Journal of Aquatic Food Product Technology

ISSN: 1049-8850 (Print) 1547-0636 (Online) Journal homepage: https://www.tandfonline.com/loi/wafp20

Isolation, Screening, and Identification of

Proteolytic Lactic Acid Bacteria from Indigenous
Chao Product

Agussalim Matti, Tyas Utami, Chusnul Hidayat & Endang S. Rahayu

To cite this article: Agussalim Matti, Tyas Utami, Chusnul Hidayat & Endang S. Rahayu (2019):
Isolation, Screening, and Identification of Proteolytic Lactic Acid Bacteria from Indigenous Chao
Product, Journal of Aquatic Food Product Technology, DOI: 10.1080/10498850.2019.1639872

To link to this article: https://doi.org/10.1080/10498850.2019.1639872

Published online: 24 Jul 2019.

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JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY
https://doi.org/10.1080/10498850.2019.1639872

Isolation, Screening, and Identification of Proteolytic Lactic Acid

Bacteria from Indigenous Chao Product
Agussalim Mattia, Tyas Utamib, Chusnul Hidayatb, and Endang S. Rahayu b

a
Department of Fisheries products processing technology, Pangkep State Polytechnic of Agricultural, Pangkajene
dan Kepulauan, Indonesia; bFaculty of Agricultural Technology, Gadjah Mada University, Yogyakarta, Indonesia

ABSTRACT KEYWORDS
The aim of the study was to isolate, select, and identify proteolytic lactic 16S rRNA gene; Chao; fish
acid bacteria (LAB) from chao, a traditional fermented fish from Pangkajene fermented; lactic acid
and Kepulauan Regency, South Sulawesi, Indonesia. LAB was isolated by bacteria; proteolytic
poured plate method. Proteolytic LAB were selected using agar skim milk
media. Protease activity of LAB was determined based on the amount of
tyrosine released in unit/mL. Proteolytic LAB were identified using API 50
CHL kit and 16S rRNA gene sequence analysis. The result showed that
a total of 60 isolates were obtained from chao, and 57% of them were cocci-
shape. Fifteen isolates were halotolerant proteolytic LAB. Their R values and
protease activity were 2.11–3.39 and 0.267–0.304 U/mL, respectively.
Identification by API 50 CHL kit showed that four rod-shape isolates were
Lactobacillus plantarum, and two others were Lactobacillus curvatus. Cocci-
shape isolates could not be identified as cocci bacterium. Rep-PCR results
showed that there were two kinds of bands, namely thick and thin. Two
isolates were selected from two types of bands that had the highest R for
the analysis of 16S rRNA gene sequences, namely Ags1-3 and Ags7-3. The
results showed that Ags1-3 isolate was identified as Lactobacillus plantarum
and Ags7-3 as Pediococcus acidilactici.

Introduction
Lactic acid bacteria (LAB) has been used as probiotic strains in food and food supplements in order
to benefit health (Han et al., 2017; Rúa et al., 2018; Shehata et al., 2016; Tallapragada et al., 2018).
LAB is closely related to fermented food because it produces lactic acid and lowers pH and inhibits
the growth of pathogenic bacteria (Gadallah and Hassan, 2017; Reis et al., 2012). Sugiharto (2016)
reported that probiotic LAB inhibit the growth of pathogenic bacteria by producing lactic acid.
Recently, LAB has been shown to produce uricase (Handayani et al., 2018), reduce acrylamide
formation in bread (Nachi et al., 2018), reduce ochratoxin A (Luz et al., 2018), produce exopoly-
saccharides (Abid et al., 2018), remove tannins (Shang et al., 2019). Local strains have also been
reported to increase folic acid levels in fermented milk (Purwadani et al., 2017).
Proteolytic LAB play an important role in traditional fermented fish because fish contains high
protein. It is a halophile strain and produces extracellular proteinase. This enzyme degrades protein
to produce bioactive peptide compounds and other metabolite products in fermented foods. Najafian
and Babji (2018) reported that bioactive peptide sequences contain hydrophobic, aspartic, and
glycine amino acids that play a role in increasing antioxidant activity in budu products. Bioactive
peptides and metabolite products have benefits for the body, such as inhibiting the activity of
angiotensin converting enzyme (Li et al., 2004), increasing the immune system (Dos Santos et al.,

CONTACT Endang S. Rahayu endangsrahayu@ugm.ac.id Faculty of Agricultural Technology, Gadjah Mada University,
Flora Street No. 1 Bulaksumur, Yogyakarta 55281, Indonesia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/wafp.
© 2019 Taylor & Francis Group, LLC
2 A. MATTI ET AL.

2016; Gildberg, 2012; Lestari et al., 2013), lowering cholesterol (Tsai et al., 2014), preventing cancer
and degenerative diseases (Kwak et al., 2014), and increasing iron absorption in the body (Hoppe
et al., 2015). Lactobacillus plantarum from bekasam milkfish have also been reported to be able to
inhibit angiotensin-I converting enzyme activity (Wikandari et al., 2012).
Indonesia has many traditional fermented foods, such as salted fermented fruit, vegetables, fish,
and shrimp (Rahayu, 2003). Some researchers have also reported LAB from dangke (Syah et al.,
2017) and bekasam (Afriani et al., 2018; Desniar et al., 2013). Chao is a traditional fermented food
from Pangkajene and Kepulauan Regency, South Sulawesi, Indonesia. This product is fermented
from fish and white rice. Tembang fish (Sardilla gibbosa) is one type of fish that is often processed.
Prior research has determined the characteristics of tembang fish as a raw material for chao
fermentation (Agussalim and Kumalasari, 2017). However, many Indonesian fermented foods are
still not explored as a source of proteolytic LAB. The current study explored proteolytic LAB from
chao. Characterization and selection were performed based on morphological and biochemical
characteristics, proteolytic activities, phenotype, and repetitive sequence-polymerase chain reaction
(Rep-PCR) amplification. The similarity of isolates was compared based on 16S rRNA gene sequence
and phylogenetic analysis.

Materials and methods

Sampling
Chao was obtained from two producers, Fishery Product Processing Group, Katojowa, in Labakkang
District (Fin code) and Home Industry in Bungoro District (Ags code), in the Pangkajene and
Kepulauan Regency, South Sulawesi. Samples were taken on the first day of fermentation and carried
out every 2 days for 14 days. The sample was packed in a cooler box with ice cubes and transported
to the Microbiology Laboratory, Center for Food and Nutrition Studies, Center for Inter University,
Gadjah Mada University of Yogyakarta to isolate the LAB content.

