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characterization, budu, Pediococcus, antimicrobial, exopolysaccharide
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Yusra Y, Syandri H, Efendi Y and Huda N. Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides [version 1; peer review: 1 not approved]. F1000Research 2022, 11:1139 (https://doi.org/10.12688/f1000research.109401.1)
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Research Article
Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides
[version 1; peer review: 1 not approved]
PUBLISHED 05 Oct 2022
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This article is included in the Agriculture, Food and Nutrition gateway.
Abstract
Corresponding author:
Yusra Yusra
Competing interests:
No competing interests were disclosed.
Grant information:
The authors wish to express their gratitude to Bung Hatta University for funding through the Professor’s Research Scheme Grant for the Acceleration of Professor candidates with Contract No: 003/ LPPM-Penelitian/Hatta/IV-2021.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:
© 2022 Yusra Y et al.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Yusra Y, Syandri H, Efendi Y and Huda N. Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides [version 1; peer review: 1 not approved]. F1000Research 2022, 11:1139 (https://doi.org/10.12688/f1000research.109401.1)
First published: 05 Oct 2022, 11:1139 (https://doi.org/10.12688/f1000research.109401.1)
Latest published: 05 Oct 2022, 11:1139 (https://doi.org/10.12688/f1000research.109401.1)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
West Sumatra has many foods that are processed through a natural fermentation process with or without the addition of microbes or a microbial inoculum. Foods derived from fermented fish are locally known as budu, tukai, and pado fish.1,2 Budu is processed through spontaneous fermentation of large pelagic fish such as Tenggiri (Scomberomorus guttatus) and Talang (Chorinemus spp.) because their flesh is white.3 Lactic acid bacteria found in budu fish can be used as probiotics and biopreservatives. One of the potential bacteria obtained from budu fish is Bacillus cereus strain HVR22, which possesses antimicrobial properties against Escherichia coli, Salmonella thypi, and Listeria monocytogenes bacteria.4,5 A bacteriocin produced by the bacterium B. cereus strain HVR22 can be used to preserve tuna fillets.6 Lactic acid bacteria that have been isolated from budu fish can produce gamma-aminobutyric acid (GABA) and exert antistress activity in broilers.7 Lactobacillus sp. has also been isolated from budu fish and can produce glutamic acid.8 Furthermore, Saccharomyces sp. strains SC 11, SC 12, and SC 21 have been isolated from budu and can be used as potential probiotics.9
Probiotics are microbes that benefit health only if used appropriately.10,11 It has been proven that probiotics can help with metabolism, the immune system, protein absorption, and inflammation reduction.12 Commercially, strains of lactic acid bacteria (LAB) are widely available in utilitarian meals as probiotics.13 LAB have the following features: a coccus shape, thick cell walls, are non-spore-forming, and are capable of fermenting carbohydrates.14 For probiotic bacteria to survive in the intestine and have a constructive effect on the body, they must have the fundamental property of being tolerant to the acidic pH of the gut and intestines.15 Digestive fluid is comprised of HCl(aq), which gives the bowels a pH value between 2.5-3.5.16 As a result, it is critical to characterize the probiotic candidate bacteria for their ability to pass through the acidic gastric surroundings and reach the jejunum.
Other essential criteria for selecting probiotic organisms are their antimicrobial properties, such as their ability to produce bacteriocins, oxidizers, and organic acids. These properties are vital for their ability to inhibit the pathogenic bacteria commonly present in the intestine.17 LAB have been identified in a variety of fermented foods that can be used as probiotics and are beneficial for human and animal health.18 Several LAB groups are known to produce secondary metabolites, namely, exopolysaccharides (EPSs) or extracellular polysaccharides. EPS is a polymer that is secreted by bacteria under unfavorable conditions. Conditions of environmental stress induce the bacterial cells to secrete exopolysaccharides as a form of self-protection. In addition, EPS also protects bacterial cells against bacteriophages, phagocytosis, and osmotic pressure. This polymer improves product texture, can act as an emulsifier, gelling agent, or stabilizer, and can provide viscosity and taste.19 EPS can organize the microbial cycle, participate in some symbiotic processes by increasing bacterial colonization through its presence between cells, and protect against dangerous substances such as poisonous materials and pathogenic bacteria.20
Many studies on the ability of LAB to produce exopolysaccharides have been carried out.21–28 Nevertheless, little is known about the potential of exopolysaccharides produced by LAB from budu fish. Therefore, this research is vital to determine the presence of LAB in budu fish, the physiological characteristics of the isolated LAB and the potential of these LAB to produce exopolysaccharides.
