymolysis conformed to first-order kinetics within the limits of the studied parameters.Michaelis constant?K_M?in ultrasonic pretreated enzymolysis decreased by 32.7%over the traditional enzymolysis.Enzymolysis thermodynamic parameters Ea,?H and?S of enzymolysis with SFCCU pretreatment significantly?p<0.05?reduced by 8.48%,8.99%and 7.52.0%respectively as compared with the traditional method.The highest polypeptide concentration of 24.12 mg/ml was obtained with the lowest energy requirement at improved conditions of 50 g/L of TRP,alcalase concentration of 2000 U/g,time of 10 min and temperature of 50 oC for the ultrasonic treated enzymolysis.The values of reaction rate constant?k?for TRP enzymolysis increased by78%,40%,82%and 60%at 20,30,40 and 50 o C,respectively.?5?The preparation and antioxidant activity of tea slag polypeptide were studied.Albumin,globulin,gliadin and glutelin were then fractionated from tea residue and their antioxidant activities were compared.Upon having a significantly?p<0.05?higher yield and antioxidant activity,glutelin was further hydrolyzed and the effect of hydrolysis time on the antioxidant activity using DPPH radical-scavenging activity?DRSA?,hydroxyl radical scavenging activity?HRSA?,reducing power?RP?and Iron ion chelating activity?FICA?were assessed.Glutelin hydrolysate that exhibited higher activities at the specific hydrolysis duration were fractionated using an ultrafiltration membrane with a molecular weight cut off of 3 kDa.The ultrafiltrated samples were subjected to size exclusion chromatography and four fractions?T₁,T₂,T₃,T₄?were obtained.The fraction from the size exclusion chromatography with the highest activity?T₂?had EC50 values signifiantly?p<0.05?higher than the positive controls,however,their antioxidant activities were comparable upon increase in concentration.?6?The preparation of antihypertensive polypeptides from tea slag protein was studied.Optimal condition for attaining the highest angiotensin I-converting enzyme ACE inhibition of 68.5%were:7.17 mg/mL concentration,pH of 8.0,enzyme/substrate?E/S?ratio of 2939 U/g and temperature of 52.7 ^o C.Glutelin hydrolysate after having the highest ACE inhibition rate were fractionated using an ultrafiltration membrane with a molecular weight cut off of 3 kDa.Upon further purification,a fraction?T3.3?with the lowest IC₅₀ of0.26 mg/mL was obtained.Stability test of the T3.3 fraction indicated that the antihypertensive peptides were stable at 4 and 25 oC.