| Summary: | 碩士 === 國立中興大學 === 食品暨應用生物科技學系 === 96 === 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.
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