Reduction of Trimethylamine Off-Odor by Lactic Acid Bacteria Isolated from Korean Traditional Fermented Food and Their In Situ Application
1Institute of Food Science, Pukyong National University, Busan 48513, Republic of Korea
2Department of Food Science and Technology, Pukyong National University, Busan 48513, Republic of Korea
3Department of Food and Nutrition, Changwon National University, Changwon 51140, Republic of Korea
J. Microbiol. Biotechnol. 2020; 30(10): 1510-1515
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Trimethylamine (TMA) is an organic and volatile fatty amine known to cause acute health problems such as pulmonary stimulation, eye irritation, and liver failure . The source of TMA is a nitrogenous organic compound named trimethylamine
Materials and Methods
Screening of LAB from Korean Traditional Fermented Food
Various Korean traditional fermented foods were screened for LAB. Samples were obtained from local markets in Busan, Republic of Korea. The samples included one type of meju (fermented soybean), 2 types of makgeolli (rice wine), 1 type of kimchi, and fermented kelp. For screening of LAB, 25 g of samples were added in a 250 ml Erlenmeyer flask containing 125 ml of 0.1% (w/v) peptone water. The mixture was homogenized and serially diluted up to 10-5 dilution. After that, serially diluted samples were spread plated on bromo cresol purple agar (BCP, plate count agar with 0.006% bromo cresol purple) plates and incubated at 30°C. The LAB produced several organic acids that reduced the pH and turned the medium yellow. We then used these yellow colonies for further testing .
Identification of Isolated LAB
The isolated colonies were identified by using the 16S rRNA sequencing and biochemical method. Genomic DNA of LAB isolates was prepared using the AccuPrep Genomic DNA Extraction Kit (Bioneer, Republic of Korea). Amplification of 16S rRNA genes from the LAB isolates was done by PCR using universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGYTACCT TGTTACGTACTT-3’) . The obtained 16S rRNA gene sequences of the LAB isolates were deposited in the GenBank nucleotide sequence database. The sequences were determined with the Basic Local Alignment Search Tool (http://ncbi.nlm.nih.gov/BLAST), which was used to find homologous sequences. Furthermore, a phylogenetic tree was constructed to determine the closest bacterial species using the neighbor-joining method of MEGA 7 (ver. 7.0.26). The carbohydrate fermentation pattern and biochemical identification of the LAB isolates were studied using the API 50 CH and API 50 CHL system (Bio-Merieux, France) following the manufacturer’s protocol.
TMA Reduction Efficacy of LAB Isolates
To prepare the LAB cell-free culture, the LAB was cultivated in DeMan-Rogosa-Sharpe (MRS) medium at 37°C up to about 9 log CFU/ml. The LAB cell-free culture was obtained by aseptic filtration of the supernatant using a 0.2 μm membrane (Toyo Co., Japan) after low centrifugation (10,000 g, 20 min). TMA reduction efficacy of LAB was carried out by mixing cultured LAB cell-free medium and TMA (0.3% of final concentration, v/v). We measured the TMA reduction efficacy of this LAB/TMA mixture after 5 and 24 h reactions at 25°C. TMA reagent was purchased from Junsei Co. (Japan). The LAB cell-free medium and TMA mixture was moved into 22 ml, clear glass vials with polytetrafluoroethylene (PTFE)-silicone septa and open-top phenolic caps and vortexed before TMA extraction and gas chromatography (GC) analysis.
