两种芽孢杆菌微胶囊制剂的研制及其对中华绒螯蟹免疫功能的影响
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摘要
本文报道了枯草芽孢杆菌和蜡状芽孢杆菌海藻酸钠微胶囊的制备工艺及其对中华绒螯蟹免疫功能及抗病力影响的研究结果,该结果对水产动物饲用益生菌制剂剂型研究和中华绒螯蟹益生菌类免疫增强剂的研发具有重要的参考价值。试验包括两种芽孢杆菌微胶囊制备工艺研究(试验一)和芽孢杆菌微胶囊制剂对中华绒螯蟹免疫功能影响的研究(试验二)两部分。
试验一:以生物相容性良好的海藻酸钠为壁材,以枯草芽孢杆菌和蜡状芽孢杆菌为芯材,应用乳化法制备芽孢杆菌微胶囊。通过单因素试验和正交试验,确定了两种芽孢杆菌微胶囊制备的较佳工艺:海藻酸钠浓度2%,乳化时间15 min,搅拌速度600 r/min,菌胶比例1:6。以该工艺制备的枯草芽孢杆菌和蜡状芽孢杆菌微胶囊的包封率分别为59.91%和58.33%,两种芽孢杆菌微胶囊均具有良好的耐酸性和肠溶性。
试验二:在基础饲料中分别添加0(对照)、1、5 g/kg的枯草芽孢杆菌、蜡状芽孢杆菌微胶囊及两种芽孢杆菌1:1混合微胶囊制剂的冻干粉(活菌含量均约为2.2×1011 cfu/g),制成颗粒饲料后投喂中华绒螯蟹7 d,然后所有试验组均改投对照组饲料。在停喂含益生菌的试验组饲料后0、1、2、3周,分别采样测定中华绒螯蟹的血细胞总数(THC)、不同血细胞数量(DHC)、血细胞呼吸爆发活性、血细胞胞内受益生菌激活的酚氧化酶(POS)和总酚氧化酶(POT)活性以及血清酚氧化酶(PO)、过氧化物酶(POD)、溶菌酶(LSZ)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)活性,并在停喂试验组饲料后第2周,以1.2×107 cfu/kg体重的剂量,体腔注射致病性嗜水气单胞菌CL99920菌株,记录接种10 d后中华绒螯蟹的累积死亡率。结果显示:枯草芽孢杆菌、蜡状芽孢杆菌微胶囊制剂及两种芽孢杆菌1:1混合微胶囊制剂均能不同程度地增加中华绒螯蟹的THC和HC数量、血细胞的呼吸爆发活性、血细胞胞内POS与POT活性以及血清PO、POD、LSZ、ACP和AKP活性,在多数采样时间点,各实验组的上述免疫指标活性均显著高于对照组(P<0.05),并且以5 g/kg剂量的刺激效果为佳。同时,饲喂试验用芽孢杆菌微胶囊制剂能明显地提高中华绒螯蟹对嗜水气单胞菌的抗感染能力,其中饲喂5 g/kg饲料剂量的枯草芽孢杆菌微胶囊制剂对中华绒螯蟹的免疫保护率最高(71.43%)。试验表明,在饲料中添加一定量的枯草芽孢杆菌、蜡状芽孢杆菌微胶囊及两种芽孢杆菌1:1混合微胶囊制剂均能明显地增强中华绒螯蟹的免疫功能和抗病力。
The results of the preparation technology of alginate microcapsules of Bacillus subtilis and B. cereus and theirs effects on immunological function of Chinese mitten crab Eriocheir sinensis are first reported in the paper. The results have important reference value to study on the preparation form of feed probiotics of aquatic animals and immunostimulants of E. sinensis. The experiment is composing of two parts as follows:
Experiment one: The sodium alginate which has good biology consistent was used as shell-material and the B. subtilis or B. cereus was used as core-material, then preparing microcapsulation with emulsification method. The optimal technological conditions of making the microcapsule were determined through single factor test and orthogonal experiment of L9 (34). The results were as follows: the concentration of sodium alginate was 2%, the emulsification time was 15 min, the stirring speed was 600 r/min, the proportion of B. subtilis or B. cereus and sodium alginate was 1:6. This method can prepare microcapsules which have good enteric solubility and acid resistance, embedding rate was 59.91% and 58.33%, respectively.
Experiment two: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of alginate microcapsules of living B. subtilis, B. cereus, B. subtilis and B. cereus (1:1) (about 2.2×1011 cfu/g powder) dosed at 0, 1 or 5 g/kg dry diet for 1 week, respectively. The total haemocyte count (THC), different haemocyte count (DHC), respiratory burst (release of superoxide anion) of haemocytes, the simulated PO activity by Bacillus (POS) and total phenoloxidase (POT) in haemocyte lysate supernatant (HLS), phenoloxidase (PO), peroxidase (POD), lysozyme (LSZ), acid phosphatase (ACP), alkaline phosphatase (ALP) activities in serum were examined at 0,1,2,3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results indicated that experimental groups of feeding microcapsules of Bacillus showed all higher haemocyte count (THC), hyaline cells (HC), higher respiratory burst (release of superoxide anion) of haemocytes, while boosted up POS and POT activities in haemocyte lysate supernatant (HLS), phenoloxidase (PO), peroxidase (POD), lysozyme (LSZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in serum. The activities of these immune parameters of test groups were significantly higher than control group at many sampling time points (P<0.05). The stimulate effects of the 5 g/kg group is more superiority than the 1 g/kg group at many sampling time points. Crab of test groups were the more resistant. It was concluded that E. sinensis that were fed with diets concluding microcapsules of Bacillus showed increased immune ability as well as resistance to A. hydrophila infection.
