| Pecan is a nutritious nut whose production has gradually increased in recent years.Pecan protein is an important component in pecan kernels.Defatted pecan powder has a high content of protein and a reasonable amino acid ratio,making it an ideal protein nutritional supplement.But at present,there are few reports on the properties of pecan protein and the research on the deep processing of pecan fruit.In addition,the large number of phenolic substances contained in the endocarp of pecan can reduce the quality of protein and limit its application.In this study,the effect of endocarp removal on polyphenol reduction and protein properties were investigated.Furthermore,enzymatic hydrolysis of pecan protein and activity screening of pecan protein peptides were performed.And active polypeptide sequences were separated and identified,and screened by molecular docking.At last,pecan protein beverages and pecan protein peptide beverages were prepared.The main research contents and results were as follows:The effects of different peeling methods,namely alkaline peeling(AP),enzymatic peeling(EP),and mechanical peeling(MP),on the removal of polyphenols and properties of pecan proteins were investigated.The Folin-phenol method was used to quantify polyphenols,HPLC was used to determine amino acids,colorimeter was used to determine chromaticity,FTIR and fluorescence spectroscopy were used to determine protein secondary and tertiary structures,scanning electron microscopy was used to characterize microstructure,and to evaluate protein oil absorption,emulsifying properties,and foaming properties were evaluated.The results showed that compared with the unpeeled samples,the three peeling methods could all significantly reduce the polyphenol content in the defatted powder(removal rate>90%),improve the solubility and purity of the protein(69.53%-74.71%),increase the sample brightness,and improve the oil absorption,emulsifying,and foaming properties of protein.Among them,the alkali peeling showed the most significant effect.The solubility of the AP sample was 4 times as higher as that of the unpeeled sample,the L*value reached 65.18,the oil absorption reached 3.73 g oil/g protein,and the emulsification exceeded 300 m~2/g.Based on the above indicators,alkaline peeling was selected as the raw material pretreatment method for subsequent experiments.Alkaline peeled protein was enzymatically hydrolyzed to prepare polypeptides with screening of the bioactivities of the prepared polypeptides and corresponding technological optimization.The degree of proteolysis and peptide yield were determined by OPA(1,2-phthalic dicarboxaldehyde)method and biuret method,and theα-glucosidase inhibition activity,pancreatic lipase inhibition activity,cholesterol micelle inhibition activity,and antioxidant activity of hydrolysates were evaluated.The results showed that:At 10 mg/m L,papain was the best enzyme for inhibition ofα-glucosidase and pancreatic lipase(44.58%,54.78%),and pepsin was the best enzyme for cholesterol micelle inhibition and antioxidant activity(34.24%,DPPH·33.82%,ABTS~+·32.26%,·OH 35.31%),among which,the pancreatic lipase inhibitory activity of papain hydrolysate was the highest.In addition,the solubility(84.88%),degree of hydrolysis(18.21%),and peptide yield(67.58%)of papain hydrolysate were relatively high among the seven proteases hydrolysates.Therefore,papain was preferred as the enzyme for hydrolysis,and the inhibitory activity of pancreatic lipase was used as the activity indicator to further optimize the hydrolysis conditions by single factor and orthogonal experiments.The optimal enzymatic hydrolysis conditions were:a solid-liquid ratio of 1:40(w/v),an enzyme addition amount of 4000 U/g,and an enzymatic hydrolysis time of 2 h.The pancreatic lipase inhibition rate of the obtained enzymatic hydrolysis product was 38.64%(5 mg/m L).Isolation,purification,and identification of pecan hydrolysate(PPH)and screening of peptides with lipid-lowering activity through molecular docking.Ultrafiltration and Sephadex were used to separate and purify the enzymatic hydrolysis products.HPLC was used to determine the molecular weight distribution,HPLC-MS/MS to identify peptide fragments,Auto Dock Vina for molecular docking,and Discovery Studio to analyze interactions.The results showed that PPH-Ⅰwith a molecular weight of less than 3000 Da accounted for 87.49%of PPH,and had the highest inhibitory activity against pancreatic lipase.It was further purified by glucan gel and two components,PPH-Ⅰ-Ⅰand PPH-Ⅰ-Ⅱ,were obtained.The polypeptide sequences of the component PPH-Ⅰ-Ⅱwith stronger activity were analyzed and identified.The18 peptides with relatively high content were used as ligands,and pancreas lipase was used as the target for virtual screening of bioactive polypeptides.According to the binding energy,3peptides with possibly higher pancreatic lipase inhibitory activity were obtained,and their amino acid sequences were FREYYA(-8.6 kcal/mol),VIESW(-8.6 kcal/mol),and LRVF(-8.2kcal/mol),respectively.Among them,FREYYA and VIESW can form a more stable complex structure with enzyme protein molecules,and after database comparison,they were found to be new lipid-lowering peptide sequences.Through synthesis verification,the pancreatic lipase inhibitory activity IC50 values of FREYYA and VIESW were 0.25 mmol/L and 1.43 mmol/L respectively.Preparation and storage stability of pecan protein and pecan peptide beverages.The particle size was measured by a particle size analyzer,the viscosity was measured by a rotational rheometer,and the suspension stability and p H were measured.The single-factor experiment and uniform experimental design were used to optimize the production process.Using alkali-peeled pecan kernels as raw materials,the optimal process parameters of pecans protein beverage were:solid-liquid ratio 1:5(w/v),refining time 20 min,adding xanthan gum 0.15%(w/v),monoglyceride 0.06%(w/v),SE-11 sucrose ester 0.06%(w/v),white sugar 6%(w/v).Pasteurization and low-temperature storage were chosen as the sterilization method and storage conditions.The storage cost of the prepared pecan protein drink was relatively high.Therefore,the enzymatic hydrolysis product was used as the raw material to optimize the parameters of the pecan peptide beverage.The optimal process parameters were:adding peptide powder 3%(w/v),sucrose 8%(w/v),xanthan gum 0.10%(w/v).High-temperature sterilization and normal ambient temperature storage were chosen. |
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