酶解海藻产物对刺参（Apostichopus japonicus）肠道菌群和免疫相关因子的影响
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摘要
本研究将0.1%的褐藻寡糖(ADO)和5%经褐藻胶裂合酶酶解的海藻粉添加到刺参基础饲料中进行周期40d的投喂实验,实验期间每隔10d取样一次,取样后检测刺参肠道内细菌总数和弧菌总数,体腔液和体壁中免疫相关因子的活性和免疫相关酶同工酶的变化,研究海藻粉和褐藻寡糖对刺参非特异性免疫的影响和初步作用机制,具体实验内容和结果如下：
1.采用平板计数的方法测定刺参肠道内细菌总数和弧菌总数。对照组刺参肠道细菌总数波动较大,海藻粉组和褐藻寡糖组细菌总数波动较小；实验期间海藻粉组刺参肠道细菌总数均小于对照组,但未形成显著性差异(P>0.05)。海藻粉组和褐藻寡糖组刺参弧菌总数占细菌总数的比值均大于对照组,而褐藻寡糖的体外抑菌实验中褐藻寡糖对副溶血弧菌没有抑制作用,推测海藻粉和褐藻寡糖对刺参肠道内优势茵群弧菌属部分细菌有一定的促进作用。
2.采用分光光度技术测定了海藻粉和褐藻寡糖对刺参体腔液和体壁组织中免疫相关因子溶菌酶(LSZ)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和溶血素活性的影响。结果表明：实验期间海藻粉组和褐藻寡糖组刺参免疫相关因子活性均高于对照组。海藻粉组和褐藻寡糖组刺参体腔液中ACP、AKP、溶血素和LSZ活性分别在第20d、20d、30d和40d达到最高,并且均与对照组形成显著性差异；海藻粉组和褐藻寡糖组刺参体壁中ACP、AKP和溶血素在其活性最高时均与对照组形成显著性差异。刺参体腔液中免疫相关因子活性的提高幅度要比体壁大,这可能是因为体腔液包含体腔细胞和各种体液免疫因子,是机体免疫反应发生的主要场所,其对免疫刺激物的免疫刺激反应比较强烈,因此活性的提高幅度较大。
3.海藻粉组和褐藻寡糖组刺参体腔液和体壁组织中抗氧化相关酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性实验期间均高于对照组。体腔液和体壁组织中抗氧化酶均呈现先增高后降低的趋势,体腔液中酶活性达到峰值的时间比体壁组织提前10d并且酶活性提高幅度较大,推测是由于体腔液中免疫刺激反应比较强烈所致。同工酶结果表明：褐藻寡糖和海藻粉对刺参抗氧化相关酶的诱导提高作用不仅体现在增加酶的浓度,同工酶酶带的增多也是增加酶活性的途径之一
海藻粉和褐藻寡糖对刺参肠道的稳定性和优势菌群的生长均有一定的促进作用,并且可以诱导刺参体腔液和体壁组织中免疫相关因子活性的提高,对刺参非特异性水平有一定的提高作用,因此将海藻粉和褐藻寡糖开发为水产动物尤其是刺参的免疫增强剂具有广阔的前景。
The effects of enzymatic hydrolysis algae powder by alginate lyase and alginate derived oligosaccharide (ADO) on the non-specific immune system of sea cucumber (Apostichopus japonicus) and the preliminary mechanism were studied by supplying 5% algae powder and 0.1% ADO in diets. The total numbers of the bacteria and the vibrio.spp, the activities of immune-relative factors in the coelomic fluid and the body wall and the changes of the isozymes were determined every 10 days during the experiment period (40 days). The specific content and results are as follows:
1.The total numbers of bacteria and vibrio.sp in the intestinal tract of sea cucumber were measured with the plate counting method. The fluctuation range of the total number of bacteria of the control was wider than those of the algae powder group and the ADO group. The total numbers of bacteria of the algae powder group were all smaller than those of the control, but did not have significant differences (P>0.05). The ratios of the total numbers of vibrio.sp to bacteria of the algae powder and ADO groups were higher that of the control. The ADO did not have antibacterial activity to vibrio parahaemolyticus in vitro experiment, so the algae powder and ADO could promote the growth of some bacteria belonging to the vibrio.sp which is one of the dominant flora in the intestinal tract.
2.Effects of algae powder and ADO on the activities of lysozyme (LSZ), acid phosphatase (ACP),alkaline phosphatase (AKP) and hemolysin in the coelomic fluid and the body wall were studied by the use of spectrophotometry. The results indicated that all the activities of immune-relative factors of the algae powder and ADO groups were higher than those of the control. The activities of the ACP, AKP, hemolysin and LSZ reached their maximum values on the 20th,20th,30th and 40th day respectively, and they all had significant differences compared with the control.The highest activities of ACP, AKP and hemolysin also had significant differences compared with the control. The coelomic fluid contains a variety of humoral immune factors and immune cells and the immune reactions take place here. So the reactions of the immune-relative factors in the coelomic fluid to the immunologic stimulant were stronger than those of the body wall and the enhancements of the activities of immune-relative factors were also higher.
3. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) of the algae powder and ADO groups were all higher than those of the control during the experiment period, and they all increased firstly and then decreased. The time that the activities of enzymes in the coelomic fluid reached the peak was 10 days ahead of that in the body wall and the enhancements of the activities were also higher than those in the body wall. The reason may be that the reactions of the immune-relative factors in the coelomic fluid to the immunologic stimulant were stronger than those of the body wall too. The results of the isozymes indicated that algae powder and ADO could not only increase the concentration of the enzymes, but also increased the bands of the isozymes.
