| Glutamine peptides are defined as a class of peptides contained non-N terminal glutamine, as steady carrier of free glutamine which can provide the glutamine supplement. Therefore the activity of glutamine peptides is presented by determining the non-N terminal glutamine content (Gln %). Glutamine is a conditional essential amino acid in stress situations and has multiply physiological function. Nevertheless the common usage of glutamine is restricted by its aqueous instability and low solubility. While it is reported that the stability and solubility of glutamine could be improved if the N-terminal is substituted.Wheat gluten is rich in glutamine high to 30%, and is a good source for preparation of glutamine peptides. Therefore, research on glutamine peptides prepared by enzymatic hydrolyzing wheat gluten, furthermore on purification and identification with ultrafiltration, chromatography and mass spectra, followed on analysis of In vitro and In vivo activities of glutamine peptides, which can obtain the good carrier of free glutamine and can provide the theory basis for the common use of glutamine peptides, meanwhile, can increase the economic value of wheat gluten.A new method of quantitative analysis of non-N terminal glutamine in peptides was first investigated, since binding glutamine could not be detected by common amino acids analysis for aqueous instability of glutamine. The new method contained four steps: BTI derivation into DABA, HCl hydrolysis, DNFB pre-column derivation and UPLC determination. Preparation of glutamine peptides from Alcalase hydrolyzing wheat gluten was optimized by response surface method in the next place. The optimal conditions were pH 7.5, temperature 50℃and Alcalase doses of 0.033 AU/gProtein, when Gln % was indexed and substrate concentration and hydrolysis time was settled at 6 g/100mL and 1 h respectively. Under the optimal conditions, Gln % was 15.85 g/100g and DH was 11.8%, and the average chain length was 8 amino acids and protein recovery reached 83%.Pretreatments of microwave, ultrasonic, dry heat, aqueous heat and adding cysteine on wheat gluten were done in the aim of improving Gln%. Results showed that both adding cysteine and aqueous heat at 90℃can highly improve Gln%, especially with cysteine Gln % increased by 20% to 18.81 g/100g. The effect of pretreatments on Alcalase hydrolysis and Gln % was further investigated through comparative analysis of SEM, secondary structure of protein, -SH, -S-S, hydrophobicity, hydration and solubility of wheat gluten protein before and after pretreatments. The SEM showed the formation of amyloid fibrils especially after microwave treatment, and the sheets of protein torn by all the treatments. The effect of pretreatments on secondary structure of protein was mainly presented the increase ofβ-sheet content. Hydrophobicity and hydration varied as different pretreatments. Base on that structural information, the mechanism of pretreatments affected Alcalase hydrolysis and Gln % was deeply discussed by Partial Least Square Regression (PLSR) method. The results showed that the degree of hydrolysis and Gln % both were highly dependent on secondary structures of intramolecularβ-sheet, hydratedβ-sheet,β-sheet at 1675 cm-1, totalβ-sheet, extended structure,α-helices,β-turn contents, and on hydrophobicity and SH- content. Glutamine peptides were conteniously purified by ultrafiltration, DA201-C macroporous resin, the ion exchange chromatography and gel chromatography, and the final Gln % was increased to 29.8 g/100g. Thereafter, the glutamine peptides were separated into four peaks by RP-HPLC. The four peaks were collected and identified by mass spectrum, which were LLEQRFLVDYL, QQPDESQQ, ENSPQSGGWNQT, and CLEYDWMDEQSDS. Among these four peaks, QQPDESQQ has the highest Gln% of 60.94 g/100g.Considering economy and practical application, UF-E of 20.1 g/100g was selected and UF-E’s physicochemical and physiological activities were investigated. The results indicated that, UF-E had good solubility with NSI higher than 80% within pH 2-12; the stable viscosity of 1 mpa·s was obtained with UF-E concentration among 1-20 g/100g; the denatured temperature was (85±3)℃detected by DSC; UF-E can resist the digestion of gastrointestinal enzymes; UF-E was stable to the conditions of heating from 50℃to 90℃and of autoclaving at 121℃at 30 min, respectively. In the terms of physiological activity, the inhibition of linoleic acid autoxidation and chelating ferrous activity were detected, which are typically representative of electron transfer and proton transfer oxidation mode respectively. The results showed UF-E had better inhibition of linoleic acid autoxidation, and the EC50 of UF-E on chelating ferrous was 0.46 mg/mL.Effect of UF-E (Gln%=20.1 g/100g) on glutamine supplement and physiological functions of weight-loading swimming rats was researched through intragastric feeding administration, and free glutamine was selected as control. Results demonstrated that UF-E can effectively supply glutamine for sportive fatigue rats and can be a good substitute of free glutamine, and UF-E had better antifatigue effect than free glutamine.The effect of UF-E on proliferation of normal rat splenocyte, human gastric cancer cell (SGC7901) and mice lymphoma cell (YAC-1) was innovatively studied by MTT method and inverted microscope, which was compared with Ala-Gln. The results revealed that UF-E had dose-effect relationship with three kinds cell observed. At UF-E concentration of 2 mmol Gln/L, the proliferation rate was the highest to 575%, meanwhile the inhibition rates of SGC7901 and YAC-1 reached 30% and 80% respectively, when compared with drug blank group. Ala-Gln had somewhat inhibition on both SGC7901 and YAC-1, but with no obvious dose-effect relationship. In general, it can be speculated that UF-E was selective in inhibition of cells. The inhibition effect of QQPDESQQ on SGC7901 was also studied, and the results revealed that QQPDESQQ had obvious dose-effect relationship, and it could be further conluded that QQPDESQQ is one of functional components of UF-E on inhibiting SGC7901. |
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