Methods
The study was carried out at the Integrated Research Lab, Faculty of Fisheries and Marine Sciences, the University of Bung Hatta, West Sumatra, Indonesia, from January 2021 to August 2021.
Source of LAB
Samples of budu fish were purchased from traditional processors in the Sungai Sirah and Gasan areas, Padang Pariaman and Sasak districts, Pasaman districts, West Sumatra Province. A total of three samples of budu fish were purchased from three traditional processors from the Sungai Sirah and Gasan areas, Padang Pariaman and Sasak districts, Pasaman districts, West Sumatra Province. Budu was produced from Tenggiri (Scomberomorus guttatus) and Talang fish (Chorinemus spp.), as shown in Figure 1. Samples were collected using sterile plastic for microbiological examination. Before sample analysis, our samples were stored in a refrigerator at 4 °C.
Figure 1. Fermented budu fish made from Tenggiri (Scomberomorus guttatus) and Talang (Chorinemus spp.).
Isolation and phenotypic identification of LAB
Isolation of the samples was carried out with serial dilutions of up to 10-5 using 9 ml of sterile sodium chloride solution in a beaker, which were then homogenized using a vortex shaker. From three dilutions (10–3 to 10–5), 1 ml of each was used to inoculate a pour plate containing de Man Rogosa Sharpe (MRS) agar medium supplemented with 1% CaCO3 (w/v). These inoculations were incubated at 37 °C for 24 to 72 hours.29 The isolates that produced a clear zone were then purified to obtain the pure isolates. Purification was carried out using a four-quadrant streak plate technique. Isolates were identified based on their physiological characteristics, such as the gram-staining reaction, morphology, CO2 production from glucose, motility, oxidase activity, nitrate reduction, and patterns of sugar agitation.30
LAB characterization
LAB resistance to gastric pH
The test was carried out using two test tubes containing MRS broth. One test tube served as a control, and in the other test tube, we added 37% HCl to obtain a pH of 2.5. After that, 0.5 ml of the bacterial culture containing approximately 109 CFU/ml was added to 5 ml of MRS HCl broth, followed by incubation for 36 hours at 37 °C. The absorbance was measured based on optical density (OD) using a spectrophotometer at a wavelength of 600 nm. Resistance to gastric pH values was ascertained as a percentage based on the standards.31 The pH of the culture after incubation at 37 °C was used to specify the acidulation gauge of LAB at 6, 12, 24, and 36 hours using the pH indicator PT-10 (Sartorius AG, Gottingen, Germany).32
LAB growth curve
A total of one dose of bacterial culture was inoculated into 10 ml of aseptic de Man Rogosa Sharpe Broth (MRSB) medium and incubated for 24 hours at 37 °C. The bacteria were then injected into 90 ml of aseptic MRSB medium and grown in an incubator shaker at 37°C. For the growth curve, the optical density of the culture was determined from four to 48 hours using the turbidity method with the aid of a spectrophotometer at a wavelength of 600 nm.33
Inhibitory ability of LAB against pathogenic bacteria
Paper discs were immersed in a 20 L LAB supernatant solution and then removed and attached to nutrient agar (NA) solid media containing the test bacteria (E. coli, S. thypi, and S. aureus). Then, the culture plate was incubated at 37°C for 24 to 72 hours. The width of the inhibitory area generated was determined using a caliper.34
Production of exopolysaccharides from LAB
Using MRS broth as the culture medium, exopolysaccharides were isolated and purified. LAB were cultivated for 24 hours in an incubator shaker in one litre of MRS broth with saccharose. The LAB culture was then heated at 100 °C for 10 minutes and centrifuged at 12,000 × g for 15 minutes to pellet the cells. Cold ethanol was added to the supernatant at twice the volume of the supernatant produced, and then this mixture was centrifuged at 5,000 g for 30 minutes at 4 °C. In a 50 °C oven, the pellets were dried to a consistent weight, and the EPS samples were then stored at -20 °C until they were assessed.35,36
