Optimization Of Manufacturing Key Techniques Of Pine Nut (Pinus Koraiensis) Immunoreactive Peptides

The research of the key technology on producing Pine Nut (Pinus Koraiensis)Immunoreactive Peptides (PNIP) was belonged to the National High Technology Researchand Development Program (863Program) Project (2013AA102206), stability andapplicability of nuts active protein were mainly responsible for research tasks. Four partsresearch work was carried out around the project research tasks, the conclusions wasbelow:(1) On study of controllable hydrolysis pilot process on pine nut meal,single-factortest and response surface were carried out to optimize parameters, single-factor testswere investigated the effects of temperature (45℃,50℃,55℃,60℃,65℃), pHvalue (6.00,7.00,8.00,9.00,10.00,11.00), enzyme amount (2%,4%,6%,8%,10%,12%)on the rate of hydrolysis. Based on the single-factor test, the response surface optimizationtest was carried out.Build a regression equation: Y=32.48+5.38X1+2.13X2+2.17X3. R2=0.6901Adjusted.R2=0.6056Pred R-Squared=0.4122Adequate.Precsion=8.278,The optimalprocess parameters of controllable hydrolysis pilot process on pine nut meal: pH9.85, thetemperature was49.61℃, enzyme dosage was10.89%, degree of hydrolysis predictivevalue of39.97%. Four kinds of pine nut poly peptides were obtained after theultrafiltration, molecular size of each component were>30,10-3,3-1,<1k Da.(2) On immunoreactive evaluation of4kinds of pine nut polypeptides,immuneactivity of4kinds of pine nut polypeptides was evaluated separately from the innateimmune system and the adaptive immune system using animal testing methods, includingthe tests: Determination of NK cell activity, carbon clearance test, peritonealmacrophages engulf fluorescent microspheres test, factor levels were measured mousecells (IL-2, IL-4, IL-6, IFN-γ), lymphocyte proliferation test, delayed metamorphosisreaction, determination of serum hemolysin. All test results show that ICR mice orallyadministered pine nut polypeptide at100mg/(kg d) the10-3kDa for four weeks, the testresults were positive (P<0.05). According to the "health food function evaluation procedures and test methods" requirement, the determination pine nut peptides at100mg/(kg d)10-3kDa could enhanced immune function.(3) On study of genotoxicity evaluation of PNIP, sperm abnormality test andmicronucleus test of bone marrow (PCE) were adopted. The results showed that gavageorally to ICR mice with four kinds of PNIP for four weeks, the sperm abnormality andPCE rate within the normal range. So there were no genotoxic effects of PNIP.(4) In the study of granulating process of PNIP microcapsule, the experiment selectedseparated screening immune active peptide as raw material, selected sodium alginate as asingle wall for embedding, and selected the embedding rate as the main test indicators. Bymeans of the single factor experiment and the response surface design experiments to getthe regression equation of red pine immunoreactive peptide microcapsule, that is Y=11.36+5.26X1+41.08X2+20.00X3—0.35X1X2+0.10X1X3—0.72X2X3—1.75X12—10.60X22—2.41X32，X1is calcium chloride concentration (%), X2for sodium alginateconcentration (%) X3for peptide liquid concentration (%) The optimal process parametersof microencapsulation crafts: concentration of calcium chloride1.48%, sodium alginateconcentration1.71%, peptide liquid concentration3.68mg/mL, the embedding rate90.20%.