Isolation of LAB
LAB isolation was performed by modifying the Sumarsih et al. (2014) and Purwandhani et al. (2018)
methods. A total of 10 g of chao was diluted into 90 mL of sterile 0.85% NaCl solution. The sample
was mixed using a stomacher (Seward 400, England). The solution was diluted from 10−2 to 10−8
using sterile 0.85% NaCl. As much as 1 mL of the dilutions was administered to a petri dish
containing de Man Rogase and Shape (MRS) agar (Oxoid), calcium carbonate (CaCO3) 1%, NaCl
2%, and Na Azida 10 ppm, following the Rahayu (2003) method. LAB was grown at 37°C for 48 h.
Colonies surrounded by clear zones were isolated based on their shape, color, and size. Isolate was
purified using a scratch method on MRS agar and grown at 35°C for 48 h. The purification process
was carried out until a pure culture was obtained.

Morphological and biochemical characterization of LAB

Morphological and biochemical LAB characteristics were performed by modifying the Rahayu
(2003) method using MRS agar media. Morphological characteristics of the LAB tested, namely
Gram staining using the painting method and cell shape, were observed using a 40x magnification
microscope (Nikon Alpha hot YS, Japan). The biochemical characteristics of the LAB tested included
catalase enzyme production, motility, and spore formation. The catalase test was carried out by
dropping 3% hydrogen peroxide (H2O2) to culture isolate on the glass slides (Purwandhani et al.,
2018). Motility test was performed by inoculating the culture into soft MRS (containing agar 5%)
using a straight needle. Spore formation was done by spore staining using a 5% malachite green
solution and 0.5% safranin. Isolates that were LAB were selected for further analysis. The isolates
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 3

were stored at −40°C using 20% glycerol and 10% skim milk (1:1 v/v). Culture isolates were activated
in MRS broth and grown at 37°C for 24 h before being analyzed.

Test for proteolytic activity and gas production of LAB

Qualitative test of proteolytic activity of LAB
The qualitative test of proteolytic characteristics of LAB was performed using 2% agar skim milk
(ASM) media by modifying the method of Jini et al. (2011) and Nespolo and Brandelli (2010). The
LAB isolate was activated in MRS broth media at 37°C for 24 h. Cells were suspended into the ASM
media well. Cells were grown at 37°C for 24 h. Proteolytic activity of LAB in ASM media was shown
by the formation of clear zones around bacterial colonies. The proteolytic activity of LAB is
expressed by the R value. The R value was the ratio between the diameter of the clear zone and
the diameter of the colony.

Quantitative test of protease activity

LAB protease activity was tested quantitatively based on Bergmeyer and Grass (1983) method. LAB
isolates were grown in MRS broth at 37°C for 24 h. Protease production was carried out by
suspending LAB isolates in MRS broth media and 1% casein. Isolates were grown at 37°C for
24 h. Extracellular protease extraction was carried out using cold centrifugation at a speed of
3000 rpm for 15 s. Measurement of protease activity was carried out by mixing 100 µL supernatant,
200 µL of casein 500 ppm, and 300 µL of phosphate buffer solution pH 7. The mixture was allowed
to stand for 60 s at 37°C on a water bath. Then, 400 µL of 4% TCA solution was added and allowed
to stand for 30 s at 27°C. The supernatant was separated using centrifugation at a speed of 3500 rpm
for 10 s. As much as 100 µL of the supernatant was diluted five times using phosphate buffer.
Absorbance was measured using a spectrophotometer at a wavelength of 275 nm. The blank was
made the same as the procedure for determining protease activity, but the addition of TCA was done
immediately after the addition of the enzyme solution. Standard tyrosine curves were made by
preparing 10 volumetric flasks. All volumetric flasks were filled with 20-ppm tyrosine solution with
series 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mL. Then, distilled water was added to the tera mark. One
protease activity unit indicates the amount of enzyme needed to produce 1 mL of tyrosine for
one minute of reaction. Protease enzyme activity was determined by the formula:
Enzymeactivity ¼ ðTirosin=Mr:TirosinÞxðv=ðpxqÞÞxfp; where v = sample volume (mL); p = number
of enzymes (mL); q = incubation time (s); fp = dilution factor.

Gas production test

Gas production from glucose of LAB isolates was carried out by modifying the method of Rahayu
(2003) using MRS broth media. LAB isolates were inoculated into a test tube containing MRS broth
and inverted Durham tube. Then, it was grown at 37°C for 48 h. The presence of air bubbles in the
Durham tube showed that the isolates positively produced gas.

Physiological characteristics of proteolytic LAB

The physiological characteristics of LAB were their ability to grow at various salt conditions, pH, and
temperature. The test was carried out by modifying the Rahayu (2003) method using MRS broth
media. Growth at various salt concentrations was carried out by inoculating LAB isolates on MRS
broth media containing 10% and 6.5% NaCl and without NaCl. Then, it was grown at 37°C for 48 h.
Growth at various pH conditions was carried out by growing LAB isolates on MRS broth media with
a pH of 4.2, 8.4, and 9.6. Furthermore, isolates were grown at 37°C for 48 h. Growth at various
temperature conditions were carried out by inoculating LAB isolates on MRS broth media. Then, the
isolates were grown at temperatures of 10°C, 37°C, 45°C, and 50°C for 48 h. MRS broth without
treatment was used as control for each type of test.
4 A. MATTI ET AL.

Phenotypic characterization of proteolytic LAB

The phenotype characteristics of proteolytic LAB were identified using the API 50 CHL v5.2 kit
(Biomerieux, France). LAB isolates were grown in MRS broth media at 37°C for 24 h. Cells were
separated by centrifugation of 3000 rpm for 10 s. Bacterial cells were washed with sterile 0.85% NaCl.
Furthermore, cells were suspended with 2 mL of sterile 0.85% NaCl. The cell solution was suspended
on medium API CHL v5.2 strips containing 49 kinds of carbohydrates and their derivatives. The
surface of the strip was covered with sterile liquid paraffin and then grown at 37°C for 48 h. Changes
in color from dark blue to yellow were expressed as positive changes. The results obtained were
processed using the Apiweb™ Identification software on the website https://apiweb.biomerieux.com
to determine the type of bacteria from each isolate.