Molecular identification of LAB
Lactic acid bacteria isolates were cultured in MRS broth at 37°C for 24 hours. Genomic DNA isolation was carried out using a Promega KIT (USA) following the manufacturer’s instructions. To break down the bacteria cell wall we used lysozyme at a concentration of 20 mg/ml to improve protein or nucleic acid extraction efficiency.37 Genomic DNA of LAB was used for amplification of 16S rRNA gene. Amplification was done using forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and primer 1492R (5′-GGTTACCTTGTTACGACTT-3′) for reverse. The reaction was carried out in a volume of 50 μl. The PCR mixture contained 22 μl of MQ, 25 μl DreamTaq Green DNA Polymerase (Thermo Fisher Scientific, USA), 1 μl of each forward and reverse primer (10 μM each, IDT synthesized) and 1 μl template. Amplification conditions were 5 minutes of preheating at 95°C, 30 seconds denaturation at 95°C, 30 seconds primer annealing at 58°C, one minute extension step at 72°C and post cycling extension of five minutes at 72°C for 35 cycles. The reactions were carried out in a thermal cycler (Biometra’s T-Personal Thermal Cycler, USA). PCR products were stored at 4°C for further examination using 1% (w/v) agarose electrophoresis in 1× TAE, 100 V for 30 minutes. The DNA bands formed from the electrophoresis process were visualized using a UV transluminator. The marker used was 1 Kb Plus DNA ladder (ThermoFisher Scientific). Sequencing of the 16S rRNA gene was performed at the Laboratory of Microbiology, Biological Research Center, Indonesian Institute of Sciences, Cibinong, Bogor, Indonesia. The products of the sequencing cycle were purified again with the ethanol purification method. Analysis of the nitrogen base sequence readings was performed using an automated DNA sequencer (ABI PRISM 2130 Genetic Analyzer) (Applied Biosystems). The BioEdit version 7.2.5 tool (RRID:SCR_007361) was then used to trim and combine the sequence data in its raw form. The assembled sequence data were then used in a BLAST search against genomic data registered with the NCBI to determine the microbe strain with the greatest similarity and other close relatives.
Results
LAB morphology
From three samples of budu fish, 56 LAB were identified. LAB isolation was conducted using MRSA CaCO3 medium (Figure 2). Twenty-nine of the LAB species identified were suspected to be Pediococcus sp. This is based on the identified physiological characteristics (Table 1), which matched the information in Cowan and Steel (1975).38 To further confirm the strain of LAB, subsequent characterization was carried out.
Figure 2. Morphology of bacterial colonies on MRSA CACO3 media.
Table 1. Physiological characteristics of Pediococcus sp. isolated from budu fish.
LAB resistance to gastric pH
After incubation for 0 to 36 hours, the pH of the supernatant decreased incrementally to 6.10, 5.08, 4.52, 4.50, and 4.49. The results obtained after incubation for three and six hours showed that LAB isolates could survive at pH 2.5. LAB generated from budu fish had a minimum resistance of 50%, indicating that they can be utilized as probiotics. The results of the LAB isolate cultures at various pH values using MRS broth showed that the isolates could grow at pH 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. The bacterial culture that was initially clear became cloudy as a sign of bacterial growth. From this research, it is clear that Pediococcus sp. from budu fish had a good acidification ability. The pH of the supernatant produced after incubation for 24 hours decreased for up to 36 hours.
LAB growth curve
The growth curve of Pediococcus sp. incubated on MRS broth medium for 36 hours is presented in Figure 3. The four stages of the growth phase of Pediococcus sp. namely the lag, exponential, stationary, and death phases.
