Bacteria isolation from Wikau Maombo was performed on MRS-Agar-specific media to obtain fermented lactic acid bacteria (LAB). This is because MRS-A serves as a source of food and nutrition for LAB [28]. Furthermore, this media aims to optimize growth, obtain the desired colonies, and inhibit the growth of unwanted bacteria [29]. In MRS media, glucose is used as a carbon source [30]. The primarily needed nutrients are carbon and nitrogen sources. Lactic acid bacteria use carbon as an energy source and lactic acid-forming material. Meanwhile, nitrogen is a material in the formation of cell biomass [31].
Morphological Characteristics of Bacterial Isolates from Wikau Maombo
Cell Shape and Characteristics of Microorganisms Colonies
Table 1 shows that the successfully grown Wikau Maombo isolates were bacilli and cocci-shaped bacteria. This finding follows the research conducted by Putri et al. [32]. During the fermentation of sweet potato in the Growol (traditional food from Kulon Progo) making process, 231 bacterial isolates were discovered. There were 63 lactic acid bacteria with Gram-positive characteristics and dominated by rod shapes with different cell lengths. Table 1 shows that the macroscopic observation of bacterial colonies on MRS-A media was punctiform, circular, irregular, and spindle-shaped with a convex elevation. The edges of the bacterial colonies are entire and undulate. This finding follows research by Putri and Kusdiyantini [27] against LAB from fermented foods having circular and punctiform shapes with convex, flat, and raised elevations. Furthermore, the edges of the colony are entire and undulate with white and cream colors.
The growing colonies' color is different because it is affected by bacteria pigments. The results show that the colonies that grew on MRS-A media were white and milky white. This fact indicates that these bacteria contain carotenoid pigments. This finding is appropriate to the research by Kasi et al. [12] in LAB from sago wastewater which stated that the morphology of the colonies is milky white in color and round in shape with flat edges.
Characteristics of Gram Microorganisms
Gram characteristics were conducted to observe the colony morphology and distinguish between Gram-negative and Gram-positive bacteria against crystal violet, safranin dyes, and the KOH string test [28]. The presence of bacteria can be observed microscopically through specific media [3]. Gram characteristics are determined by Gram staining and are usually confirmed by the 3% KOH test. The Gram method with a 3% KOH test is a good identification technique for determining the dominant type of active bacteria [33]. The successfully grown Wikau Maombo isolates were Gram-positive bacteria. During the Gram staining process, Gram-positive bacteria are marked purple, which is negative (Gram-positive) when demonstrating a non-sticky/no mucus colony with a needle [21], [22]. This is because their cell walls contain low lipids, causing dehydration and a reduced pore size when alcohol is added. It also causes the dye to remain bound, and the cell remains purple as it retains the purple color of the primary dye [34]. Barus et al. [13] stated that the LAB isolates from cassava tapai showed positive results on Gram staining (purple color). The research by Wikandari et al. [35] showed that there were three genera of LAB, namely Lactobacillus, Pediococcus, and Leuconostoc, with Gram-positive characteristics. According to Arini and Wulandari [36], Gram-positive bacteria with high peptidoglycan content differ from Gram-negative bacteria with high lipid content.
Table 1
Cell shape and colony characteristics of bacterial isolates from Wikau Maombo
Isolate code | Cell Shape | Color | Shape | Edge | Elevation |
UM 24.2 | Basil | milky white | Circular | Entire | Convex |
UM 48.1 | Cocci | White | Irregular | Entire | Convex |
UM 48.2 | Cocci | milky white | Irregular | Entire | Convex |
UP 24.1 | Cocci | White | Spindle | Undulate | Convex |
UP 24.2 | Basil | White | Circular | Entire | Convex |
UP 24.4 | Cocci | milky white | Punctiform | Entire | Convex |
UP 48.1 | Basil | milky white | Punctiform | Entire | Convex |
Characteristics of Bacterial Isolate Enzyme Activity
The enzyme activity of bacterial isolates from Wikau Maombo was characterized to determine their ability to qualitatively produce enzymes by forming a clear zone. Furthermore, a clear zone around the colony showed that the bacterial isolate had enzyme activity. Table 2 present the results of qualitative enzyme characterization.
