Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51093
標題: Identification and quantification of probiotics in the animal feed by PCR-DGGE and real-time quantitative PCR
應用聚合酶連鎖反應-變性梯度膠體電泳與即時定量聚合酶連鎖反應於飼料中益生菌之定性及定量檢測
作者: Lin, Tzu-Hsing
林姿杏
關鍵字: Probiotics;益生菌;PCR-DGGE;Real-time quantitative PCR;聚合酶;連鎖反應-變性梯度膠體電泳;即時定量聚合酶;連鎖反應
出版社: 食品暨應用生物科技學系
摘要: 
目前使用添加抗生素的飼料餵養家禽畜的情形相當普遍,抗生素可以維持動物健康以及促進生長,但是這些抗生素的使用可能會破壞腸道菌相平衡,或是造成人類和動物致病菌對抗生素之抗藥性,因此歐盟從2006年起禁止畜禽養殖業者在飼料中使用促進生長用抗生素,為因應歐盟的腳步以及安全、自然農業的發展趨勢,農委會預計逐年禁止製造、輸入和使用含抗生素的飼料添加物。
聯合國農糧組織(Food and agriculture organization of the united nations, FAO)(2001)報告指出:益生菌為添加適當量的活微生物有益於宿主之健康。益生菌被證實主要作用是改善腸道微生物菌相:對腸道菌相之正面效益可以使動物趨向健康,尤以新生動物效果為佳;促進動物生長,如增加飼料轉換率(攝取之飼料總量和動物生長之比值)。當益生菌在腸道內有大量的活菌才可達到改善動物腸道菌相之功效,因此動物飼料中含有的益生菌之菌量非常重要。應用在動物飼料添加劑之微生物以含乳酸菌的生菌劑做為飼料添加物之應用最具潛力,其中又以乳酸桿菌最常被使用,但是目前對於混合微生物菌相中乳酸菌種之檢測,尚無標準及準確可靠的方法。
本研究目的擬應用PCR-DGGE與即時定量PCR於檢測飼料益生菌產品中之乳酸菌菌種,以期建立一有效且可靠的乳酸菌定性及定量檢測方法。本研究以脫脂大豆粕混合標準菌株模擬市售之益生菌產品,定性的部分:先以乳酸菌引子組Lac1及Lac2GC進行PCR反應,擴增產物約為380 bp,再配合DGGE區分出不同菌種,無法區分之菌株,以各別專一性引子進一步鑑別。定量的部分:使用個菌種專一性引子組進行即時定量PCR,以得到之Ct對應菌液濃度繪製標準曲線,將未知樣品之Ct代入標準曲線方程式,可得知模擬之益生菌飼料中所含之乳酸菌菌量。使用Student’s t-test比較即時定量PCR得到之菌量與培養基培養之結果(p > 0.05),表示兩方法間並無顯著差異,利用culture-independent的菌量計數具有應用之潛力。
本研究建立一快速鑑定及定量檢測飼料中乳酸菌之方法,以PCR-DGGE分析並結合菌種專一性PCR,可應用於區分常用於動物飼料中之乳酸桿菌,再以菌種專一性之即時定量PCR可用於檢測動物飼料中之乳酸桿菌菌量。

In poultry and livestock industry, growth-promoting antibiotics have been used in animal feed widely in order to improve the health and well-being of animals. However, these substances have led the beneficial intestinal flora to imbalance and the antibiotic resistant of pathogenic bacteria of humans or animals. Most of the antibiotics used as growth promoters in animal feeding have been banned by European laws, alternative strategies are needed. In recent years, consumers care for the problem of antibiotic residence in meat and poultry products, and therefore the development of substitute to feed antibiotics is becoming important.
The probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO, 2001). Their use established the efficacy of intestinal population. This positive effect on intestinal flora resulted in improved health status, especially for young animals, but also improvement in animal performance such as growth or feed conversion rate (the ratio between the amount of feed consumed and the animal growth). Probiotic bacteria may be effective when they reach the intestines without loss of viability. The microbial viability and authenticity are prominent criteria to be verified for the probiotics in the animal feed. A lot of probiotic products containing lactic acid bacteria have been commercialized and used in feed industry. Among the lactic acid bacteria, lactobacilli are mostly commonly used in animal feeds and human foods. However, the standard and reliable detection methods for lactic acid bacteria in feed are still not available.
This study investigated the detection of simulated feed containing lactobacilli by PCR-DGGE and real-time PCR. The simulated feeds are composed of defatted soybean meal and lactobacilli reference strains. For qualitative, PCR-DGGE analysis of 8 lactobacilli in simulated feed was developed using universal primers Lac1/Lac2GC. By generating the fragments of 16S rDNA, these eight species could be separated into six groups and the undifferentiated groups could be separated by further PCR reaction with specific primers. By PCR-DGGE, it is allowed for identification of species in the probiotic feed without prior isolation. In spite of the length homogeneity of amplicons, the technique provided a distinguishable separation of fragments, showing a great detection and identification potential for these PCR products.
For quantitative, real-time PCR analysis of 8 lactobacilli reference strains was developed using specific primers. By using the Ct (cycle threshold) and the concentration of bacteria cells, could generate the standard curve of lactobacilli reference strains. Substitution Ct value of simulated feed into standard curve could evaluated the concentration of bacteria. The Student's t-test was used to compare each quantitative analysis between plating enumeration and real-time PCR. The result (p > 0.05) indicates that there were no significant differences between the two methods at a confidence level of 95 %.
This study describes a detection method for lactic acid bacteria in feed products. The PCR-DGGE analysis combined species -specific PCR is showing a great detection and identification potential using for eight species of lactobacilli. The species -specific real-time PCR for evaluating the concentration of bacteria is no significant differences with the plating methodologies for enumeration. The result indicates that it has potential for developing a culture-independent bacteria enumeration procedure.
URI: http://hdl.handle.net/11455/51093
Appears in Collections:食品暨應用生物科技學系

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