The Controllable Preparation Of Pollock Immunomodulating Peptide And Its Immunity Activity

Immunomodulating peptide can enhance the immunity of organism, the phagocytose ability of macrophages, and ability of anti-infection, which exhibited the many advantages such as the low molecular mass, good stability, weak immunogenicity, high biological activity and so on. Recently the study of bioactive peptides mainly focused on the optimization of bioactivities and kinetic model, while neither of them could confirm that the hydrolysis reaction was conducted as expected. In order to achieve the accurate prediction, dynamic monitoring and controlled hydrolysis during the preparation process of immunomodulating peptide, biotechnology, biosensors, mathematical model, and artificial neural network were used in the study. The bioactive peptides were refined by the resin. It could provide a theoretical basis and technological support for industrialization of immunomodulating peptide. In addition, the immune mechanism of bioactive peptides was also studied.The research results of this study were as follows:1,The Alaska Pollock frame (APF) was used as research materials and the main compositions were determined. The crude protein level of APF was high, with the content of 18.4%, which mainly consisted of alkali-soluble protein (35.49%) and stromatin (30.49%). It exhibited the high level of Gly (26.51%) and Glu (12.57%), followed by Ala, Asp, Ser, Leu and Lys. In order to make full use of APF, high- pressure cooking was set at 120℃for 30 min2,Degree of hydrolysis values, molecular weight distribution, and peptide levels were the important factors that reflected the characteristics of the hydrolysates, but it was difficult to monitor them online. Free amino acid levels of APF hydrolysates showed regular changes and can be used as response factor for indicating DH. They can be detected by biosensor, monitor the hydrolysis reaction. The advantage of the biosensor response factor is fast, high accuracy, good stability. To the measurements of glutamic acid and lysine, the relative error were 1.5% and 1.0%, coefficient of variation were 3.85% and 3.03%, the standard deviation are 0.78 and 0.60, respectively. Although seven enzymes were used for hydrolysis, free glutamic acid and lysine were changed regularly, and positively related to degree of hydrolysis, which can be used as hydrolysates response factor, while within a certain degree of hydrolysis, free glutamate and lysine acid concentration and the degree of hydrolysis showed a good linear relationship. It's feasible to using the concentration of free glutamic acid and lysine as the response factors to monitor the degree of hydrolysis.3,It was confirmed that APF trypsin hydrolysates could enhance spleen lymphocyte proliferation, T lymphocyte proliferation, and phagocytic activity of macrophages (p<0.05) significantly. The molecular mass and degree of hydrolysis (DH) were important factors effecting on spleen lymphocyte proliferation. The highest proliferation ratio was reached in the range of 15-18%. APF was hydrolyzed by trypsin treatments to obtain immune activity polypeptides. The optimum parameters of hydrolysis were obtained by response surface methodology (RSM) as follows: fish protein concentration of 25g/L, pH 8.0, 50.0℃, time 290min, and [E]/[S] 24 U/mg, respectively. The average DH of five verification tests was 16.87% and the average spleen lymphocyte proliferation was 28.45%, which was in agreement with theoretical value. The immune activity peptide (PFH) was characterized in this study. It exhibited a maximum absorbance at 220 nm and 280nm and was rich in PRO levels (15.69%). PFH showed good solubility over a wide pH range and low viscosity in the range of 20-60℃; It showed significantly effect of pH on foaming properties and emulsifying characteristics. Although PFH had a little change which was treated by pepsin, trysin, and complex enzymes, the main composition was very similar to that of PFH before treatment.4,The kinetic model can reflect the hydrolysis process when the hydrolysis condition was constant. The kinetic model of APF and parameter of controlled- enzymatic hydrolysis were obtained by mathematic deduction and experimental analysis. The kinetic formulas were as follows: R= (0.1693 E0– 0.2816 S0) exp│-0.357 * DH│, DH= 2.801 ln│1 + (0.06044 E0/S0– 0.1005)t│, deactivation rate constant Kd was 0.0512 min-1. The kinetic model of PFH was as follows: DH= 2.801 ln [1+ 1.35006t].The preparation time of PFH can be obtained by its kinetic model, so it was very important for its preparation. The verification tests showed that the kinetic model could predict the hydrolysis process of PFH under constant condition. However, the hydrolysis conditions such as changes of stirring rate and temperature, were varied, and had significantly effect on final hydrolysates. Therefore, it was necessary to monitor the hydrolysis process online for the maximum immune activity peptide content in finally hydrolysates.5,The biosensor was used in the study for monitoring preparation of the immunodulating peptide of Alaska pollock frame