The lipase enzyme activity test was conducted to determine the ability of bacterial isolates from Wikau Maombo to hydrolyze triacylglycerol, releasing free fatty acids and glycerol. The test results showed that the bacterial isolates did not have lipase enzyme activity which was indicated by the absence of a clear zone around the colony. Meanwhile, the catalase test was performed to determine the ability to produce the catalase. The results showed that three bacterial isolates dripped with 3% H2O2 solution were catalase-negative, indicated by the absence of gas bubbles, while four were catalase positive.
Starch and protease hydrolysis tests were conducted to determine the ability of the seven bacterial isolates to produce amylase and protease enzymes. Those producing these enzymes characterized the formation of clear zones in the media containing starch and skim milk. A clear zone around the colony indicated that the bacterial isolate had extracellular amylase enzyme activity. Sari et al. [38] stated that a clear zone was formed in the amylase test against LAB Leuconostoc sp. Aa8. According to the results, the highest clear zone was discovered in isolate UM 24.2, with a diameter of 5.51 mm.
Meanwhile, isolates UM 48.1 and UM 48.2 had values of 4.88 mm and 4.27 mm, respectively. That formed around the colony results from starch hydrolysis activity by extracellular amylase [39]. Furthermore, Suciati et al. [39] showed that isolates of Pediococcus sp., Lactobacillus sp., and Streptococcus sp. could produce extracellular amylase.
Table 2
Enzyme activity of bacterial isolates from Wikau Maombo
Isolate code | Lipase | Catalase | Amylase | Protease |
UM 24.2 | - | - | + | + |
UM 48.1 | - | - | + | + |
UM 48.2 | - | - | + | + |
UP 24.1 | - | + | - | - |
UP 24.2 | - | + | - | - |
UP 24.4 | - | + | - | - |
UP 48.1 | - | + | + | - |
Bacterial Isolate Amylase Enzyme Activity from Wikau Maombo
The observation on the curve shows the effect of fermentation time on bacterial growth and the production of amylase enzymes. Its impact on bacterial isolate UM 24.2 (Fig. 1a) showed a significantly increased growth at 18 hours with an OD value of 1.023 and enzyme activity of 551 mU/mL. Enzyme activity and maximum growth were observed at the 27th hour with an OD value of 1.938 and an enzyme activity of 1053 mU/mL. A fermentation time of 21 hours significantly increased the growth of bacterial isolates UM 48.1 and UM 48.2 with OD values and enzyme activity of 1.076 and 414 mU/mL (UM 48.1) as well as 1.057 and 435 mU/mL (UM 48.2), respectively. Meanwhile, enzyme activity and maximum growth were observed at the 33rd hour with OD and activity values of 2.041 and 446 mU/mL (UM 48.1) and 1.887 and 499 mU/mL (UM 48.2), respectively. This finding indicates that the enzyme activity is directly proportional to the OD Value.
The growth curves of UM 24.2, UM 48.1, and UM 48.2 started with a lag phase (adaptation) at 0 to 3 hours. Sharah et al. [40] on Weisella confuse (Lactobacillus Brevis I) showed that the adaptation phase is relatively short, lasting up to the 3rd hour of incubation. In this stage, the bacteria adapt to the new environment. Furthermore, Puspawati et al. [41] stated that the biomass and bacteria cells increase during the lag phase. There was also a change in the chemical composition of the bacteria. The adaptation phase is short because the growth and rejuvenation media are the same.
The next phase, the logarithmic (growth phase), occurred from the 3rd to the 27th hour for UM 24.2 bacteria (Fig. 1a). Research by Hidayatulloh et al. [42] showed that the logarithmic phase of L. Plantarum ATCC 8014 occurred from the 3rd hour to the 24th hour. Meanwhile, it happened from the 3rd to the 33rd hour for UM 48.1 and UM 48.2. Bacteria growth occurs quickly in this stage because it multiplies rapidly and constantly to double the previous population. Energy requirements increase in this phase than in the adaptation.
Furthermore, the growth curve was relatively constant from the 27th to the 48th hour. This period is a stationary phase where the number of cells that grow is the same as those that die. The cell size becomes smaller because the nutrients are reduced [43].