Genotypic characterization of proteolytic LAB

DNA extraction
LAB isolates were grown in MRS broth media at 37°C for 18 to 24 h. Cells were separated using
centrifugation at 13,000 rpm for 10 s. Genomic DNA extraction was carried out based on the
method of Suhartatik et al. (2014) with several modifications. The bacterial cells obtained were
suspended in 1000 µL lysis buffer. The solution was mixed using vortex and activated with
a shaker for 20 s. Furthermore, the solution was added to 20 mg/mL of proteinase K. The
solution was mixed using vortex and activated with a shaker for 20 s to accelerate cell destruc-
tion. The solution was added to 100 mg/mL lysozyme. The solution was mixed using vortex and
activated at 55°C for 30 s. Supernatant was separated using centrifugation at 13,000 rpm for 15 s.
Then, it was transferred to a new 2-mL micro tube, and phenol 1:1 (v/v) was added. The solution
was mixed using vortex and incubated with a shaker for 30 s. Furthermore, supernatant was
separated by centrifugation at 13,000 rpm for 10 s. Then, the upper solution was transferred to
a new 2-mL micro tube, and 1:1 (v/v) cold chloroform was added to clean phenol. The solution
was mixed using vortex and activated with a shaker for 20 s. Supernatant was separated using
centrifugation at 13,000 rpm for 10 s. The upper supernatant was transferred to a new 1.5-mL
micro tube and 1:1 (v/v) of cold ethanol was added. Genomic DNA was separated after being
stored at −20°C for one night. The genomic DNA was separated using centrifugation at
13,000 rpm for 10 s and then added to 500 mL of cold 70% ethanol. The solution was separated
using centrifugation at 13,000 rpm for 5 s to obtain pure genomic DNA. The genomic DNA was
dried for 2 h, added to 50 µL TE buffer and 3 µL RNAse, and incubated at 37°C for 1 h before
further application.

Amplification Rep-PCR
Rep-PCR amplification was carried out following the method of De Bruijn (1992) using the PCR mix
Ready to Go (RTG) kit. RTG was reacted by adding 1 µL of DNA, 23-µL nuclease-free water
(ddH2O), and 1 µL of BOX AIR primer (5ʹCTACGGCAAGGCGACGCTGACGCTGACG-3ʹ)
(Tacão et al., 2005). Rep-PCR product was detected by 1.5% agarose gel electrophoresis following
the method of Al-Saleh et al. (2014). An electrophoregram was obtained with photographs above
UV-vis.

16S rRNA gene sequencing

16S rRNA gene amplification was carried out by modifying the method of a previous study using
two universal primers, namely 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1492R (5ʹ-
GGTTACCTTGTTACGACTT-3ʹ) (Sulistiani et al., 2014). PCR mix contained 25 µL of ingredients,
consisting of 12.5-µL Green Go Tag Flexi, 8.5-µL nuclease-free water, 1-µL primer 27F, 1-µL primary
1492R, 2-µL genomic DNA. PCR products were detected by 1.5% agarose gel electrophoresis
following the method of Al-Saleh et al. (2014). An electrophoregram was obtained with photographs
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 5

on UV-vis. PCR amplification products were sequenced with the Dideoxy Sanger method using
automatic DNA.

DNA sequence and phylogenic analysis

The results of 16S rRNA gene sequencing of LAB isolates were analyzed for similarity with 16S
rRNA nucleotide base sequences of other bacteria in the GenBank database using the BLAST
program on the NCBI website (http://www.ncbi.nlm.nih. Gov/genbank). The double alignment of
the nitrogen base sequence was analyzed using the clustalW program to construct phylogenetic trees.
The similarity between nucleotide base sequences was analyzed using neighbor-joining method with
1000x Bootstrap method in MEGA7 software.

Results and discussion

Isolation of LAB
Sixty pure isolates were isolated from 20 chao samples (Table 1). Fin and Ags codes both produced
30 isolates. These isolates grew in the MRS media under anaerobic conditions. All isolates were
identified as LAB based on their morphological and biochemical characteristics. Those isolates were
gram positive and negative catalase bacteria, which are characteristic of LAB. This means that all
samples taken from two chao producers in Pangkajene and Kepulauan Regency contained LAB
strains.

Table 1. Morphological and biochemical characteristics of LAB isolates.

MBC MBC
SC IC GS S CR SF M SC IC GS CF CR SF M
Fin1 Fin1-1 + Cocci − − − Ags1 Ags1-1 + Cocci − − −
Fin1-2 + Cocci − − − Ags1-2 + Cocci − − −
Fin1-3 + Cocci − − − Ags1-3 + Rods − − −
Fin2 Fin2-1 + Rods − − − Ags2 Ags2-1 + Rods − − −
Fin2-2 + Rods − − − Ags2-2 + Cocci − − −
Fin2-3 + Rods − − − Ags2-3 + Cocci − − −
Fin3 Fin3-1 + Rods − − − Ags3 Ags3-1 + Cocci − − −
Fin3-2 + Cocci − − − Ags3-2 + Cocci − − −
Fin3-3 + Rods − − − Ags3-3 + Cocci − − −
Fin4 Fin4-1 + Rods − − − Ags4 Ags4-1 + Rods − − −
Fin4-2 + Rods − − − Ags4-2 + Cocci − − −
Fin4-3 + Cocci − − − Ags4-3 + Cocci − − −
Fin5 Fin5-1 + Rods − − − Ags4-4 + Cocci − − −
Fin5-2 + Cocci − − − Ags5 Ags5-1 + Cocci − − −
Fin6 Fin6-1 + Cocci − − − Ags5-2 + Rods − − −
Fin6-2 + Rods − − − Ags6 Ags6-1 + Cocci − − −
Fin6-3 + Rods − − − Ags6-2 + Cocci − − −
Fin7 Fin7-1 + Cocci − − − Ags7 Ags7-1 + Cocci − − −
Fin7-2 + Rods − − − Ags7-2 + Cocci − − −
Fin7-3 + Cocci − − − Ags7-3 + Cocci − − −
Fin7-4 + Rods − − − Ags7-4 + Cocci − − −
Fin8 Fin8-1 + Rods − − − Ags8 Ags8-1 + Cocci − − −
Fin8-2 + Cocci − − − Ags8-2 + Cocci − − −
Fin8-3 + Rods − − − Ags8-3 + Rods − − −
Fin9 Fin9-1 + Rods − − − Ags9 Ags9-1 + Rods − − −
Fin9-2 + Rods − − − Ags9-2 + Rods − − −
Fin9-3 + Cocci − − − Ags9-3 + Rods − − −
Fin10 Fin10-1 + Cocci − − − Ags10 Ags10-1 + Rods − − −
Fin10-2 + Cocci − − − Ags10-2 + Rods − − −
Fin10-3 + Cocci − − − Ags10-3 + Cocci − − −
SC: sample code; IC: isolate code; MBC: Morphological and biochemical characteristics; GS: gram staining; S: shape; CR: catalase
reaction; SF: spore formation; M: motility; (+) Positive; (−) Negative
6 A. MATTI ET AL.