Figure 3. P. pentosaceous growth curve in MRS broth.
The inhibitory ability of LAB against pathogenic bacteria
Pediococcus sp. also showed a high antimicrobial activity, with inhibition zones reaching 20.5 mm against E. coli, 23.0 millimeters against S. aureus, and 21.0 millimeters against S. thypi (Figure 4).
Figure 4. Antimicrobial activity of Pediococcus sp. in inhibiting the growth of the bacteria E. coli, S. aureus, and S. thypi.
Production of exopolysaccharides from LAB
Pediococcus sp. produced exopolysaccharides at concentrations ranging from 870–1910 mg/l. The average EPS production was 2,700 mg in wet conditions and 400 mg/l in dry conditions (Figure 5). The bacteria Pediococcus sp. isolated from budu fish was able to produce a large amount of EPS on MRS agar modified with the addition of 20% saccharose.
Figure 5. Isolate of Pediococcus sp. producing exopolysaccharide.
Molecular identification of LAB
To determine the strain of the bacterium Pediococcus sp., identification was carried out based on the 16S rRNA gene. Based on the BLAST results, Pediococcus sp. isolated from budu fish had a 16S rDNA gene that was 1514 bp in length, which showed the highest percentage of similarity (100%) with the Pediococcus pentosaceous strain 4942. The sequence homology results of Pediococcus sp. using BLAST in NCBI can be seen in Table 2, and the phylogenetic tree can be seen in Figure 6.
Table 2. Results of the sequence homology analysis of Pediococcus sp.
| Sample code | Description | GenBank accession number | Homology |
|---|---|---|---|
| 2.1 | Pediococcus pentosaceus strain 4942 | MT512069.1 | 100% |
Figure 6. Phylogenetic tree of the bacterium Pediococcus pentosaceous strain 4942.
Discussion
MRSA medium is a selective medium for detecting lactic acid bacteria. CaCO3 was added to the MRSA medium that was used. The addition of CaCO3 was used as an indicator of bacterial colonies capable of producing acid by showing the growth of colonies that formed a clear zone (Figure 2). From the MRS agar medium to which CaCO3 was added, the colonies obtained were characterized by a clear zone around them.39 Pediococcus is a lactic acid bacterium that is often found in fermented foods, including budu fish. The Pediococcus pentosaceus strain has been isolated from kombucha, fermented fish products, “idli”, a traditional food from South India, the Korean liquor “omegisool”, and “dadih”, a fermented buffalo milk.40–45 Furthermore, Weissella, Pediococcus, and Lactobacillus bacteria are prevalent LAB during the food fermentation process based on their habitus.46 Similar claims by other researchers have also reported that P. pentosaceus LBM 18 was prevalent during the corn silage fermentation process and had antibacterial and antifungal properties.47 Recently, Pediococcus activity was demonstrated, which was shown to have properties that can make this bacterium useful in food preparation.48 P. pentosaceus microbes can be used as food additives because of their abilities to improve taste, food nutrition, and preservation of animal products and to exert antibacterial and probiotic activities (anti-inflammatory, cancer-fighting, oxidation-inhibiting, hypolipidemic, and detoxifying activities).49
For the use of LAB strains as cultures to produce some fermented foods, it is important to determine the bacterial features such as acidifying activity, exopolysaccharide synthesis ability, and pathogen-killing bactericidal activity.50 Furthermore, acidulation activity is also a significant factor in characterizing bacteria, especially as a potential starter culture. These findings support research in which it was shown that LAB isolated from fermented food products in Ethiopia had a survival rate of 90.13% at pH 2.5 with an incubation of two hours.51 The resistance of LAB isolates from budu fish had a value of 60.15% with a three-hour incubation period and reached 60.68% after six hours, a small decrease of only 0.53%. This indicates that LAB from budu fish had a high survival rate, as seen from the increased resistance from three to six hours of incubation. Probiotics have a high survival and growth rate. At pH 2.5, L. brevis, L. plantarum, and P. ethanolidurans isolated from conventional pickles had viability ranging from 33–64%, 35–85%, and 40–76%, respectively.31 Furthermore, L. fermentum bacteria isolated from fermented milled flour had a survival rate of approximately 80% and a pH of 2.5 after a four-hour incubation period.52
