The Isolated Microbial Strains of the Hepatopancreas from Laminarin-Fed Prawn, Macrobrachium rosenbergii, Identified as Probiotics

本研究于喂食褐藻多醣后的淡水长脚大虾(Macrobrachium rosenbergii)之肝胰脏分离并评估因喂食出现的特定微生物作为益生菌之可行性。实验以30 µg褐藻多醣喂食每尾虾子连续喂食10天期间，喂食第5天的虾子肝胰脏分离到喂食前和对照组虾子都出现的梅奇酵母菌(Metschnikowia bicuspidata)菌株MB58及MB550。毒性测试发现，菌株MB58及MB550分别注射处理虾子后造成的死亡率分别为12%和22%，均低于已知为益生菌的Lactococcus plantarum的致死率87%。将致病菌分别培养于菌株MB550和MB58之胞外液的结果显示，两菌株都可以抑制致病菌Aeromonas hydrophila和A. veronii的生长；但仅MB550能抑制Lactococcus garvieae。连续口服MB58、MB550及混合菌株(MB58 + MB550)后五天，以L. garvieae感染的虾子存活率分别为70.6%、73.4%和100%，然控制组虾子全数死亡。免疫相关之原酚氧化酵素(prophenoloxidase, proPO)活化系统的表现显示，喂食后5天，MB550喂食组和MB58 + MB550混合喂食组的虾子血浆的活化型酚氧化酵素活性(POS)和总酚氧化酵素活性(POT)与对照组均无差异，然两组虾子的血球内POS及POT则高于对照组；MB58喂食组虾子仅血浆POS高于对照组，血浆POT和血球内POS及POT均与对照组无差异。进一步以褐藻酸(alginic acid)作为黏着剂，将菌株MB58及MB550分别包裹入饲料以制作出5种颗粒饲料，分别连续喂食虾子五天后，易感性实验显示，未包裹菌株的对照组虾子的死亡率为47.8%，仅包裹褐藻酸组23.8%，以褐藻酸包裹MB58、MB550及两株混合的三组虾子的死亡率分别为10.3%、12.0%及11.5%，均明显低于对照组。喂食5天和感染后1天和3天分别测定血浆及血球内POT的结果显示，MB58喂食组仅于感染前的血浆POT高于对照组；MB550组于感染前后的血浆和血球内的POT皆明显高于对照组；混合组则于感染前血浆及血球内POT和感染后血浆POT皆高于对照组。综合上述结果推测，单独喂食菌株MB58和MB550可能抑制致病菌生长和增强proPO活化系统，以提升虾子对感染的抗抵力而降低虾子的死亡率。因此，分离自喂食褐藻多醣之虾子的两株梅奇酵母菌MB58和MB550可能具有作为益生菌的潜力以应用于虾类水产养殖。 This study tries to isolate special microbes from the hepatopancreas of laminarin-fed prawn, Macrobrachium rosenbergii, and further evaluate whether these microbes can be used as probiotics. On days 5 after continuous feeding with laminarin (30 µg/prawn), two strains MB58 and MB550 belonged to Metschnikowia bicuspidata were isolated from the hepatopancreas, but the same species was not isolated in the domesticated prawn and control prawn. The virulence test found that, after injection with MB58 and MB550, the mortality rate of prawn was 12% and 22%, respectively; both mortalities were lower than 87% caused by what is known as probiotic Lactococcus plantarum. The results from the pathogenic strains cultured in the extracellular fluids of MB58 or MB550 showed that the growth of Aeromonas hydrophila and A. veronii could be inhibited by the both strains and that of Lactococcus garvieae only was inhibited by strain MB550. The survival of prawn challenged with L. garvieae after feeding for 5 days was 70.6%, 73.4% and 100% for prawn fed with only MB58, MB550 and both strains, respectively; however, the control group prawn all died. After feeding for 5 days, the activities of the stimulated phenoloxidase (POS) and the total PO (POT) in hemocytes of the MB550-fed group and the mix-fed group were higher than those of the control group; however, those in plasma were not significantly different from the control. For the MB58-fed group, only plasma POS was higher than the control group. Furthermore, the strains MB58 and MB550 were separately encapsulated into the feed by alginic acid as the adhesive to produce five kinds of particulate feeds. The mortality rates of prawn challenged with L. garvieae after feeding for 5 days were 10.3%, 12.0% and 11.5% for the MB58-fed, MB550-fed and mix-fed groups, respectively; these rates were lower than that of the control group (47.8%). The results of POT detected on days 5 after continuous feeding and day 1 and day 3 after challenge showed that the POT in plasma and hemocytes of the MB550-fed group was significantly higher than that of the control group either before or after infection and that the POT of the MB58-fed group was significantly higher than that in control group only in plasma before infection. For the mix-fed group, the POT in plasma and hemocytes was higher than that of the control group before infection, and that in hemocytes was also higher than that of the control after infection. These results suggest that the two strain MB58 and MB550 by themselves or mixed may enhance the resistance against infection and reduce the mortality of prawn by both the inhibition of the growth of pathogens and the in-crease of the proPO-activating system. Therefore, the both strains should have the potential as probiotics to be used in prawn aquaculture.