Morphological and biochemical characteristics of LAB

Gram staining test results showed that all isolates were Gram positive bacteria. Observations on the
shape of cell morphology showed that 34 isolates were cocci and 26 were rod-shape. This means that
chao was dominated by 57% of cocci-shaped bacteria. The biochemical characteristics test results
showed that all isolates did not produce catalase enzymes, did not form spores, and did not grow
spreading on soft MRS media. The test results showed that LAB can be isolated every day during the
fermentation of chao. Morphological and biochemical profiles of LAB isolates are shown in Table 1.

Proteolytic activities, gas production, and physiological properties of LAB

All LAB isolates were tested for their qualitative proteolytic ability using ASM media. The test results
showed that 25% of isolates formed clear zones with varying R values (Table 2). The R value of each
isolate was between 2.11 and 3.39. The results showed that all isolates had a good proteolytic activity
(Abubakr and Al-adiwish, 2017). These 15 isolates were proteolytic LAB. Eight LAB isolates were
rod-shaped, and seven other were cocci-shaped. The hydrolysis activity of a bacterium was described
by its ability to form clear zones around the walls of ASM media or bacterial colonies. The clear zone
indicated that casein in the skim was hydrolyzed to peptides and amino acids (Jini et al., 2011; Thapa
et al., 2006). Enzymes that played a role in hydrolyzing casein were extracellular proteinase enzymes
produced by LAB. The number of proteases produced by bacteria were affected by pH, temperature,
and the concentration of the substrate. The factors in this study were the same, namely neutral pH,
37°C temperature, and the same media concentration. Each bacteria requires a different pH,
temperature, and optimum substrate to produce the maximum amount of enzymes. This causes
the proteolytic activity of the isolates tested to be different. The 15 isolates were then quantitatively
tested for protease activity.
The quantitative test results showed that all isolates had different protease activity (Table 2). The
protease activity of LAB isolates ranged from 0.267 to 0.304 U/mL. Fin3-2 isolate had the lowest
protease activity, and Ags7-3 isolate was the highest. This value is greater than the protease activity
of Bacillus sp. reported by Baehaki et al. (2011). Two isolates that were highest in protease activity
were also high in R values, namely Ags7-3 and Ags1-3. However, quantitative protease activity was
not directly proportional to the high and low of qualitative proteolytic abilities. Both of these isolates
have the potential to be used to produce bioactive peptides in chao products and other fermented
foodstuff.

Table 2. Protease activities, gas production, and type of fermentation of LAB isolates.
Isolate R** Protease activities (U/mL)** Gas production Type of fermentation
Fin2-2 2.92 ± 0.006 0.285 ± 0.001 − Ho
Fin3-2 2.40 ± 0.006 0.267 ± 0.003 − Ho
Fin4-3* 3.09 ± 0.010 0.275 ± 0.002 − Ho
Fin6-1 3.22 ± 0.006 0.297 ± 0.000 + He
Fin8-3* 3.21 ± 0.021 0.282 ± 0.000 − Ho
Fin9-2 3.13 ± 0.010 0.285 ± 0.001 + He
Fin10-1* 3.04 ± 0.023 0.268 ± 0.001 − Ho
Fin10-2* 3.10 ± 0.006 0.291 ± 0.001 − Ho
Ags1-3* 3.39 ± 0.010 0.302 ± 0.001 − Ho
Ags2-1* 3.29 ± 0.006 0.295 ± 0.001 − Ho
Ags4-4 2.11 ± 0.006 0.250 ± 0.001 − Ho
Ags7-3* 3.34 ± 0.015 0.304 ± 0.000 − Ho
Ags8-3* 3.12 ± 0.006 0.298 ± 0.000 − Ho
Ags9-1* 3.24 ± 0.021 0.293 ± 0.000 − Ho
Ags10-1* 3.02 ± 0.025 0.286 ± 0.000 − Ho
*selected isolates.
Ho: homofermentative; He: heterofermentative.
**Data are means ± SD, n = 3
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 7

The differences in protease activity between isolates were caused by differences in bacterial
species. However, the different activities indicated that the 15 isolates were not varied. Other factors
influence differences in protease activity, such as the ability of bacteria to adapt to their environment.
Species that are tolerant of the environment produce high protease enzyme (Desniar et al., 2013).
The factors that influence protease production are the time and availability of the substrate. Each
bacterial isolate has a different optimum time for producing enzymes. Time production of the
enzyme in these studies for all isolates was 24 h, so the amount of protease produced was not the
same. Protease in isolates were constitutive enzymes, namely enzymes in microbial cells in relatively
constant amounts. However, after leaving the cell, it will be inductive and the amount will increase if
there is a substrate in the media.
The difference in protease activity between isolates was also influenced by temperature and pH of
the analysis (Afriani et al., 2018). The temperature and pH of the analysis used was the same, namely
37°C and neutral. Each protease had a different optimum temperature and pH to be active (Baehaki
et al., 2011). Protease activity increased with increasing temperature until it reached the optimal
temperature. Higher temperatures caused the structure of enzyme proteins to be damaged so that
non-covalent interactions were denatured. Denaturation causes the structure and active side of the
enzyme to change, so that enzyme activity decreases. A stable pH will maintain the stability of the 3D
structure of the enzyme. Ion groups play a role in maintaining the composition of active enzymes in
binding to the substrate (Afriani et al., 2018).
The results of gas production testing showed that two isolates produced gas from glucose
fermentation, namely Fin6-1 and Fin9-2 isolates (Table 2). This means that 13% of proteolytic
isolates are heterofermentative types. The isolates ferment glucose through the phosphoketolase
producing lactic acid, ethanol, and CO2. Gas was produced after experiencing phosphorylation and
dehydrogenation. Fin6-1 isolate had similar characteristics to the heterofermentative Lactobacillus
genus, whereas Fin9-2 was similar to Leuconostoc. Yuliana et al. (2018) reported that Leoconostoc was
a LAB that produced gas in rusip. The two gas-forming isolates were not suitable for use in food
because they can affect the quality of the packaging, so both these LAB were not analyzed further.
Thirteen isolates including homofermentative do not produce gas from glucose fermentation. These
strains ferment glucose through the glycolysis pathway to produce lactic acid as the only final
product. Isolates Fin2-2, Fin8-3, Ags1-3, Ags2-1, Ags8-3, Ags9-1, and Ags10-1 had the same
characteristics as the genus Lactobacillus homofermentative. Fin3-2, Fin4-3, Fin10-1, Fin10-2,
Ags4-4, and Ags7-3 had the same characteristics as the genera Streptococcus, Lactococcus, and
Pediococcus. Proteolytic isolates with R > 3.00 and homofermentative characteristics were selected
for further analysis. Ten selected isolates were tested for physiological properties and identified
species (Table 2).