Molecular Characteristics of Lactic Acid Bacteria Isolates from Wikau Maombo
The three stages of bacterial genome isolation include cell destruction, DNA extraction, and purification [44]. The PCR is conducted to amplify specific DNA fragments [45]. The next step is electrophoresis, which separates cellular molecules based on size and movement. It uses an electric field applied to a media [46]. The genomic DNA was tested quantitatively and qualitatively using a nanophotometer (Table 3) and electrophoresis (Fig. 2).
Table 3 indicates that the average isolates with different concentrations had a purity of 1.8–2.0. The difference in concentrations could be affected by the amount of each bacteria before extraction. According to Sambrook and Russell (1989) in Ningsih et al. [47], DNA is pure when it has an absorbance ratio of λ 260/280 nm ranging from 1.8 to 2.0. A purity value lower than 1.8 shows that the sample is contaminated with protein, while a value higher than 2.0 implies that it is contaminated by RNA.
Table 3
The purity and concentration level of DNA of lactic acid bacteria isolates from Wikau Maombo quantitatively
Isolate | Purity | Concentration (ng/µL) |
UM 24.2 | 2.03 | 18.34 |
UM 48.1 | 1.89 | 30.22 |
UM 48.2 | 1.90 | 43.63 |
The next stage is DNA amplification using the PCR method with universal primers. DNA bands measuring 1400 bp were successfully amplified from all isolates. Therefore, the base sequence of each bacterial isolate could be determined from the sequence of a single band obtained by electrogram from PCR results of highly pure genomic DNA. Figure 2 shows the amplicon of genomic DNA amplification using the PCR (Polymerase Chain Reaction) method.
The results of 16S rRNA gene sequencing with primer pairs 8F and 1492R obtained nucleotide data which was used to determine its identity and function. The advantage is that it can detect similarities between bacterial species, which is 99% and can be grouped into relatively high and reasonably distant (low) similarities [48]. Table 4 shows the Basic Local Alignment Search Tool (BLAST) results.
The sequencing results showed the closeness between the bacterial isolates from Wikau Maombo and others in GenBank. Table 4 shows that the three isolates had the closest homology to lactic acid bacteria. Furthermore, UM 24.2, UM 48.1, and UM 48.2 had the closest homology of 99.37%, 99.17%, and 98.77%, respectively, with Lactobacillus plantarum strain CQ2017ZC, Pediococcus pentosaceus strain 1931, and Pediococcus pentosaceus strain 5583. Emmawati et al. [49] research on Mandai fermented food (from South Kalimantan) discovered nine bacteria isolate, including L. plantarum and P. pentosaceus. Puspawati et al. [50] stated that isolates with a similarity percentage of 93% − 97% could represent identities at the genus level but differed at the species level. A genus is said to be similar (different species) when it has < 97% similarity. Also, it is a species with > 97% similarity [51].
Table 4
Comparative taxonomic identification of bacteria isolated from Wikau Maombo by sequencing the 16S rRNA encoding gene according to the database
Isolate code | Type similarity | Accession | Similarity (%) | Description |
UM 24.2 | Lactobacillus plantarum strain CQ2017ZC | MH778541.1 | 99,37 | LAB |
UM 48.1 | Pediococcus pentosaceus strain 1931 | MT597748.1 | 99,17 | LAB |
UM 48.2 | Pediococcus pentosaceus strain 5583 | MT510326.1 | 98,77 | LAB |
Pediococcus pentosaceus is an acid-tolerant lactic acid bacteria with substantial development due to its ability as a starter culture to ferment various foods such as meat, vegetables, and cheese [52]. Rosyidah et al. [53] stated that P. pentosaceus could produce high lactic acid, bacteriocin, and OD values in the logarithmic phase.
Lactobacillus plantarum is a Gram-positive bacteria discovered primarily in milk, meat, fermented vegetables, and the human digestive tract. It can live in facultative anaerobic conditions. However, under aerobic conditions, these bacteria can convert oxygen into peroxides. They can ferment in anaerobic conditions by converting sugar into lactic acid or alcohol under heterofermentative states [54]. L. plantarum is a potential bio preservative because it can inhibit the growth of pathogenic and destructive bacteria. Furthermore, it can inhibit the growth of pathogenic bacteria with the most significant inhibition compared to other lactic acid bacteria [55].