试验一:以生物相容性良好的海藻酸钠为壁材,以枯草芽孢杆菌和蜡状芽孢杆菌为芯材,应用乳化法制备芽孢杆菌微胶囊。通过单因素试验和正交试验,确定了两种芽孢杆菌微胶囊制备的较佳工艺:海藻酸钠浓度2%,乳化时间15 min,搅拌速度600 r/min,菌胶比例1:6。以该工艺制备的枯草芽孢杆菌和蜡状芽孢杆菌微胶囊的包封率分别为59.91%和58.33%,两种芽孢杆菌微胶囊均具有良好的耐酸性和肠溶性。
试验二:在基础饲料中分别添加0(对照)、1、5 g/kg的枯草芽孢杆菌、蜡状芽孢杆菌微胶囊及两种芽孢杆菌1:1混合微胶囊制剂的冻干粉(活菌含量均约为2.2×1011 cfu/g),制成颗粒饲料后投喂中华绒螯蟹7 d,然后所有试验组均改投对照组饲料。在停喂含益生菌的试验组饲料后0、1、2、3周,分别采样测定中华绒螯蟹的血细胞总数(THC)、不同血细胞数量(DHC)、血细胞呼吸爆发活性、血细胞胞内受益生菌激活的酚氧化酶(POS)和总酚氧化酶(POT)活性以及血清酚氧化酶(PO)、过氧化物酶(POD)、溶菌酶(LSZ)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)活性,并在停喂试验组饲料后第2周,以1.2×107 cfu/kg体重的剂量,体腔注射致病性嗜水气单胞菌CL99920菌株,记录接种10 d后中华绒螯蟹的累积死亡率。结果显示:枯草芽孢杆菌、蜡状芽孢杆菌微胶囊制剂及两种芽孢杆菌1:1混合微胶囊制剂均能不同程度地增加中华绒螯蟹的THC和HC数量、血细胞的呼吸爆发活性、血细胞胞内POS与POT活性以及血清PO、POD、LSZ、ACP和AKP活性,在多数采样时间点,各实验组的上述免疫指标活性均显著高于对照组(P<0.05),并且以5 g/kg剂量的刺激效果为佳。同时,饲喂试验用芽孢杆菌微胶囊制剂能明显地提高中华绒螯蟹对嗜水气单胞菌的抗感染能力,其中饲喂5 g/kg饲料剂量的枯草芽孢杆菌微胶囊制剂对中华绒螯蟹的免疫保护率最高(71.43%)。试验表明,在饲料中添加一定量的枯草芽孢杆菌、蜡状芽孢杆菌微胶囊及两种芽孢杆菌1:1混合微胶囊制剂均能明显地增强中华绒螯蟹的免疫功能和抗病力。
The results of the preparation technology of alginate microcapsules of Bacillus subtilis and B. cereus and theirs effects on immunological function of Chinese mitten crab Eriocheir sinensis are first reported in the paper. The results have important reference value to study on the preparation form of feed probiotics of aquatic animals and immunostimulants of E. sinensis. The experiment is composing of two parts as follows:
Experiment one: The sodium alginate which has good biology consistent was used as shell-material and the B. subtilis or B. cereus was used as core-material, then preparing microcapsulation with emulsification method. The optimal technological conditions of making the microcapsule were determined through single factor test and orthogonal experiment of L9 (34). The results were as follows: the concentration of sodium alginate was 2%, the emulsification time was 15 min, the stirring speed was 600 r/min, the proportion of B. subtilis or B. cereus and sodium alginate was 1:6. This method can prepare microcapsules which have good enteric solubility and acid resistance, embedding rate was 59.91% and 58.33%, respectively.
Experiment two: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of alginate microcapsules of living B. subtilis, B. cereus, B. subtilis and B. cereus (1:1) (about 2.2×1011 cfu/g powder) dosed at 0, 1 or 5 g/kg dry diet for 1 week, respectively. The total haemocyte count (THC), different haemocyte count (DHC), respiratory burst (release of superoxide anion) of haemocytes, the simulated PO activity by Bacillus (POS) and total phenoloxidase (POT) in haemocyte lysate supernatant (HLS), phenoloxidase (PO), peroxidase (POD), lysozyme (LSZ), acid phosphatase (ACP), alkaline phosphatase (ALP) activities in serum were examined at 0,1,2,3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results indicated that experimental groups of feeding microcapsules of Bacillus showed all higher haemocyte count (THC), hyaline cells (HC), higher respiratory burst (release of superoxide anion) of haemocytes, while boosted up POS and POT activities in haemocyte lysate supernatant (HLS), phenoloxidase (PO), peroxidase (POD), lysozyme (LSZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in serum. The activities of these immune parameters of test groups were significantly higher than control group at many sampling time points (P<0.05). The stimulate effects of the 5 g/kg group is more superiority than the 1 g/kg group at many sampling time points. Crab of test groups were the more resistant. It was concluded that E. sinensis that were fed with diets concluding microcapsules of Bacillus showed increased immune ability as well as resistance to A. hydrophila infection.
引文
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