Preparation, Properties, Bioactivities And Applications Of Giant Salamander Glyecopeptides

Giant salamander (Andrias davidianus) belongs to the class of Amphibia, order ofCaudata, family of Cryptobranchidae. It is a rare precious animal protected as Chinesenational second grade animal, mainly distributed in the upstream of Yangtze River,Zhujiang River, and Hanshui River. A large number of the domestication and breedingenterprises have been set up since1990s in Hunan, Hubei, Shanxi and Guangxi provinces.There are about35taming and breeding enterprises in Hunan province and about40,000-50,000giant salamanders are cultured every year. Deep processing and utilization ofgiant salamander need to be developed immediately. Therefore, fast creation of thetechnology for deep processing and utilization of giant salamander is an urgent task.Giant salamander is that there are many skin glands, instead of scales, on the bodysurface. These skin glands secrete a lot of mucus during the process of growth. If the mucuscan be used, sustainable utilization of giant salamander resources can be realized. Use ofmucus secreted by the skin glands of giant salamanders will exert no injuries to their growthand reproduction.As a species existed for350million years, Giant salamander has remarkablecharacteristics of longevity and self-repairs with a unique non-specific immune systempresented as in their mucus. Materials with biological activities for environmental changesare, after long-term evolution, contained in the mucus of giant salamander, which render themucus potential utilization in food industry.In this study, giant salamander glycopeptides (GSGPs) were obtained by using theAspergillus sp. acid protease from the marine organism to hydrolyze the mucus.. Thepreparation process, physical and chemical properties, biological activities of GSGPs andtheir application in juice and cosmetics are studied. The results are of theoretical andpractical significance for the processing and utilization of this mucus for the benefits ofhuman beings. Results are summerized as follows:1. The optimum conditions of marine-derived Aspergillus acid protease forhydrolyzing giant salamander skin mucus are obtained by the orthogonal test. The effectorder of hydrolysis is: hydrolysis time> E/S> temperature>pH. The optimum conditionsare:55C,pH:2.0,E/S:0.03%,,hydrolysis time:3h. Eutectic point, frozen-thaw processand freeze concentration process of the giant salamander hydrolysates are studied, and some of measures are obtained to improve the efficiency of freeze-drying of giant salamanderhydrolysates. Before freeze-drying operation, the giant salamander hydrolysates ispre-freezed in temperature range-8.6to-13.6C, meanwhile the solution concentration isimproved by the approaches of frozen-thaw and freeze concentration.2. The hydrolysates of giant salamander mucus (GSGPs) detected byMALDI-TOF-MS. Total protein and carbohydrate contents of GSGPs were estimated to be80.01%and15.15%, respectively. GSGPs contain3.39%glucosamine,0.65%glucuronicacid,2.45%galacturonic acid and0.60%sialic acid. While the sugar contents are22.38%,16.17%,4.29%and3.69%in total sugar respectively. Sulfuric acid sugar has not beendetected in GSGPs.18amino acids have been determined in GSGPs. In addition totryptophan not be detected, the other8essential amino acids (EAA) for human being arehigher. Amino acid of GSGPs was rich in Thr (13.1%), Pro, Ala, Leu, Arg and Phe.The O-glycosidic linkage of GSGPs is demonstrated by β-elimination reaction andblood cell agglutination test.GSGPs are performed peptide sequencing by nano-electrospray ionization massspectrometry/mass spectrometry(nano-ESI-MS/MS)．The database search is finished withthe Mascot search engine (http://www．matrixscience．co．