under the constant and dynamic condition. It exhibited the good linear relations between degree of hydrolysis and free lysine and free glutamic acid when DH was in the range of 11-18% under constant condition. The formulas were as follows: DH = 0.2225[Glu] - 5.3097,DH = 0.0126 [Lys] - 1.2275. The mathematical relationship between concentrations of glutamic acid and lysine and DH was not ambiguity and it showed the ruleless curve relation when the initial enzyme concentration and initial substrate concentration were varied. Although they could not depict the relationship using the uniform mathematical model, the critical value of the hydrolysis reaction exhibited the good linear relationship and the formulas were as follows: [Glu] = 0.8186 [S0] + 0.0346 [E0] + 0.4506,[Lys] = 0.7430 [S0] + 0.1370 [E0] + 5.2931.6,The three network models were established for monitoring preparation of immune activity peptide under the dynamic condition including GLU-BP-ANNs LYS-BP-ANNs and GLU-LYS- BP- ANNs. They were based on BP-ANNs and biosensor. The response factors were included free Glu,Lys,Glu-Lys and so on. Comparative analysis of sample value and simulation value showed that R2 values were 0.9967,0.9964 and 0.9967,simulation error ranges were 0-4.14%,0-4.56% and 0-4.71%,simulation average error values were1.06%,0.94% and 0.99%。The verification tests of the three network models (five times for each) showed that they had a good reliability, and the theoretical value was in agreement with experimental value. The relative error ranges were 0.23%-2.81%,0.26%-1.91% and 0.30%-2.35%, respectively. Therefore the three network model can monitor the hydrolysis of Alaska Pollock frame under the dynamic conditions. The common parameters of the three models were as follows: one hidden layer, transfer function logsig in layer one, transfer function purelin in layer two, training function trainlm, adaptation learning function learngdm. The other parameters of GLU-BP-ANNs were as follows: input layer nodes were initial enzyme concentration E0, initial substrate concentration S0, and free glutamic acid concentration [Glu]; the number of output layer nodes was 10. The other parameters of LYS-BP-ANNs were as follows: input layer nodes were initial enzyme concentration E0, initial substrate concentration S0, and free lysine concentration [LYS]; the number of output layer nodes was 11. The other parameters of GLU-LYS-BP-ANNs were as follows: input layer nodes were E0, S0, [LYS] and [Glu]; the number of output layer nodes was 8.7,Three immune activity peptides were isolated from the Alaska pollock frame trypsin hydrolysates, using the chromatographic methods including Sephadex G-25 gel filtration chromatography, SP Sephadex C-25 ion-exchange chromatography and reversed phase high-performance liquid chromatography. The purity of immunomodulating peptide was measured using RP-HPLC equipped the Zorbax SB C18 analysis column. The sequences of peptide were identified by Nano-ESI-Ms/Ms as follows. The sequence of Immunomodulating peptide Y3 was Asn– Gly– Met– Thr– Tyr with molecular mass of 583.9922Da, and the spleen lymphocyte proliferation ratio treated by Y3 was 35.92%. The sequence of Immunomodulating peptide H2 was Asn– Gly– Leu– Ala– Pro with molecular mass of 470.1422Da, and the spleen lymphocyte proliferation ratio treated by H2 was 32.96%. The sequences of peptide were identified by Nano-ESI-Ms/Ms as follows. The peptide molecular mass and sequence of S4 was Trp-Thr and 305.1622Da, with the spleen lymphocyte proliferation ratio was 31.35%.8,The effects of PFH on immunomodulatory activities in normal and immunodepression mice were investigated by oral administration. PFH enhanced spleen lymphocyte transformation(P<0.05),the delayed-type hypersensitivity (p<0.05) and the phagocytic index and rate of macrophages(P<0.05) significantly in normal mice (P<0.05), however, PFH showed the limited effects on the immune organ index and the hemolysin content (p>0.05). In hypoimmune mice, PFH increased the immune organ index (p<0.05). PFH could heighten the delayed-type hypersensitivity level (p<0.05)and spleen lymphocyte transformation (P<0.05), and it was indicated that PFH enhanced the function of cellular immunity in hypoimmune mice.In addition, PFH could increase the hemolysin content (P< 0.05) and promote the humoral immunity of hypoimmune mice. PFH also could enhance the ability of macrophages for carbon particle clearance (P< 0.01)and the phagocytic index and rate (P< 0.05, P< 0.01) significantly, and it was indicated that PFH could non-specific immunity in immunodepression mice.9,The adsorbing capacity of DA201-C resins was best. The highest adsorbing rate was 84.7% under 25℃, pH 4.0, the solution 10 mg/mL, and the ratio of resin mass to the volume of peptide 2:1 (g/mL). The highest rate of desorption (82.7%) was reached at the ethanol concentration of 50%. In dynamic adsorption and desorption, the results showed that the desalting and recovery rates of peptides were 98.1% and 90.3%, respectively. The sample desalted by DA201-C resin could enhance the splenocyte proliferation in a dose-dependent manner.