TMA Extraction and Quantitative Analysis
TMA contents were extracted and quantitatively determined using the GC Solid Phase Microextraction (SPME) method as described by Park
In-Situ Reduction of TMA by Cell-Free Supernatant of LAB Culture
To validate the TMA reduction ability of the LAB isolates, we performed in situ reduction testing by augmenting cell-free supernatant of LAB cell culture onto the spoiled fish samples. The fish samples (Ribbon fish;
All experiments were carried out in triplicates. Data were expressed as mean ± standard deviation. Obtained data were statistically analyzed by analysis of variance (ANOVA) and Duncan’s multiple range test. Statistical significance of differences between mean values of data was interpreted at a significance level of
Results and Discussion
Identification of LAB Isolates from Korean Fermented Food
Fifteen LAB isolates were isolated and identified from four types of Korean traditional fermented foods. All fifteen LAB isolates were identified by PCR amplification and sequencing of the 16S rRNA gene (1,300-1,500 bp). Sequencing results showed that the LAB isolates belonged to the genera
These five LAB isolates were also screened for their performance regarding growth characteristics in carbon source using an API 50CH and 50CHL medium system. The results of biochemical identification showed similar results with 16S rRNA gene identification results (Table 1). All LAB isolates ferment different carbon sources including D-ribose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, and salicin. Interestingly, the strain SKD 14 utilizes both D-xylose and D-tagatose, and the SKD 11 strain ferments an additional carbon source, D-lyxose. Other strains, SKD 1, 4, and 15, can utilize hexoses such as mannitol and sorbitol (Table 2). Ashmaig
In an effort to identify SKD 1, 4, 11, 14, and 15 isolates at the species level, molecular phylogenic analysis was conducted and the phylogenetic tree was constructed based on the 16S rRNA gene sequence from evolutionary distances by the neighbor-joining method (Figs. 1A and 1B). Based on the analysis result, (Fig. 1A), SKD 1, 4, and 15 strains belonged to the genus
Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences.The neighbor-joining phylogenetic tree shows the relationship between LAB isolates. (A) SKD 1, 4, and 15 (○, ●, and △) belong to Lactobacillusspecies. (B) SKD 11, and 14 (○, ●) belong to Pediococcusspecies. Numbers at behind strain names are accession numbers of the published sequences at NCBI.
LAB Exhibiting TMA Reduction Efficacy
All five LAB strains, SKD 1, 4, 11, 14, and 15, showed significant reduction of TMA within 5 h of incubation and the values were 88.2%, 88.1%, 89.86%, 92.14%, and 91.33%, respectively. Furthermore, after 24 h of incubation, the reduction of TMA by strains SKD 11, 14, and 15 was found to be similar as reported during 5 h of incubation. However, the reduction of TMA by strains SKD 1 and 4 was found to be slightly decreased during 24 h of incubation (Table 1). Based on the above results, we concluded that the maximum reduction of TMA occurred by each LAB isolate within 5 h of incubation; however, there was no significant reduction during prolonged incubation. This might suggest that the enzyme present in the cell-free supernatant gets fully saturated with the available substrate and showed maximum catalytic activity within 5 h of incubation.
Boraphech and Triravetyan  screened several plant materials having TMA reduction activity and they reported
In Situ Reduction of TMA Content from Spoiled Fish by LAB Strains
As shown in Table 3, TMA contents in spoiled fish were decreased by the treatment of five LAB strains (SKD1, 4, 11, 14, and 15). The five LAB strains showed TMA reduction activity and have stable performance and activity as compared to other LAB strains. The TMA reduction efficiency of the five LAB cell-free cultures’ supernatant was reported as 45%, 62%, 60%, 59%, and 52% by SKD 1, 4, 11, 14, and 15, respectively. The in situ results indicate that sample treated with LAB cell-free supernatant showed significant TMA reduction in the range of 45-62%, which is lower as compared to the LAB-treated TMA reduction (in the range of 88.17-92.87%.). The lesser amount of TMA reduction from fish treated with LAB cell-free supernatant might be due to the interaction of TMA with the secondary metabolites produced by LAB , which might counter the action of TMA degrading enzymes present in the supernatant. However, a future study is required to explain this mechanism. Although in situ reduction of TMA is lower as compared to the LAB-treated TMA reduction, a significant in situ TMA reduction suggests that these LAB isolates can be applied in the seafood industry to effectively reduce the off-odor of spoiled fish by decreasing TMA content.
Researchers in several studies have tried to remove TMA using various methods. Among them, it was reported that cellulose-based active carbon fiber, charcoal particles, and ortho-phosphoric acid were able to remove gases such as TMA and ammonia [7, 10, 21]. It was also reported that a continuous flow extraction using microbubbles combined with supercritical CO2 removed volatile compounds released from the fish sauce . However, these previous methods have a limitation in that they are applicable only for liquid and not solid samples. In contrary to the previous methods, the present results obtained in this study strongly suggest LAB isolates and their cell-free supernatant are applicable to remove volatile compounds such as TMA from both liquid and solid samples. To the best of our knowledge, this is the first report on TMA reduction by metabolic activity of LAB isolates and its in situ application using spoiled fish samples.
In conclusion, the present study reported a new approach for the deodorization of TMA or fish off-odor by microbial treatment especially using LABs. In addition, LAB treatment was effective in the deodorization of spoiled fish samples. LAB strains that were isolated from Korean traditional fermented foods have been characterized by biochemical and 16S rRNA gene sequencing approaches. The identified LAB isolates were characterized as
This work was supported by a research grant from Pukyong National University (2019).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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