利用Bacillus subtilis E20做為南美白蝦飼料益生菌之研究

Use of Bacillus subtilis E20 as probiotic in white-leg shrimp (Litopenaeus vannamei) feed

何珮琳(Pei Lin Ho)

指導教授 ： 劉俊宏

國立屏東科技大學/農學院/水產養殖系所/碩士(2009年)

https://doi.org/10.6346/NPUST.2009.00311

摘要

本研究之目地係由納豆 (醱酵黃豆) 中分離及鑑定具蛋白酶分泌功能的Bacillus subtilis E20，並將其添加於白蝦飼料中，以促進白蝦之成長。由納豆中分離具產蛋白酶能力的菌株E20經由API 50 CHB 及16S rDNA 鑑定為Bacillus subtilis，故將其命名為B. subtilis E20。B. subtilis E20可在廣範圍的溫度 (10-50℃)、pH (5-10)及鹽度 (0-9%) 下生存，但最佳分泌蛋白酶條件為40℃、pH 6-8及鹽度0%。分析B. subtilis E20之安全性試驗顯示，白蝦感染B. subtilis E20 (最高至109 cfu/shrimp)後，並沒有任何的白蝦死亡，顯示其對白蝦為安全菌株。另外，在抑菌試驗也發現B. subtilis E20對水產養殖病原菌Aeromonas hydrophhila (ATTC 7966) 有抑制之效果。將B. subtilis E20添加於飼料 ( 0 (控制組)、106 cfu/kg、107 cfu/ kg及108 cfu/kg)中，分析其對白蝦成長的影響。結果顯示白蝦攝食含益生菌108 cfu/kg飼料後，相對於控制組有較好的成長及產量，推估原因為B. subtilis E20可改善白蝦腸道內蛋白酶活性，進而增加食物的消化及吸收。經PCR及real-time PCR分析顯示，白蝦在攝食含B. subtilis E20之飼料後，可於腸道中檢測到B. subtilis E20的存在，且菌量在攝食後第14天會明顯增加，此意謂B. subtilis E20可通過消化道，而於蝦子腸道活存及增殖。此外，分析室溫或4℃貯存條件對B. subtilis E20飼料品質的影響，結果顯示放置於室溫或4℃經過2個月後，B. subtilis E20之濃度並沒有下降之趨勢，且雜菌出現的時間亦無差異，但含有益生菌B. subtilis E20之飼料中的雜菌出現時間明顯較控制組飼料慢。綜合上述結果可知，B. subtilis E20為蝦類良好之益生菌，其可改善蝦類消化系統之蛋白酶量，以促進白蝦腸道消化吸收能力，進而增進成長，其建議添加量為108 cfu/kg。

關鍵字

南美白蝦 ； Bacillus subtilis E20 ； 益生菌 ； 蛋白酶 ； 成長

並列摘要

The purpose of this study was to isolate and identify a beneyicial bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp (L. vannamei) feed to promote shrimp (L. vannamei) growth performance. A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50℃), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40℃, a pH of 6–8 and 0% NaCl. No shrimp (L. vannamei) died after being injected with B. subtilis E20 [up to 109 colony-forming units (cfu) per shrimp]. The analysis of bacterial growth inhibitory showed that B. subtilis E20 has an ability to supress the growth of Aeromonas hydrophila. B. subtilis E20 was incorporated in diets at the levels of 0 (control), 106, 107, and 108 cfu/ kg) for shrimp (L. vannamei) grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (108 cfu/kg) of diet), shrimp (L. vannamei) had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20. According the results of PCR and real-time PCR analysis, positive result was recorded in intestine of shrimp (L. vannamei) fed with B. subtilis E20 containing diets, and the concentration of B. subtilis E20 in shrimp (L. vannamei) intestine was significant increased at the 14 days of feeding. It is suggested that B. subtilis E20 is able to survivial and colon in shrimp (L. vannamei) intestine. In order to understand the effect of storage on the quality of B. subtilis E20 containing diets, the experimental diets was placed at 4℃ and room temperature for 2 months. The results showed that the concentration of B. subtilis E20 was not statistical decreased in all experimental diets, but the contaminated bacteria in diets was increased as the increase of storage time. However, the time of contaminated bacteria occurring in B. subtilis E20 containing diets was significant postponed compared to control diet. In conclusion, Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp (L. vannamei) growth performance through increasing the digestibility of food. Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp (L. vannamei) growth performance, and consequently reduce feed costs.

並列關鍵字

Litopenaeus vannamei ； Bacillus subtilis E20 ； probiotic ； protease ； growth performance

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