Studies On Aqueous Enzymatic Extraction Of Oil And Protein Hydrolysates From Peanut

Aqueous enzymatic extraction technology has been studied for a long time. However, it could not be popularized and applied in several oilseeds in industrial scale because the obtained emulsion in the system limiting the free oil yield. The aqueous enzymatic process extraction is not similar for different oilseeds. In this dissertation, the optimum technology suitable to peanut has been studied. The parameters of the extraction step, the condition and mechanism of demulsification operation, the recovery, purification and the function of peanut protein hydrolysates (PPH) and isolation and purification of the functional peptides in the PPH were then studied.First the aqueous enzymatic extraction suitable for peanut was developed. Blanched peanut was ground and then dispersed in five volume times of water. At pH 8.50, 60℃, the mixture was stirred for 30min and then treated with four different enzymes at their optimum temperature and pH for 8h. The enzymatic reaction system was centrifuged and separated into free oil-I , emulsion, hydrolysates solution and solid phase. The solid phase was dissolved into two volume times of water of peanut and stirred for 30 min at 60℃, and recentrifuged. The centrifuged suspension was separated into the cream layer, liquid layer and solid. Hydrolysate solution and liquid were combined and heated at 90℃ for 10min to denature the enzyme hence PPH was obtained. The emulsion and cream layer were collected and demulcificated by freezing and thawing.In the technology, peanuts were ground dry in order to avoid formation of a stable emulsion to some degree. After alkaline extraction, total oil and protein yields were 97.14% and 96.37%, respectively. In the enzymatic step, four kinds of proteases (Alcalase 2.4L, As1398, Protease-N and Papain) were used, but Alcalase 2.4L had the best efficiency. The percent of the protein hydrolysates with molecular weight less than 2000Da was 90.2%. Under the condition of excessive grinding Alcalase 2.4L had the most effect in the enzymatic step. Adding cellulase, pectinase, α-amylase or endo-protease (Flavourzyme) couldn't increase the yield of free oil and hydrolysates. The results show that Alcalase 2.4L at 1.5% (1mL/100g protein) at original pH 8.50, temperature of 60℃ and extraction 5h presented the most effective conditions with DH 21.6%, total free oil yield of 91.3% and hydrolysates yield of 83.3%. The percent of the protein hydrolysates with molecular weight less than 2000Da was 87.01%.The emulsion formed in the aqueous enzymatic extraction technology was a system reflecting an essentially elastic nature. Emulsion viscosity decreased with increasing temperature and shearing rate. There was a liquid crystal phase on the emulsion interface. The percent of the hydrophobic amino acid in protein from emulsion interface was 38.94%. The protein adsorbing at emulsion interface was partly spread and the random curl percent was high. Therefore its EAI and ES were higher than that of the protein in the liquid and peanutprotein.Comparing the effects of different demulsification methods, the results showed the effect of freezing and thawing to be the best. During the freezing and thawing demulsification, the oil recovery was increased with decrease in freezing temperature. The SFC was so different with different freezing time that the mean particle diameter of the emulsion after thawing was different. The demulsification effect could not be increased by increasing the thawing temperature. The gelatinization of starch in the emulsion resulted in the increase of the emulsion viscosity and decrease of demucification effect when the thawing temerature ranged between 50~80°C.The optimum demusification conditions were: freezing the emulsion for 15h at -16°C, thawing for 2h at 35°C, and then centrifugation at 3500rpm for lOmin. At these condition the oil recovery in the emulsion was 92.16%.The PPH solution was concentrated by nanofiltration (NF) with crimped membrane module and aroma polyamide. The optimum NF operating parameters were obtained as follows: operating pressure 0.9MPa, temperature 35°C and runoff 75L/h. The concentrated solution was spray dried with inlet temperature 170-180 "C and outlet temperature 75-85 °C, hence the PPH power was obtained. It had better solubility, hygroscopicity and moisture retention and lower viscosity at high concentration.The antioxidative activities of PPH were studied in vitro using five models of antioxidation. The results showed that PPH had the property of reduction, and the antioxidation in lecithin liposome system was good. It had hydroxyl radicals, DPPH and superoxide scavenging activities. The PPH had the ACE inhibiting activity and the effects increased with its concentration.The carbohydrate content of the PPH was 28.51%. In this dissertation macroporous adsorption resins were used to remove the carbohydrates. The adsorption behavior of PPH on different macroporous adsorption resins was investigated. Static adsorbing and desorbing tests showed that DA201-C resin had the best adsorption behavior to PPH among the five kinds of macroporous adsorption resins. The effects of different eluents on PPH desorbing from macroporous adsorption resins were different and 75% ethanol was the best. Dynamic adsorbing and desorbing tests showed that the pH of the PPH solution influenced the adsorption capacity. The result showed that 40mg/mL PPH dissolved with pH 3 HAc solution was carried out to remove carbohydrates using a DA201-C column in 0.75BV/hr flow rate, then the column was washed using water and PPH desorbed by 75% ethanol. Carbohydrates (63.6%) were removed and the peptide recovery was 79.24%. After removing carbohydrates, the protein content in sample increased from 65.12% to 84.46% and the carbohydrates content decreased from 28.51% to 10.35%.The peanut peptides obtained after removal of carbohydrates from PPH had more significant effect of inhibiting trauma by oxidation, protecting the cell membrane and inhibiting haemolysis occurring than that of PPH. The effect of peanut peptides on delay of fatigue was measured. In vivo study with mice showed that in the physical stamina swimming, the experimental mice drowned after a longer period of time than the control mice. TheIVactivity of lactate dehydrognase (LDH) and hepatic and muscular glycogen content in the experimental mice were evidently higher than that in the control mice. After the exercise, the accumulation of blood lactic acid and the increase in serum urea nitrogen in the experimental mice were significantly lower than that in the control mice. In conclusion peanut peptides at the dosage of 250 and 500mg/kg-d had a significant effect on raising the physical stamina and delaying fatigue in mice.In this dissertation the ACE inhibitory and hydroxyl radical scavenging activities of the peptides in the PPH were studied in order to realize the potential effects of PPH to antioxidation and decrease blood pressure and the relationship between function and its structure completely. A component F-60-6-1 with higher hydroxyl radical scavenging and ACE inhibitory activities was isolated from PPH by size-exclusion chromatography, macroporous adsorption resin and semi-preparative RP-HPLC. The identification by size-exclusion high performance liquid chromatography (SE-HPLC), LC/MS and amino acid analysis showed that its molecular weight was 628.7Da, while its amino acid sequence was Tyr-Gly-Asn-Leu-Tyr. Its function was related to its structure. Another component F-60-3 with higher hydroxyl radical scavenging and ACE inhibitory activities was mainly composed of peptides with molecular weight 286Da. Amino acid analysis showed that the amino acids were Ser, Gly, Tyr, Phe and He.