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Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

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Abstract

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 °C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44 × 103 copies/μL) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47 ± 0.73) × 103 colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.

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Acknowledgments

This work was supported by Shantou Fish Sauce Factory Co., Ltd.

Conflict of Interest

Yun-Zhu Xiao declares that he has no conflict of interest. Si-Yang Zhao declares that he has no conflict of interest. Duan-Kai Wu declares that he has no conflict of interest. Wei-Min Lin declares that he has no conflict of interest. Xiao-Yong Zhang declares that he has no conflict of interest. Xiang-Yang Gao declares that she has no conflict of interest. This article does not contain any studies with human or living animal subjects.

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Authors and Affiliations

  1. College of Food Science, South China Agricultural University, NO.483 Wushan road, Guangzhou, 510642, People’s Republic of China

    Yun-Zhu Xiao, Si-Yang Zhao, Wei-Min Lin & Xiang-Yang Gao

  2. Guangdong PRB Bio-Tech Co., Ltd, Guangzhou, 510100, People’s Republic of China

    Duan-Kai Wu

  3. Key Laboratory of Tropical Marine Microbiology and Ecology, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Material Medical, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, People’s Republic of China

    Xiao-Yong Zhang

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  1. Yun-Zhu Xiao
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Correspondence to Xiang-Yang Gao.

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Xiao, YZ., Zhao, SY., Wu, DK. et al. Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce. Food Anal. Methods 7, 1634–1642 (2014). https://doi.org/10.1007/s12161-014-9799-5

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