Physiological properties of proteolytic LAB

The results of the growth test in various concentrations of salt, pH, and temperature are presented in
Table 3. All isolates grew on media containing 6.5% NaCl and without NaCl. No isolates grew on the
media with 10% NaCl concentration. These isolate were the halotolerant proteolytic LAB because
they grew in non-halophile and mild halophile media. Udomsil et al. (2010) reported that halophilic
LAB plays an important role during the fermentation of fish sauce. These isolates have the potential
to be applied as probiotics to salty foods. All isolates grew in media with pH 4.2 and 8.4. This
condition was characterized by the genera of Lactobacillus and Pediococcus. All isolates did not grow
at pH 9.6, indicating that the isolates were not the genera of Aerococcus and Enterococcus (Rahayu,
2003). All isolates did not grow at temperatures of 10°C and 50°C, which is characteristic of the
genera of Lactobacillus and Pediococcus. Five rod-shaped isolates (Fin8-3, Ags1-3, Ags8-3, Ags9-1,
and Ags10-1) grew at a temperature of 45°C, which is characteristic of thermophiles Lactobacillus;
and one isolate (Ags2-1) did not grow at a temperature of 45°C, which is a feature of mesophilic
Lactobacillus. Two cocci-shaped isolates (Fin10-1 and Ags7-3) grew at a temperature of 45°C,
8 A. MATTI ET AL.

Table 3. Physiological properties of proteolytic LAB isolates.

Growth at
NaCl (%) pH Temperature (°C)
Isolate 0 6.5 10 4.2 8.4 9.6 10 37 45 50
Fin4-3 + + − + + − − + − −
Fin8-3 + + − + + − − + + −
Fin10-1 + + − + + − − + + −
Fin10-2 + + − + + − − + − −
Ags1-3 + + − + + − − + + −
Ags2-1 + + − + + − − + − −
Ags7-3 + + − + + − − + + −
Ags8-3 + + − + + + − + + −
Ags9-1 + + − + + + − + + −
Ags10-1 + + − + + + − + + −
(+) growth; (−) no growth

characteristic of the thermophile Pediococcus genus, and two other isolates (Fin 4-3 and Fin10-2)
were mesophile Pediococcus (Rahayu, 2003). All isolates grew at a temperature of 37°C, indicating
that these isolates were LAB.

Phenotypic characterization of proteolytic LAB

The phenotypic characterization of the 10 selected isolates was carried out with API 50 CHL kit.
Characterization results showed that all isolates can ferment sugars L-Arabinose, D-Ribose,
D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetyl glucosamine, D-cellobiosa,
and gentiobiose. The ability of a bacterium to metabolize arabinose and gluconate is characteristic of
Lactobacillus plantarum, Lactobacillus pentosous, Lactobacillus curvatus, and Lactobacillus murinus
based on Bergey’s Manual Identification.
Identification results (Table 4) showed that six rod-shaped isolates were identified as Lactobacillus
curvatus and Lactobacillus plantarum, two (20%), and four (44%) isolates, respectively. Lactobacillus
plantarum was the dominant strain grown in fermented chao compared to Lactobacillus curvatus.
This result is consistent with that reported by Rahayu (2003) that Lactobacillus plantarum was
a dominant LAB in Indonesian traditional fermented food. Lactobacillus species were also reported
to be dominant in traditional fermented shubat (Shori, 2012). Four cocci-shape isolates could not be
detected as cocci bacteria. Nurhidayu et al. (2012) reported that the identification of bacteria
phenotype using a commercial kit tends to cause errors. Therefore, analysis of 16S rRNA gene
sequences was carried out for more accurate identification. This method can identify the bacteria to
the species level (Amqizal et al., 2017; Veˇtrovsky and Baldrian, 2013).

Table 4. Identification of proteolytic LAB isolates using API 50 CHL kit.

Isolate LAB significant taxa Identity (%)
Ags10-1 Lactobacillus curvatus 83.7
Ags9-1 Lactobacillus curvatus 82.7
Ags8-3 Lactobacillus plantarum 55.2
Fin4-3 ND
Fin8-3 Lactobacillus plantarum 93.5
Fin10-1 ND
Fin10-2 ND
Ags1-3 Lactobacillus plantarum 99.8
Ags2-1 Lactobacillus plantarum 99.8
Ags7-3 ND
ND: not detected
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 9

Genotypic characterization of proteolytic LAB

Amplification Rep-PCR
Rep-PCR amplification was carried out to differentiate genetic types from several bacteria (Beaz-
Hidalgo et al., 2008). Genetic differences were indicated by the banding pattern formed. The isolates
of Rep-PCR amplification were grouped based on differences in banding patterns formed (Akabanda
et al., 2013). Differences in band-shape indicated differences in types of DNA and species of bacteria.
Rep-PCR results showed that there were two kinds of bands, namely thick and thin (Figure 1). The
difference in band thickness shows differences in DNA concentrations and bacterial species. This
indicates that the 10 isolates consisted of two types of DNA. Six thick isolates were rod-shaped LAB,
and four thin isolates were cocci-shaped LAB. One isolate from each group was selected for 16S
rRNA gene sequencing. The selected isolates were those with the highest R value of each type of
band, namely Ag1-3 (R = 3.39) and Ags7-3 (R = 3.34).

16S rRNA gene sequence and phylogenetic analysis

The PCR product of Ags1-3 and Ags7-3 isolates showed the formation of a single band (Figure 2(a)).
This illustrates that the 16S rRNA gene sequences of both isolates were successfully amplified at
1500 bp. The results of the comparison of 16S rRNA gene sequences of Ags1-3 isolate with 16S
rRNA sequences in GenBank database are presented in Table 5. Ags1-3 isolate had a genetic
similarity of 99.79% with Lactobacillus plantarum strain JCM 1149, Lactobacillus plantarum CIP
strain 103151, and Lactobacillus pentosus strain 1242. The relationship between Ags1-3 isolate and
the three LAB strains was described in phylogenetic trees (Figure 2(b)). Ags1-3 isolate was in the
same branch with Lactobacillus plantarum JCM 1149 strain. This result was identified using API 50
CHL kit. It indicated that both isolates had very close genetic proximity. Lactobacillus plantarum
species was also reported to have been isolated from fish and other aquatic products (Ida Muryany
et al., 2017; Kouakou et al., 2012; Le and Yang, 2018). This species was reported to increase the
activity of ACE inhibitors in fermented fish, act as probiotic in the digestion of fish, and inhibit the
growth of Vibrio parahaemolyticus in the fish culture industry (Kang et al., 2016; Le and Yang, 2018;
Wikandari et al., 2012). Therefore, Lactobacillus plantarum isolated from chao is potentially a source
of probiotics in local food and aquaculture.
Analysis of 16S rRNA gene sequences of Ags7-3 isolate with the other 16S rRNA gene sequence in
GenBank database showed that this isolate had a genetic similarity of 99.38% with Pediococcus
acidilactici strain DSM 20284 (Table 5). This isolate also had 98.06% similarity with Pediococcus
pentosaceus strain DSM 20336. The kinship relationship of Ags7-3 isolate and the two LAB strains
was described in phylogenetic trees (Figure 2(b)). Ags7-3 isolate had genetic closeness with
Pediococcus acidilactici strain DSM 20284. The same species was also reported to have been obtained

Figure 1. DNA band-shape produced by Rep-PCR amplification using AIR BOX primer.
10 A. MATTI ET AL.