The algae powder and ADO could sustain the stability of the intestinal flora, promote the growth of the dominant flora and enhance the activities of immune-relative factors suggesting that the presence of algae powder and ADO improved the non-specific immunity of sea cucumber significantly and applying them as non-specific immunostimulants for sea cucumber will have a broad foreground.
1.采用平板计数的方法测定刺参肠道内细菌总数和弧菌总数。对照组刺参肠道细菌总数波动较大,海藻粉组和褐藻寡糖组细菌总数波动较小；实验期间海藻粉组刺参肠道细菌总数均小于对照组,但未形成显著性差异(P>0.05)。海藻粉组和褐藻寡糖组刺参弧菌总数占细菌总数的比值均大于对照组,而褐藻寡糖的体外抑菌实验中褐藻寡糖对副溶血弧菌没有抑制作用,推测海藻粉和褐藻寡糖对刺参肠道内优势茵群弧菌属部分细菌有一定的促进作用。
2.采用分光光度技术测定了海藻粉和褐藻寡糖对刺参体腔液和体壁组织中免疫相关因子溶菌酶(LSZ)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和溶血素活性的影响。结果表明：实验期间海藻粉组和褐藻寡糖组刺参免疫相关因子活性均高于对照组。海藻粉组和褐藻寡糖组刺参体腔液中ACP、AKP、溶血素和LSZ活性分别在第20d、20d、30d和40d达到最高,并且均与对照组形成显著性差异；海藻粉组和褐藻寡糖组刺参体壁中ACP、AKP和溶血素在其活性最高时均与对照组形成显著性差异。刺参体腔液中免疫相关因子活性的提高幅度要比体壁大,这可能是因为体腔液包含体腔细胞和各种体液免疫因子,是机体免疫反应发生的主要场所,其对免疫刺激物的免疫刺激反应比较强烈,因此活性的提高幅度较大。
3.海藻粉组和褐藻寡糖组刺参体腔液和体壁组织中抗氧化相关酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性实验期间均高于对照组。体腔液和体壁组织中抗氧化酶均呈现先增高后降低的趋势,体腔液中酶活性达到峰值的时间比体壁组织提前10d并且酶活性提高幅度较大,推测是由于体腔液中免疫刺激反应比较强烈所致。同工酶结果表明：褐藻寡糖和海藻粉对刺参抗氧化相关酶的诱导提高作用不仅体现在增加酶的浓度,同工酶酶带的增多也是增加酶活性的途径之一
海藻粉和褐藻寡糖对刺参肠道的稳定性和优势菌群的生长均有一定的促进作用,并且可以诱导刺参体腔液和体壁组织中免疫相关因子活性的提高,对刺参非特异性水平有一定的提高作用,因此将海藻粉和褐藻寡糖开发为水产动物尤其是刺参的免疫增强剂具有广阔的前景。
The effects of enzymatic hydrolysis algae powder by alginate lyase and alginate derived oligosaccharide (ADO) on the non-specific immune system of sea cucumber (Apostichopus japonicus) and the preliminary mechanism were studied by supplying 5% algae powder and 0.1% ADO in diets. The total numbers of the bacteria and the vibrio.spp, the activities of immune-relative factors in the coelomic fluid and the body wall and the changes of the isozymes were determined every 10 days during the experiment period (40 days). The specific content and results are as follows:
1.The total numbers of bacteria and vibrio.sp in the intestinal tract of sea cucumber were measured with the plate counting method. The fluctuation range of the total number of bacteria of the control was wider than those of the algae powder group and the ADO group. The total numbers of bacteria of the algae powder group were all smaller than those of the control, but did not have significant differences (P>0.05). The ratios of the total numbers of vibrio.sp to bacteria of the algae powder and ADO groups were higher that of the control. The ADO did not have antibacterial activity to vibrio parahaemolyticus in vitro experiment, so the algae powder and ADO could promote the growth of some bacteria belonging to the vibrio.sp which is one of the dominant flora in the intestinal tract.
2.Effects of algae powder and ADO on the activities of lysozyme (LSZ), acid phosphatase (ACP),alkaline phosphatase (AKP) and hemolysin in the coelomic fluid and the body wall were studied by the use of spectrophotometry. The results indicated that all the activities of immune-relative factors of the algae powder and ADO groups were higher than those of the control. The activities of the ACP, AKP, hemolysin and LSZ reached their maximum values on the 20th,20th,30th and 40th day respectively, and they all had significant differences compared with the control.The highest activities of ACP, AKP and hemolysin also had significant differences compared with the control. The coelomic fluid contains a variety of humoral immune factors and immune cells and the immune reactions take place here. So the reactions of the immune-relative factors in the coelomic fluid to the immunologic stimulant were stronger than those of the body wall and the enhancements of the activities of immune-relative factors were also higher.
3. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) of the algae powder and ADO groups were all higher than those of the control during the experiment period, and they all increased firstly and then decreased. The time that the activities of enzymes in the coelomic fluid reached the peak was 10 days ahead of that in the body wall and the enhancements of the activities were also higher than those in the body wall. The reason may be that the reactions of the immune-relative factors in the coelomic fluid to the immunologic stimulant were stronger than those of the body wall too. The results of the isozymes indicated that algae powder and ADO could not only increase the concentration of the enzymes, but also increased the bands of the isozymes.
The algae powder and ADO could sustain the stability of the intestinal flora, promote the growth of the dominant flora and enhance the activities of immune-relative factors suggesting that the presence of algae powder and ADO improved the non-specific immunity of sea cucumber significantly and applying them as non-specific immunostimulants for sea cucumber will have a broad foreground.
引文
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