Study On The Functional Components In Haliotis Discus Hannai Ino Gonad And Their Structure-activity Correlations

In recent years, the breeding scale of abalone has expanded unceasingly in China and the production has increased year by year. During the processing, a large number of low-value by-products, such as abalone gonad and shell, were normally discarded as industrial wastes, which accounted for 20～30% of the weight of abalone. Such processing method resulted in a waste of resources and environmental pollution. Abalone gonad is in a large proportion of organic contents, which is rich of functional components, such as proteins, polysaccharide.Hence, it is necessary to focus on the effective use of bioactive molecules in the gonad of abalone.In this study, the enzymatic hydrolysis technology has been used to obtain the polysaccharides and proteins (or peptides) from abalone (Haliotis discus hannai Ino) gonad, and their structure-activity relationship had been investigated. The main research contents and results are as follows:(1) Protease-assisted extraction and successive ion-exchange and gel-filtration chromatography technology were combined to isolate and purify the abalone gonad polysaccharides (AGPs).The composition of AGPs were then analyzed. The results illustrated that three homogeneous polysaccharides were obtained, named as AGP-31, AGP-32 and AGP-33, and the molecular weight (MW) is 37.8, 32.2 and 27.5 kDa, respectively. The chemical composition analysis indicated that all the three AGPs were sulfated polysaccharides without glycosaminoglycan-like structure, containing a great amount of neutral sugar 53.10～58.82%, uronic acids 17.02～18.26%, sulfated groups 8.30～9.48% and a small amount of proteins 0.68～1.04%. The percentage compositions were slightly different for the three AGPs: AGP-32 had the highest percentage of neutral sugars and the lowest percentage of uronic acids, whereas AGP-33 had the highest percentages of sulfated groups. They all contained mannose, rhamnose, glucuronic acid, glucose,galactose, xylose, arabinose and fucose, with very similar monosaccharide profile.(2) The structure of AGP-33 was revealed by chemical methods, Fourier transform infrared (FT-IR) spectroscopy, methylation and nuclear magnetic resonance (NMR) spectroscopy. The backbone fraction of AGP-33 (BAGP-33)was prepared by partial acid hydrolysis and eluted from Sephadex G-75.BAGP-33, with an average molecular weight of 17.0 kDa, has shown to be homogeneous by HPLC and composed of mannose, glucose and galactose.According to methylation and NMR spectroscopy, the backbone of AGP-33 was identified as mainly consisting of 1→3-linked, 1→4-linked, and 1→6-linked monosaccharides in a proportion of 1:1:3. Based on the experimental data above,it could be deduced that the backbone of AGP-33 may be consisted of(1→4)-linked galactose and (1→6)-linked glucose, linked together as a disaccharide unit of [→4)-α-Galp-(1→)-α-Glcp-(1→] n.(3) The biological activity of the AGPs was also investigated, including CCK-inducing activity, intracellular signaling pathways involved in AGP-induced CCK secretion in STC-1 cells and anticoagulant activity. The results showed that three polysaccharides extracted from abalone gonads were able to stimulate CCK secretion in intestinal endocrine STC-1 cells. The AGPs significantly increased the secretion of CCK at the concentration of more than 2 mg/mL. It was shown that MW of the AGP significantly influenced the stimulating effects, and that pathways Ca2+/CaM/CaMKII, cAMP/PKA and MAPK-p38 seemed to play roles in the AGP-induced CCK secretion. Anticoagulant activity of AGPs was evaluated by the classical coagulation assays APTT, PT and TT, by using heparin as a standard reference. It was concluded that all the AGPs showed significantly higher inhibition of thrombin, relative to normal saline at a sulphated polysaccharide concentration of 25 μg/mL. AGPs can prolong the activated partial thromboplastin time (APTT) and thrombin time (TT). The anticoagulant effects of AGPs were considered to inhibit the intrintic and/or common pathway, especially the last coagulant step of the thrombin-mediated fibrin formation. At a concentration of 50μg/mL, AGP-33, BAGP-33 and the desulphurization product (AGP-33-deS)showed that polysaccharide with higher molecular weight and sulfate content demonstrated greater anticoagulant activity .(4) The neutral protease was chosen as the tool enzyme, and the hydrolysis effects of neutral protease to the defatted female abalone gonads (AFG) and male abalone gonads (AMG) were investigated. The crude protein contents (on dry basis) of defatted abalone gonad were increased to 50.46±0.53% (female gonad)and 58.44±0.44% (male gonad), respectively. The results showed that both female and male abalone gonad can be effectively hydrolyzed by neutrase. The enzymatic hydrolysis of female and male abalone gonad with neutrase for 3 h resulted in degree of hydrolysis (DH) values of 37.12% and 35.78%, peptides yield values of 51.20% and 44.56%, respectively. The hydrolysis effect of neutrase on female gonad was better than that of male. The hydrolysis of female abalone gonad by neutrase contained more than 90% of components with MW below 3 kDa. The total amino acid composition of defatted abalone gonad power showed that gonad contained eighteen kinds of amino acids and the flavor amino acids, essential amino acids accounted higher than 77% for the total amino acids. Abalone gonad is the good raw materials for the development of bioactive peptides by hydrolysis.(5) The functional properties of protein hydrolysates from male abalone gonad, hydrolyzed by neutrase (AMGHs) with different DH were investigated.The gelation property were improved and the elastic modulus G’, viscous modulus G" and complex viscosity η* of AMGHs were raised, with the increase of hydrolysis degree. The elastic modulus G’ was always higher than that of viscous modulus G". Hydrolysates showed a significant shear thinning phenomenon,which should be non-Newtonian pseudoplastic fluid. For the functional properties,protein solubility was increased to above 65% over a wide pH range 2～10 after hydrolyzed for 50 min. Meanwhile, water holding capacity (WHC) and oil holding capacity (OHC) were significantly improved. When the DH increased, the interfacial activities (surface hydrophobicity, foaming capacity and foam stability)of hydrolysates increased firstly and then (after 50 min) showed a slight stable trend. While the emulsion activity index and emulsion stability index decreased during extensive hydrolysis.(6) Female abalone gonad was hydrolyzed with four commercially available proteases, including alkali protease, papain, neutrase and flavourzyme. The hydrolysis of female abalone gonad by neutrase (AFGHs-Neu) contained more than 65% of components with molecular weight below 3 kDa and was riched in antioxidant-related amino acids. The AFGHs-Neu obtained was fractionated by using gel-filtration chromatography technology. Five fractions were obtained from AFGHs-Neu on sephadex G-25 column, of which fraction 4 (F4) with the highest DPPH radical scavenging activity was further analyzed by ESI-QTOF-MS/MS,and a hepta-peptide His-Gly-Asp-Gly-Trp-Trp-Gln (884 Da), named as P1, was identified. P1 showed DPPH and hydroxyl radical scavenging activities with IC50 values of 6.50 and 7.48 mmol/L, respectively.