For Pediococcus sp., the lag stage lasts from one to four hours. The exponential phase occurs from the fifth to the twentieth hour. This is because the nutrient content of the medium is frequently used as a source of power for growth of the cells. The stationary phase occurs from the twenty-first to the forty-eighth hour, and in this phase, there is no increase in the number of bacterial cells because the number of growing cells is equal to the number of dying cells. The final phase is the death phase, during which the number of bacterial cells begins to decline as the nutrients in the media begin to be depleted. Primary metabolites are also formed by this time, which accumulate in this medium and inhibit bacterial development. This phase occurs after the bacteria have been incubated for 72 hours. The results of this study were in accordance with the optimization of the growth of the P. pentosaceus 2397 strain, in which the exponential phase occurs at 12–48 hours and begins to decrease at 72 hours of incubation.53 Likewise, research on the bacterium P. pentosaceous ATCC 43200 found an exponential phase starting at four to 10 hours, after which the bacteria entered a stationary phase until 48 hours.54 The bacteria P. pentosaceus 63, P. pentosaceus 145, and P. pentosaceus 146, isolated from cheese made in Minas, Brazil, reached a stationary phase at 15 hours.55 The bacteria L. lactis PFC77 and P. acidilactici PFC69, isolated from fermented milk, a fermented soup originating from Anatolia, were able to inhibit the enlargement of S. aureus ATCC 29213 and B. cereus ATCC 11778 because they could produce bacteriocins and could be used as food preservatives.56 Optimal bacterial growth depends on the medium used.
LAB’s antibacterial activity against harmful bacteria has been extensively researched in food.57,58 The broad antibacterial range is associated with the LAB’s capability to generate antibacterial agents, i.e., acetic acids, H2O2, and antimicrobial compounds, that can inhibit the growth of other dangerous organisms.59 P. pentosaceus bacteria are lactic acid-producing bacteria that can create antibacterial compounds, hydrogen peroxide, and bacteriocins that are effective against S. enteritidis and E. casseliflavus and have the highest inhibitory activity compared to other LAB isolates.60 Several species of Pediococcus sp. have been reported to produce bacteriocins capable of inhibiting the growth of infectious agents.61,62 Pediococcus sp. can cause a decrease in the pH matrix of fermented foods, which leads to inhibition of the growth of unwanted bacteria.59 The acidifying activity of Pediococcus sp. strains has been observed, which causes a decrease in the pH matrix of food preservation and pathogenic microorganisms.63 Pediococcus spp., which were isolated from a fermented kombucha drink, were able to inhibit all test bacteria, namely, L. monocytogenes, S. enterica, B cereus, P hauseri, and L. ivanovii.48 Pediococcus acidilactici BK01 bacteria isolated from tamarind showed inhibition against S. aureus and E. coli during storage for a month at various temperatures.64 Furthermore, P. acidilactici M76 bacteria isolated from a black raspberry fermented drink showed antimicrobial activity against the pathogenic bacteria S. aureus, S. epidermidis, S. xylosus, P. aeruginosa, P. putida, B. cereus, B. subtilis, B. vallismortis, E. coli, and P. acnes.65
The ability of LAB to produce EPS is an important consideration when choosing an LAB culture to be utilized in dairy manufacture because these compounds function as texturizers and stabilizers, which are needed to create a smooth and creamy product.66 Furthermore, when utilized as a supplementary culture to speed up food fermentation time, the synthesis of a large amount of EPS by Pediococcus strains is regarded as a benefit for LAB from other varieties. Exopolysaccharides are usually secreted by microbes to the outside of cells and are generally found outside of the bacterial cellular structure. EPS is connected to the cell in the form of a capsule or mucus that is present on the cell surface. Lactic acid bacteria have been identified in Nigerian fermented food products (ogi, gari, and fufu) that are capable of producing exopolysaccharides and can be used as starter cultures to make functional foods.32 Furthermore, it was also found that the bacterium P. acidilactici M76 isolated from a fermented black raspberry drink could produce EPS and be used as a functional probiotic.65 L. plantarum JLAU103, isolated from hurood, a Chinese soured food, produced 75 mg/l exopolysaccharide after incubation for 24 hours.67 L. fermentum MC3 bacteria isolated from bamboo shoot fermentation products produced 88.776 mg/l exopolysaccharide after incubation for 48 hours using MRS medium supplemented with 4% glucose and 0.3% yeast extract.68