Figure 2. Electroforegram of 16S rRNA gene sequence Ags1-3 and Ags7-3 isolate in fragment 1500 bp (a) Neighbor-joining
phylogenetic tree for Ags1-3 and Ags7-3 isolates based on the 16S rRNA gene sequences (b).

Table 5. Identification of Ags3-3 and Ags7-3 isolates from chao by 16S rRNA gene sequencing.
Isolate Access number on NCBI Identify (%) Suggested species
Ags1-3 NR_117813.1 99.79 Lactobacillus plantarum strain JCM 1149
NR_104573.1 99.79 Lactobacillus plantarum strain CIP 103151
NR_029133.1 99.79 Lactobacillus pentosous strain 124-2
Ags7-3 NR_042057.1 99.38 Pediococcus acidilactici strain DSM 20284
NR_042058.1 98.06 Pediococcus pentosaceus strain DSM 20336

from the fishery products and by-products of fish (Amin et al., 2017; Jini et al., 2011; Lim, 2016;
Siddegowda et al., 2017). The species generated from indigenous chao has the potential to improve
the microbiological and chemical quality of food, especially similar products. Kusmarwati et al.
(2011) reported that Pediococcus acidilactici was able to improve the quality of rusip.
Two proteolytic LAB that have been explored from indigenous chao have the potential to be
applied as starter culture and probiotic in fermented food and non-food. Shori (2015) reported that
the food industry is currently trying to produce various types of probiotic foods in addition to dairy
products that have the potential to provide health benefits.

Conclusions
Sixty isolates of LAB were isolated from chao. Fifteen isolates were halotolerant proteolytic LAB.
Ags1-3 and Ags7-3 isolates were analyzed in 16S rRNA gene sequence based on Rep-PCR product.
The results showed that the Ags1-3 isolate has a genetic closeness to the Lactobacillus plantarum
strain JCM 1149 species, whereas the Ags7-3 isolate has a genetic proximity to the Pediococcus
acidilactici strain DSM 20284 species. Both of these proteolytic strains have the potential to be
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 11

applied to produce bioactive peptides in chao products and other fermented foodstuff, such as
bekasam, surip, and fish sauce. In further studies, these two strains will be analyzed for their
probiotics properties and application to produce ACE inhibitors in chao fermented products.

Funding
This work was supported by the Ministry of Research, Technology, and Higher Education, Republic of Indonesia
under Grant of Doctoral Dissertation Program 2018 [No. 3/E/KPT/2018, 2018].

ORCID
Endang S. Rahayu http://orcid.org/0000-0002-6101-3433

References
Abid, Y., Casillo, A., Gharsallah, H., et al. 2018. Production and structural characterization of exopolysaccharides from
newly isolated probiotic lactic acid bacteria. Int. J. Biol. Macromol. 108: 719–728. doi: 10.1016/j.
ijbiomac.2017.10.155
Abubakr, M. A. S., and Al-adiwish, W. M. 2017. Isolation and identification of lactic acid bacteria from different fruits
with proteolytic activity. Int. J. Microbiol. Biotechnol. 2: 58–64. doi: 10.11648/j.ijmb.20170202.12
Afriani, A., Marlida, Y., and Yuherman. 2018. Isolation and characterization of lactic acid bacteria proteases from
bekasam for use as a beef tenderizer. Paki. J. Nutr. 17: 361–367. doi: 10.3923/pjn.2018.361.367
Agussalim, M., and Kumalasari, T. 2017. Karakteristik ikan tembang (Sardinella gibbosa) sebagai bahan baku
pembuatan produk fermentasi Chao. J. Galung Trop. 6: 72–80.
Akabanda, F., Owusu-kwarteng, J., Tano-debrah, K., Glover, R. L. K., Nielsen, D. S., and Jespersen, L. 2013.
Taxonomic and molecular characterization of lactic acid bacteria and yeasts in nunu, a Ghanaian fermented milk
product. Food Microbiol. 34: 277–283. doi: 10.1016/j.fm.2012.09.025
Al-Saleh, A. A., Ismail, E. A., and Metwalli, A. A. 2014. Autolysis detection and evaluation of some lactic acid bacteria
by renaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and polymerase chain reaction assays.
Int. J. Dairy Technol 67: 290–296. doi: 10.1111/1471-0307.12113
Amin, M., Adams, M., Bolch, C. J. S., and Burke, C. M. 2017. In vitro screening of lactic acid bacteria isolated from
gastrointestinal tract of Atlantic salmon (Salmo salar) as probiont candidates. Aquacult Int. 25: 485–498. doi:
10.1007/s10499-016-0045-6
Amqizal, H. I. A., Al-kahtani, H. A., Ismail, E. A., Hayat, K., and Jaswir, I. 2017. Identification and verification of
porcine DNA in commercial gelatin and gelatin containing processed foods. Food Control 78: 297–303.
doi:10.1016/j.foodcont.2017.02.024
Baehaki, A., Rinto, and Budiman, A. 2011. Isolasi dan Karakterisasi Protease dari bakteri tanah rawa Indralaya,
Sumatera Selatan. J. Teknol. Dan Ind. Pangan. 12: 40–45.
Beaz-Hidalgo, R., López-Romalde, S., Toranzo, A. E., and Romalde, J. L. 2008. Polymerase chain reaction amplification
of repetitive intergenic consensus and repetitive extragenic palindromic sequences for molecular typing of pseu-
domonas anguilliseptica and aeromonas salmonicida. J. Aquat. Anim. Health. 20: 75–85. doi: 10.1577/H07-007.1
Bergmeyer, H. U., and Grass, M. 1983. Method of Enzymatic Analysis Third. Weinheim, Germany: Verlag Chemie.
De Bruijn, F. J. 1992. Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric
consensus) sequences and the polymerase chain reaction to fingerprint the genomes of rhizobium meliloti isolates
and other soil bacteria. Appl. Environ. Microbiol. 58: 2180–2187.
Desniar, Rusmana, I., Suwanto, A., and Mubarik, R. 2013. Characterization of lactic acid bacteria isolated from an
Indonesian fermented fish (bekasam) and their antimicrobial activity against pathogenic bacteria. Emir. J. Food
Agric. 25: 489–494. doi: 10.9755/ejfa.v25i6.12478
Dos Santos, T. F., Melo, T. A., Almeida, M. E., Rezende, R. P., and Romano, C. C. 2016. Immunomodulatory effects of
lactobacillus plantarum Lp62 on intestinal epithelial and mononuclear cells. Biomed Res. Int: 1–8. doi: 10.1155/
2016/8404156
Gadallah, M. G. E., and Hassan, M. F. Y. 2017. Quality properties of Kishk (a dried fermented cereal-milk mixture)
prepared from different raw materials. J. Saudi Soc. Agric. Sci. doi: 10.1016/j.jssas.2017.02.003
Gildberg, A. 2012. Journal of aquatic food enzymes and bioactive peptides from fish waste related to fish silage, fish
feed and fish sauce production. J. Aquat. Food Prod. Technol. 3: 3–11. doi: 10.1300/J030v13n02_02
Han, Q., Kong, B., Chen, Q., Sun, F., and Zhang, H. 2017. In vitro comparison of probiotic properties of lactic acid
bacteria isolated from harbin dry sausages and selected probiotics. J. Funct. Foods. 32: 391–400. doi: 10.1016/j.
jff.2017.03.020
12 A. MATTI ET AL.

