Analysis of Chemical and Microbiological Changes During Over-fermentation of Naem by Using Biochemical and Molecular Biology Methods

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1 Analysis of Chemical and Microbiological Changes During Over-fermentation of Naem by Using Biochemical and Molecular Biology Methods Trilert Chaicherdsakul, Worrawalan Phoonsawat and Vethachai Plengvidhya * Faculty of Biotechnology, Assumption University Bangkok Thailand 1 Abstract Naem is a traditional Thai fermented sausage regularly fermented at room temperature for approximately hours. However, many producers and consumers prefer keeping the products at room temperature. This practice can lead to unacceptable products due to continuous fermentation. This period can be described as over-fermentation. Previous studies using traditional biochemical and molecular biology methods to study the ecology of commercial Naem fermentations revealed that various lactic acid bacteria species such as Leuconostoc mesenteroides, Lactobacillus plantarum, L. pentosus, L. curvatus, L. sakei, L. bravis and Pediococcus pentosaceus were involved (Kunawasen 2000). However, no attention has been paid to the overfermentation period (more than 3 days after the start of the fermentation). The objective of this research was to study the microbial diversity and community structure of Naem during the over-fermentation period by using PCR of rrna genes methods. Naem samples from commercial processing plant were sampled during the overfermentation period of 120 and 168 hours after the start of the fermentation. The samples were plated on MRS agar and selected numbers of isolates for each time point was recovered. DNA was isolated for PCR fingerprinting and 16S rdna sequence analysis. A total of two hundred isolates were collected for analysis. A database of PCR fragment pattern has been generated by amplifying the intergenic transcribed spacer (ITS) regions between the 16S and 23S rdna genes. The 16S rdna gene has been sequenced to further identify selected isolates. According to ITS-PCR banding patterns, the number of bacterial species involved in the over-fermentation was four on days five and five on days seven. The use of ITS-PCR and 16S rdna analysis indicated that L. plantarum was the dominant species in over-fermented Naem. We recovered only a small number of isolates belonging to L. brevis, L. paracasei, L. curvatus, L. fermentum and P. pentosaceus. The combination of ITS-PCR for isolate grouping and further characterized by 16S rdna sequencing can offer and alternative tool for describing the important bacterial species involving in over-fermented Naem samples. This study reveals that minimizing the number of L. plantarum might be one alternative in prolonging the shelf life of Naem when stored at room temperature by delaying the over-fermentation period. Keywords: Polymerase chain reaction (PCR), Over-fermented Naem, Lactic acid bacteria, rrna genes, intergenic transcribed spacer (ITS), 16S rdna sequencing, restriction digestion * Food Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand 217

2 Introduction Naem is one of the most famous Thai traditional fermented food. Normally, Naem is prepared from minced pork, boiled pork rinds, cooked rice, garlic, salt, sugar, pepper, chilli and sodium nitrite. The mixture is then packed in banana leaves or plastic sheet and allowed to ferment for approximately hours. (Valyasevi and Rolle 2002). However, many producers favor to keep Naem at room temperature, which can cause overfermentation leading to unacceptable products. The over-fermented Naem causes changes in texture and sensory, leading to unacceptable characteristics from consumers. This study aims to study the chemical and microbiological changes of Naem during over-fermentation period. In order to identify microorganisms responsible for the over-fermentation using molecular biology techniques based on PCR of rrna genes. The combination of using PCR to analyze intergenic transcribe spacers (ITS) and 16S rrna gene, which remains presently one of the most accurate and easiest way to routinely analyze the genetic diversity of bacterial populations at the chromosome level (Nesme and Normand 2004), can facilitate us to describe the important bacterial species involving in over-fermented Naem samples. Methods Sampling: Naem samples were commercially produced at Product Development Division, Department of Livestock prior to being sampled every 12 hours to the first 24 hours followed by every 24 hours up to 7 days. 100 well isolate colonies were randomly collected from 120 th and 168 th hours MRS plate. Chemical Analysis: Sugars, and organic acids were determined by HPLC and GC-FID respectively. The ph of the Naem samples was measured by a ph meter. Microbiological Analysis: The total LAB populations were determined by plating on MRS. Isolate Identification: Isolates were identified by Rsa I restriction digestion of intergenic transcribed spacer (ITS)-PCR and 16S rdna sequencing. Results Microbiological and Chemical Analysis The LAB population increased continuously during the regular period of Naem fermentation (up to days 3) followed by the period of decline from 96 th hour to 168 th hour after the start of the fermentation which is contrast to continuously decreases of Naem ph from days 1 into the over-fermentation period. The organic acid analysis allowed that most of organic acid found in Naem were lactic acid. The increasing of lactic acid production was correlated to the decrease in ph as well as the declining in total fructose and glucose concentration at 120 th and 168 th hour into the fermentation. Glucose was possibly the only remaining carbon source available at days 7 of the fermentation due to the fact that total fructose concentration was less than 30mg/100g. Investigation of Microbial Community Changes during Naem Over-fermentation Twelve unique ITS-PCR patterns were obtained from over-fermented Naem isolates. Representative isolates which belong to each unique ITS-PCR patterns have been subjected to 16S rdna analysis for genus and species identification. Six species of lactic acid bacteria namely L. plantarum, L. brevis, L. paracasei, L. fermentum, L. curvatus, as well as P. pentosaceus have been found to associate with over-fermented Naem. L. plantarum is a major organism associated with the over-fermentation period comprised of almost 84% (81 out of 97 isolates) of total isolates from 5 th day. The number of bacterial species increased from four in days 5 to five in days 7. L. plantarum remained the major species found in overfermented Naem sample on days 7 but it contribution has been increased to almost 90% (89 from 99) of total isolates. Other LABs that has been found in significant numbers were L brevis and P. pentosaceus. L. curvatus, L. paracasei, and L. fermentum were also recovered from over-fermented Naem samples but in less proportion. 218