Molecular techniques based on DNA restriction fragments have been widely used to classify LAB isolated from various food products.69 This was validated through gene sequencing of the 16S rRNA genes from samples of bacteria. P. pentosaceus is an LAB that has thick cell walls, produces no spores, is negative for catalase, has a coccus shape, does not have cytochromes, and has facultatively anaerobic growth. P. pentosaceus can be isolated from the gastrointestinal tract. P. pentosaceus 4I1, a known LAB, was isolated once from the intestinal microorganisms of a sample of Zacco koreanus.70 LAB can also be isolated from fermented buffalo milk known as “dadih”, cereal fermentation, digestive organs of Bali cattle, tamarind fermentation, dried sausage, fermented “kombucha”, fermented freshwater fish and corn silage.41,45,47,48,71–74
Conclusions
In testing 56 LAB isolates from budu fish, as many as 29 isolates were demonstrated to be Pediococcus sp. Based on bacterial characterization, one potential isolate was determined to be Pediococcus pentosaceus. These bacteria showed resistance to gastric acid conditions, with a survival rate of 60.68% at pH 2.5. These bacteria inhibited dangerous microbes such as S. aureus, E. coli, and S. thypi from multiplying and were able to produce exopolysaccharides. After being identified molecularly, this bacterium was shown to have 100% similarity with Pediococcus pentosaceous strain 4942.
Data availability
Underlying data
Figshare: Underlying data for ‘Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides’. https://doi.org/10.6084/m9.figshare.19612677
This project contains the following underlying data:
• Table 1. Pedicoccus sp. bacteria resistance to gastric pH (2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) using MRS broth fter incubation 36 hours
• Table 2. Row data: Growth curve of Pediococcus sp. bacteria incubated on MRS broth medium for 36 hours
• Table 3. Row data: Inhibitory ability of Pediococcus sp. bacteria against pathogenic bacteria (Escherichia coli, Staphylococcus aureus and Salmonella thypi)
• Table 4. Row data: The sequence of nitrogen bases from the sequencing of Pediococcus sp.
• Supplementary Figure 1. PCR gel image.
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)
Accession numbers
NCBI Gene: Pediococcus pentosaceus strain 4942 16S ribosomal RNA. Accession number MT512069.1. https://www.ncbi.nlm.gov/nuccore/MT512069.1
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how to cite this article
Yusra Y, Syandri H, Efendi Y and Huda N. Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides [version 1; peer review: 1 not approved]. F1000Research 2022, 11:1139 (https://doi.org/10.12688/f1000research.109401.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Kamiloğlu A. Reviewer Report For: Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides [version 1; peer review: 1 not approved]. F1000Research 2022, 11:1139 (https://doi.org/10.5256/f1000research.120896.r203606)
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https://f1000research.com/articles/11-1139/v1#referee-response-203606
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In this study, the characterization and exopolysaccharide production abilities of lactic acid bacteria isolated from West Sumatra fermented fish were examined. However, when the study is evaluated, its purpose cannot be fully understood. What was wanted, what was sought, what
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Kamiloğlu A. Reviewer Report For: Characterization of lactic acid bacteria isolated from budu, a West Sumatra fish fermentation product, and their ability to produce exopolysaccharides [version 1; peer review: 1 not approved]. F1000Research 2022, 11:1139 (https://doi.org/10.5256/f1000research.120896.r203606)
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