uk) using the data processedthrough MasSeq． Amino acid sequences of3peptides are obtained as follows:KAPILSDSSCKSC, KLQGTVSWGSGCQAKNC and VVHSLVQVTANKVMVRM,respectively.3. The hydroxyl radical, DPPH free radical and superoxide anion radicals scavengingactivities are detected in GSGPs. The free radical scavenging capacity is graduallyenhanced with GSGPs concentration increasing. GSGPs at the concentration of1.0mg/mlexhibited54.69%scavenging activity against hydroxyl radical,92.25%against DPPHradical and52%against superoxide radical.GSGPs have showed an ACE inhibitory activity by HPLC. An ACE inhibitory activityratio attains90.82%at GSGPs concentration of40mg/mL.4. The impacts of different doses of GSGPs on immune functions of mice indicate that:the mice were divided into three groups (0.01g/kg bw,0.05g/kg bw,0.1g/kg bw)compared with control group (0g/kg bw) according to the body weight, and fed withGSGPs at three levels for8d, respectively. GSGPs at three levels obviously improved theamounts of antibody, enhanced the phagocytic capacity of macrophages and increased the amounts of positive ANAE cell compared with the control. Therefore, the immunologicalfunction of mice is improved by GSGPs.GSGPs were injected into the stomach of the mice once a day for15consecutive days.The mice are divided into2groups according to different GSGPs concentrations (lowconcentration of100mg/mL, high concentration of150mg/mL). Each group daily givendose is0.1mL/16g bw compared with control group with the same volume of saline bygavage. After intragastric administration for15d,the mice were forced swimming to deathwith5%body weight loaded. The results show that compared with control group,theswimming time prolonged significantly, up to103min (P <0.01); the concentration ofhepatic glycogen increased100mg/mL, by30.3%up to; the level of lactic acid in musclereduced43.2mg/mL, by39.2%; the level of urea reduced26mg/mL, by68.4%.Kunming male mice are divided into6groups: blank control group, model controlgroup, DDB control group [200mg/(kg d)], low GSGPs concentration [200mg/(kg d)],middle GSGPs concentration [400mg/(kg d)] and high GSGPs concentration [800mg/(kg d)]. GSGPs are given to mice once a day for7consecutive days by gavage. Theprotection of mice liver damaged by CCl₄has been detected. The results indicate that theactivity increases of AST and ALT caused by CCl₄are significantly inhibited by middleGSGPs concentration and high GSGPs concentration. In mice liver, the MDA content ofhigh GSGPs concentration has significantly been lowered, as well as SOD activityenhanced.Microscopic observations show that: in the CCl₄model group, liver cells are markedlyswelled and deformed, liver cell cords inordinate and accompanied by the inflammatory cellinfiltration. At low, middle and high GSGPs concentration,liver cell cords are arranged inorder, liver cells are regular arranged, and the damage is significantly reduced comparedwith the model group.5. The function of the glycopeptides in resisting the ultraviolet (UV) rays was strongerunder the wavelength from280to315nm. UV damaged test in mice demonstrates that themouse ear index of smearing GSGPs group is similar with the index of smearing ediblevegetable oil group without UV irradiation reveals smearing GSGPs on the skin of mouseear may avoid UV injury.6. Caco-2cell absorption test reveals that GSGPs can be absorbed by Caco-2cell.GSGPs can be absorbed by HaCaT cell; meanwhile GSGPs have the ability of thegrowth-promoting for HaCaT cell. MTT colorimetric assay indicates that compared with control group, the livability ratio of HaCaT cell cultured for72h in the GSGPs of0.098mg/mL is145%.7. As indicators of pH, precipitation rate, sensory score, and solid content, throughsingle factor and orthogonal experiments the optimum production technology conditions ofhealth beverage containing GSGPs are: the ratio of blackcurrant juice and blueberry juice1:3(15mL:45mL), the ratio of GSGPs and oyster polysaccharide1:3(1.5mL:4.5mL),lemonjuice10mL, pectin0.1%, sterilization temperature95C,sterilization time10min. Theproduct manufactured by the optimum technology has better color, flavor and stability.The cream with GSGPs has capability of hygroscopicity under the low humiditycondition(RH=43%), which is3.8%; under the high humidity condition(RH=81%) thehygroscopicity rate is15.6%. The stability of the cream made of GSGPs meets therequirements of Chinese Industry Standard QB/T1684-93. There is no allergic reactionappeared on the skin of guinea pigs smeared by GSGPs cream, similar with the skin ofhuman subjects.The work is supported by National Natural Science Foundation Project (31071612),Hunan Provincial Science and Technology Plan Project (2011FJ4098) and Science andTechnology Plan Project of Zhangjiajie (2011ZD16).