Handayani, I., Utami, T., Hidayat, C., and Rahayu, E. 2018. Screening of lactic acid bacteria producing uricase and
stability assessment in simulated gastrointestinal conditions. Int. Food Res. J. 25: 1661–1667.
Hoppe, M., Önning, G., Berggren, A., and Hulthén, L. 2015. Probiotic strain lactobacillus plantarum 299v increases
iron absorption from an iron-supplemented fruit drink: A double-isotope cross-over single-blind study in women
of reproductive age. Br. J. Nutr. 114: 1195–1202. doi: 10.1017/S000711451500241X
Ida Muryany, M. Y., Ina Salwany, M. Y., Ghazali, A. R., Hing, H. L., and Nor Fadilah, R. 2017. Identification and
characterization of the lactic acid bacteria isolated from Malaysian fermented fish (Pekasam). Int. Food Res. J. 24:
868–875.
Jini, R., Swapna, H., Amit, K. R., et al. 2011. Isolation and Characterization of potential lactic acid bacteria (LAB) from
freshwater fish processing wastes for application in fermentative utilisation of processing waste. Brazilian
J. Microbiol. 42: 1516–1525. doi:10.1590/S1517-83822011000400039
Kang, C., Shin, Y., Kim, Y., and So, J. 2016. Isolation of lactobacillus strains from shellfish for their potential use as
probiotics. Biotechnol. Bioprocess Eng. 21: 46–52. doi: 10.1007/s12257-015-0518-x
Kouakou, A. C., N’Guessan, K. F., Durand, N., Thomas, D. A., Montet, D., and Dje, M. K. 2012. Molecular bacterial
characterization and free amino acids composition in Ivorian traditional fermented fish produced by two methods.
Fish Sci 78: 1125–1136. doi: 10.1007/s12562-012-0526-0
Kusmarwati, A., Heruwati, E. S., Utami, T., and Rahayu, E. S. 2011. Pengaruh penambahan Pediococcus
acidilacticiF-11 sebagai kultur starter terhadap kualitas rusip teri (Stolephorus sp). J. Pascapanen Dan Bioteknol.
Kelaut. Dan Perikan. 6: 13–26. doi:10.15578/jpbkp.v6i1.84
Kwak, S.-H., Cho, Y.-M., Noh, G.-M., and Om, A.-S. 2014. Cancer preventive potential of kimchi lactic acid bacteria
(Weissella cibaria, Lactobacillus plantarum). J. Cancer Prev. 19: 253–258. doi: 10.15430/JCP.2014.19.4.253
Le, B., and Yang, S. H. 2018. Probiotic potential of novel Lactobacillus strains isolated from salted-fermented shrimp as
antagonists for Vibrio parahaemolyticus. J. Microbiol. 56: 138–144. doi:10.1007/s12275-018-7407-x
Lestari, L. A., Setyabudi, F. M. C. S., Julia, M., and Amalia, L. D. 2013. Effect of synbiotic yogurt made with indigenous
probiotic Lactobacillus plantarum Mut7 and sweet potato fiber (Ipomoea batatas) in healthy children. Int. Res.
J. Microbiol. 4: 98–102.
Li, G., Le, G., Shi, Y., and Shrestha, S. 2004. Angiotensin I–converting enzyme inhibitory peptides derived from food
proteins and their physiological and pharmacological effects. Nutr. Res. 24: 469–486. doi: 10.1016/j.
nutres.2003.10.014
Lim, E. 2016. Inhibitory effect of bacteriocin-producing lactic acid bacteria against histamine-forming bacteria isolated
from Myeolchi-jeot. Fish. Aquat. Sci. 19: 1–10. doi: 10.1186/s41240-016-0040-x
Luz, C., Ferrer, J., Mañes, J., and Meca, G. 2018. Toxicity reduction of ochratoxin A by lactic acid bacteria. Food
Chem. Toxicol. 112: 60–66. doi: 10.1016/j.fct.2017.12.030
Nachi, I., Fhoula, I., Smida, I., et al. 2018. Assessment of lactic acid bacteria application for the reduction of acrylamide
formation in bread. LWT-Food Sci. Technol. 92: 435–441. doi:10.1016/j.lwt.2018.02.061
Najafian, L., and Babji, A. S. 2018. Purification and Identification of antioxidant peptides from fermented fish sauce
(Budu) purification and identification of antioxidant peptides from. J. Aquat. Food Prod. Technol.: 2–11. doi:
10.1080/10498850.2018.1559903
Nespolo, C. R., and Brandelli, A. 2010. Production of bacteriocin-like substance by lactic acid bacteria isolated from
regional ovine cheese. Brazilian J. Microbiol. 41: 1009–1018. doi:10.1590/S1517-83822010000400020
Nurhidayu, A., Ina-Salwany, M., Daud, H. M., and Harmin, S. 2012. Isolation, screening and characterization of
potential probiotics from farmed tiger grouper (Epinephelus fuscoguttatus). African J. Microbiol. Res. 6: 1924–1933.
doi: 10.5897/AJMR11.913
Purwadani, S. N., Utami, T., Millati, R., and Rahayu, E. S. 2017. Potensi Lactobacillus plantarum yang Diisolasi dari
Dadih dalam Meningkatkan Kadar Folat Susu Fermentasi. Agritech 37: 395–401. doi:10.22146/agritech.10493
Purwandhani, S. N., Utami, T., Milati, R., and Rahayu, E. S. 2018. Isolation, characterization and screening of
folate-producing bacteria from traditional fermented food (dadih). Int. Food Res. J. 25: 566–572.
Rahayu, E. S. 2003. Lactic acid bacteria in fermented foods of Indoesian origin. Agritech 23: 75–84.
Reis, J. A., Paula, A. T., Casarotti, S. N., and Penna, A. L. B. 2012. Lactic acid bacteria antimicrobial compounds:
characteristics and Applications. Food Eng. Rev. 4: 124–140. doi:10.1007/s12393-012-9051-2
Rúa, J., López-rodríguez, I., Sanz, J., García-fernández, M. C., Valle, M. P., and Del, García-armesto, M. R. 2018.
Improving functional properties of “Piel de Sapo” melon juice by addition of a Lippia citriodora natural extract and
probiotic-type lactic acid bacteria. LWT - Food Sci. Technol 96: 75–81. doi: 10.1016/j.lwt.2018.05.028
Shang, Y., Cao, H., Ma, Y., Zhang, C., Ma, F., and Wang, C. 2019. E ff ect of lactic acid bacteria fermentation on
tannins removal in Xuan Mugua fruits. Food Chem 274: 118–122. doi: 10.1016/j.foodchem.2018.08.120
Shehata, M. G., El Sohaimy, S. A., El-sahn, M. A., and Youssef, M. M. 2016. Screening of isolated potential probiotic
lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Ann. Agric. Sci 61: 65–75. doi:
10.1016/j.aoas.2016.03.001
Shori, A. B. 2012. Comparative study of chemical composition, isolation and identification of micro-flora in traditional
fermented camel milk products: gariss, Suusac, and Shubat. J. Saudi Soc. Agric. Sci. 11: 79–88. doi: 10.1016/j.
jssas.2011.12.001
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 13

Shori, A. B. 2015. The potential applications of probiotics on dairy and non-dairy foods focusing on viability during
storage. Biocatal. Agric. Biotechnol. 4: 423–431. doi: 10.1016/j.bcab.2015.09.010
Siddegowda, G. S., Bhaskar, N., and Gopal, S. 2017. Fermentative properties of proteolytic pediococcus strains isolated
from salt fermented fish hydrolysate prepared using freshwater fish rohu (Labeo rohita). J. Aquat. Food Prod.
Technol. 26: 341–355. doi: 10.1080/10498850.2016.1185754
Sugiharto, S. 2016. Role of nutraceuticals in gut health and growth performance of poultry. J. Saudi Soc. Agric. Sci. 15:
99–111. doi: 10.1016/j.jssas.2014.06.001
Suhartatik, N., Cahyanto, M. N., Rahardjo, S., Miyashita, M., and Rahayu, E. S. 2014. Isolation and identification of
lactic acid bacteria producing β glucosidase from Indonesian fermented foods. Int. Food Res. J. 21: 973–978.
Sulistiani, A., Sukara, E., Salamah, A., Dinoto, A., and Mangunwardoyo, W. 2014. Identification of lactic acid bacteria
in sayur asin from central java (Indonesia) based on 16S rDNA sequence. Int. Food Res. J. 21: 527–532.
Sumarsih, S., Sulistiyanto, B., Sutrisno, C. I., and Rahayu, E. S. 2014. Characteristic of Lactobacillus isolated from
Pengging duck’s intestines as probiotics. Int. J. Poult. Sci. 13: 47–51. doi: 10.3923/ijps.2014.47.51
Syah, S. P., Sumantri, C., Arief, I. I., and Taufik, E. 2017. Research article isolation and identification of indigenous
lactic acid bacteria by sequencing the 16S rRNA from dangke, A traditional cheese from Enrekang, South Sulawesi.
Pakistan J. Nutr. 16: 384–392. doi: 10.3923/pjn.2017.384.392
Tacão, M., Alves, A., Saavedra, M. J., and Correia, A. 2005. BOX-PCR is an adequate tool for typing Aeromonas spp.
Antonie van Leeuwenhoek. J. Microbiol 88: 173–179. doi: 10.1007/s10482-005-3450-9
Tallapragada, P., Rayavarapu, B., Rao, P. P., and Ranganath, N. N. 2018. Screening of potential probiotic lactic acid
bacteria and production of amylase and its partial purification. J. Genet. Eng. Biotechnol. 16: 357–362. doi: 10.1016/
j.jgeb.2018.03.005
Thapa, N., Pal, J., and Prakash, J. 2006. Phenotypic identification and technological properties of lactic acid bacteria
isolated from traditionally processed fish products of the Eastern Himalayas. Int. J. Food Microbiol. 107: 33–38. doi:
10.1016/j.ijfoodmicro.2005.08.009
Tsai, C.-C., Lin, P.-P., Hsieh, Y.-M., Zhang, Z., Wu, H.-C., and Huang, C.-C. 2014. Cholesterol-lowering potentials of
lactic acid bacteria based on bile-salt hydrolase activity and effect of potent strains on cholesterol metabolism
in vitro and in vivo. Sci. World J.: 1–10. doi: 10.1155/2014/690752
Udomsil, N., Rodtong, S., Tanasupawat, S., and Yongsawatdigul, J. 2010. Proteinase-producing halophilic lactic acid
bacteria isolated from fish sauce fermentation and their ability to produce volatile compounds. Int. J. Food
Microbiol. 141: 186–194. doi: 10.1016/j.ijfoodmicro.2010.05.016
Veˇtrovsky, T., and Baldrian, P. 2013. The variability of the 16S rRNA gene in bacterial genomes and its consequences
for bacterial community analyses. PLoS ONE 8: 1–10. doi: 10.1371/journal.pone.0057923
Wikandari, P. R., Marsono, Y., and Rahayu, E. S. 2012. Potensi Bakteri Asam Laktat yang Diisolasi dari Bekasam
sebagai penghasil angiotensin converting enzyme inhibitor pada fermentasi bekasam-like product. Agritech 32:
258–264.
Yuliana, N., Koesoemawardani, D., and Kurniati, Y. 2018. Lactic acid bacteria during fish fermentation (rusip). MOJ
Food Process. Technol. 6: 211–216. doi: 10.15406/mojfpt.2018.06.00167