3 Log CFU/g Log CFU/g 4 ph ph Fig. 1: The total LAB and ph changes during Naem fermentation Lactic acid (g/100g) Lactic acid (g/100g) Fig. 2. Acid Production during Naem fermentation Discussion In this study, chemical and microbiological analysis indicated changes in ph, lactic acid production, total sugar utilization, and total lactic acid bacteria population during over- fermentation of Naem. Total organic acid production at the over-fermentation at the overfermentation period increased continuously with correlation to the decrease of total fructose, glucose and ph. During the normal fermentation period (up to three days), the 219

4 Fructose (mg/100g Nham) Glucose (mg/100g Nham) Fructose (mg/100 g Nham) Glucose (mg/100 g Nham) Fig. 3: Total fructose and glucose changes during Naem fermentation. M M M 1000 bp 800 bp 500 bp 300 bp 200 bp 100 bp 1000 bp 800 bp 500 bp 300 bp 200 bp 100 bp Fig 4. Restriction digestion patterns of ITS-PCR products of isolates taken from overfermented Naem. Lane M contained the 100 bp DNA ladder-size standards. The lane numbers for PCR patterns are: L. plantarum (1,7,9), L. brevis (2,10,12), P. pentosaceus (3,4,8), L. paracasei (5), L. fermentum (6), and L. curvatus (11). 220

5 Table 1. Percentages of lactic acid bacteria at 120 th and 168 th of over-fermented Naem Lactic acid bacteria % of total population 120 th hour 168 th hour L. plantarum L. brevis P. pentosaceus L. curvatus L. paracasei ssp. paracasei L. fermentum increase in number of microorganisms was correlated to the decreasing in ph, sugar utilization, and the increasing of lactic acid production. However, in contrast to the regular fermentation period, the population of microorganisms decreased when Naem reached the over-fermentation stage. The LAB population decreased from 96 th to 168 th hour into the fermentation, possibly due to the inhibition by higher amount of acid production. The majority of population in overfermentation period belonged to obligated and facultative hetero-fermentative lactobacilli, representing 91.7% and 96.9% of total population at 120 th and 168 th hours, respectively. During fermentation, indigenous lactobacilli continue to grow and diversity at the end of fermentation was high s evidenced by 5 species of Lactobacillus had been found. The uses of ITS-PCR and 16S rrna analysis indicated that 6 types of lactic acid bacteria have been involved in over-fermentation namely L. plantarum, L. brevis, L. pracasei, L. fermentum, L. curvatus, and P. pentosaceus. L. plantarum has been found to be the dominant species involved in over-fermented Naem, which the proportion in population increased from 120 th (83.5%) to 168 th hour (89.9%). However, other lactic acid bacteria namely L. brevis, L. curvatus, and P. pentosaceus proportion were decreased. In addition, L. paracasei and L. fermentum were presented only at 168 th hour of the fermentation. Conclusion We believe that using the combination of ITS-PCR for isolate grouping and further characterized by 16S rdna sequencing can offer an alternative tool for describing the important bacterial species involving in overfermented Naem samples. This study has shown that minimizing the number of L. plantarum might be one alternative in prolonging the shelf life of Naem when stored at room temperature by delaying the overfermentation period. References Altschul, S.F.; Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; and Lipman, D.J Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: Available online from < Kunawasen, S Molecular typing of lactic acid bacteria isolated during Nham Fermentation. M.S. thesis, Mahidol Univ., Bangkok, Thailand. Nesme, X.; and Normand, P Easy Individual Strain and Community Typing by rdna ITS1 Analysis. In: Molecular Microbial Ecology Manual, Vol 1. (Eds. G.A Kowalchuck,., F.J. de Bruijn, I.M. Head, A.D. Akkermans, and J.D. van Elsas) Springer, New York, pp Temmerman, R.; Temmerman, R.; Huys, G.; and Swings, J Identification of lactic acid bacteria: Culture-dependent and culture-independent methods. Trends in Food Sci. Technol. 15: Valyasevi, R.; and Rolle, R.S An overview of small scale food fermentation technologies in developing countries, scope for their improvement. Int. J. Food Microbiol.. 75: