Preliminary Study On Rhythm Changes Of Glycogen Concentration And Activities Of Lysozyme From Oncomelania Hupensis

Part ⅠRhythm Changes of the Glycogen Concentration inOncomelania hupensisObjective：To evaluate the glycogen concentration seasonal changes and circadianchanges in different tissues of Oncomelania hupensis．Methods：Oncomelania hupensis snails were collected from the Qing Yi river ofWuhu city from August2012to July2013．The livers and pedal muscles were dissectedfrom body．Anthrone colorimetric method was used to evaluate the glycogen content ofthe whole body，liver and muscle in each months and the whole body within24hours．Results：The whole body’s and liver glycogen concentration decreased month by monthduring hibernation（from November to June）．The female whole body’s glycogenconcentration was the lowest in June （0.55μg/mg），but the male whole body’s glycogenconcentration was the lowest in April（0.88μg/mg）．The mean whole body’s glycogenconcentration was（♀=2.99μg/mg，♂=3.39μg/mg）．Liver glycogen concentration waslowest in May（♀=0.29μg/mg，♂=0.22μg/mg），both of them reached peak level inAugust（♀=2.49μg/mg，♂=2.78μg/mg）．The average of the liver glycogenconcentration was（♀=1.09μg/mg，♂=0.89μg/mg）．The muscle glycogenconcentration gradually decreased from February to June，the lowest was found in June（♀=0.25μg/mg，♂=0.41μg/mg），then reached peak level in December（♀=16.59μg/mg，♂=10.06μg/mg）．The average of the muscle glycogen concentration was（♀=7.99，♂=6.05μg/mg）．There was positive linear correlation between the whole body’sand liver glycogen concentration（P＜0.05），and both of them were have similar trend in their monthly change．A positive linear correlation was found among the wholebody’s，the liver and the muscle glycogen concentration（P＜0.05）．Begin with2o’clock，turn down2h’s results were5.227±1.000，4.495±0.100，7.010±0.200，6.175±0.500，6.37±0.700，7.097±0.300，8.242±0.300，5.895±1.500，5.362±1.70，6.5075±0.200，6.220±0.010，6.792±1.4μg/mg．By the single factor analysis ofvariance，the difference was statistically significant（F=3.763，P＜0.05）．Conclusion：The glycogen concentration was seasonal changes and circadianchanges in different tissues of Oncomelania hupensis． Part ⅡPreliminary Study on the Activities of Lysozyme fromOncomelania hupensisObjective：To identify the lysozyme in Oncomelania hupensis．Observe the optimalpH，temperature and inhibitory effect of the enzyme activities on bacterial growth．Toexplore the relationship between the lysozyme activities of Oncomelania hupensis snailsamong different seasons，and the difference of the lysozyme activities among the differentages of the snails．Methods：The soft tissues of Oncomelania hupensis were initially homogenated andimmersed in Tris-HCl-TritonX-114buffer solution for24hours then the supernatant wasextracted by centrifugation10000×g with10minute，the supernatant is incubated in a37℃water bath for15minute and centrifugation with2000×g，take the precipitate andcentrifugal30minute，with centrifugal filter units（Mr）=3000， at the temperature4℃for eccentricity5000×g，followed by preparation of the concentrated enzyme．Theprotein content lysozyme activity and the antibacterical effect were measured with bicinchonininc acid（BCA）method，turbidimetric method and agar diffusion（K-Bmethod），respectively．Results：The antibacterial protein lysozyme was identified in the gastropodOncomelania hupensis．The protein concentration in the July concentrated enzymeaccounted for3.428mg/ml．The total activity and specific activity arrived at（1520±240）U and（221±35）U/mg，respectively．The optimal pH and temperature againstMicrococcus lysodeikticus were pH7.0and50℃，respectively，and the enzyme wasstable at temperature35℃between60℃and pH5.0～8.0．The enzyme producedexclusive inhibitory effects on the Micrococcus lysodeikticus and Shigelladysenteriae．Their average inhibit diameter was10～12，12～15mm respectively．On thecontrary，the results showed no inhibition zone on Staphylococcus aureus，Escherichiacoli and Candida albicans．There were significant differences of lysozyme activitiesamong different seasons (spring，summer，autumn，winter) with the results of71.33±4.16U/mg，93.67±7.10U/mg，150.33±10.01U/mg，358±35.38U/mg，respectively(F=144.455，P＜0.01)．There were also significant differences of the activitiesbetween two age groups (adults and young snails)with the results of93.67±7.10U/mgand69.33±5.03U/mg，respectively (t=4.845，P＜0.05)．Conclusion：The concentrated enzyme can be extracted from the lysozyme in softtissues of Oncomelania hupensis with Tris-HCl-TritonX-114buffer solution，and theoptimal pH and temperature were pH7.0and50℃．The inhibitory effect was exhibitedon the micrococcus lysodeikticus and Shigella dysenteriae but have no inhibition zone onStaphylococcus aureus，Escherichia coli and Candida albicans．There are significantdifferences of the lysozyme activities among different seasons and ages，and the highestlysozyme activity is in winter．The lysozyme activity of adult snails is higher than that ofthe youth．...