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Assessment of Potential Probiotic Lactic Acid Bacteria from Tempe and

Tape
To cite this article: Sulistiani et al 2020 IOP Conf. Ser.: Earth Environ. Sci. 572 012026

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The 9th International Symposium for Sustainable Humanosphere IOP Publishing
IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

Assessment of Potential Probiotic Lactic Acid Bacteria from

Tempe and Tape

Sulistiani1*, I Novarina2, Inawati 2, A Dinoto1, H Julistiono1, R Handayani1 and S

Saputra1
1
Research Center for Biology, Indonesian Institute of Sciences, Cibinong Science
Center, Cibinong-Bogor 16911, Indonesia
2
Sekolah Tinggi Teknologi Industri dan Farmasi - STTIF Bogor
*
E-mail: [email protected]

Abstract. Probiotics are living organisms with many beneficial effects on the health of host if
consumed in sufficient quantities. These beneficial effects have been initiating efforts directed
towards exploring strains Lactic Acid Bacteria (LAB) as probiotic from fermented food such
tempe and tape. Although several groups of microorganism from fermented foods such as tempe
and tape were reported, the potential LAB derived from those food sources is limited. The aim
of this study was to assess probiotic candidate from LAB strains isolated from tempe and tape
based on in vitro analysis. A total of 30 LAB isolates were tested for probiotic properties
including tolerance to bile salt and acid, antimicrobial activity, simulated gastric juice (SGJ) and
simulated intestinal juice (SIJ), physiology and enzymatic properties. The results showed eight
bacterial isolates (Pediococcus pentosaceus Su-ls13, P. pentosaceus Su-ls14, Enterococcus
faecalis Su-ls15, P. pentosaceus Su-ls16, P. pentosaceus Su-ls21, P. pentosaceus Su-ls22, P.
pentosaceus Su-ls24 and Lactobacillus plantarum Su-ls29) fulfilled the criteria as probiotic
candidates, including the capability of producing antimicrobial activity by inhibiting the growth
of 12 pathogenic bacteria, have survivability under the condition of (or high tolerance to) low
pH, and being exposed to bile salt, simulated gastric juice and simulated intestinal juice. All
isolates were able to grow at NaCl 3-6.5%, 30-45C and produce phytase. In addition, six isolates
(Su-ls13, Su-ls14, Su-ls15, Su-ls16, Su-ls21, Su-ls22) were able to produce protease and two
isolates (Su-ls22, Su-ls24) were able to produce amylase.

1. Introduction
Lactic acid bacteria (LAB) are a group of gram-positive, non-spore forming, cocci or rods, which
produce lactic acid as the major end product during the fermentation of carbohydrates [1]. LAB play a
significant role in fermented foods and produce antimicrobial metabolite compounds such as lactic acid,
bacteriocins, and hydrogen peroxide [2]. Some fermented food such tempe (mold fermented soybean)
and tape (alcoholic fermented steamed glutinous rice or cassava) use a starter culture, however
microorganisms from the environment may contaminate the ferments and grow during the
fermentations. Rhizopus oligosporus is main starter culture for tempe fermentation however yeasts and
lactic acid bacteria (LAB) are also detected in tempe [3]. In tape fermentation, LAB contributes to the
development of flavor [4].

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The 9th International Symposium for Sustainable Humanosphere IOP Publishing
IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

Traditional fermented foods can be used as potential sources of probiotics as they commonly contain
LAB, including species of Lactobacillus, Pediococcus, Enterococcus, Weissella and Leuconostoc [4].
Over the past two decades, a large number of LAB isolated from various fermented food systems in
different parts of the world, have been studied for their probiotic potential and ability to produce
industrially important substances [5]. Some strains of LAB can be considered as probiotic bacteria.
Probiotics can be defined as live microorganisms, which when administered in adequate amounts, confer
a health benefit on the host. Many studies have demonstrated the efficacy of probiotics to offer a proper
alternative to the use of antibiotics in the treatment of enteric infection or to reduce the symptoms of
antibiotic-associated diarrhea [2].
Probiotic and other functional properties are strain dependent and all probiotic strains are unique and
different; therefore, their properties and characteristics need to be well defined. Several criteria have to
be met in selecting probiotic strains, including acid and bile tolerance, survival through the
gastrointestinal track, ability to adhere to intestinal surfaces, antimicrobial activity against potentially
pathogenic bacteria, and good technological properties [4].
Although several groups of microorganism from fermented foods such as tempe and tape were
reported, the potential probiotic LAB derived from those food sources is limited. This study was aimed
to assess the probiotic potential of LAB from tape and tempe which may exert beneficial effects for
mankind.

2. Materials and Methods

2.1. Bacterial strains and growth conditions
LAB isolated from tempe originally from Bali were used, including: Lactobacillus fermentum Su-ls1,
Weissella paramesenteroides Su-ls2, Lactobacillus plantarum Su-ls3, Lactobacillus plantarum Su-ls4,
Lactobacillus plantarum Su-ls5, Weissella paramesenteroides Su-ls6, Enterococcus faecalis Su-ls7,
Enterococcus faecalis Su-ls8, Enterococcus faecium Su-ls9, Lactobacillus plantarum Su-ls10,
Pediococcus pentosaceus Su-ls11, Enterococcus faecium Su-ls12, Pediococcus pentosaceus Su-ls13,
Pediococcus pentosaceus Su-ls14, Enterococcus faecalis Su-ls15, Pediococcus pentosaceus Su-ls16 and
LAB isolated from tape: Lactobacillus vini Su-ls18, Leuconostoc mesenteroides Su-ls19, Weissella
paramesenteroides Su-ls20, Pediococcus pentosaceus Su-ls21, Pediococcus pentosaceus Su-ls22,
Lactobacillus fermentum Su-ls23, Pediococcus pentosaceus Su-ls24, Lactobacillus fermentum Su-ls25,
Lactobacillus fermentum Su-ls26, Lactobacillus fermentum Su-ls27, Lactobacillus kunkeei Su-ls28,
Lactobacillus plantarum Su-ls29, Lactobacillus plantarum Su-ls30, Leuconostoc mesenteroides Su-
ls31. These bacteria were cultivated in de man, rogosa and sharpe (MRS) broth performed at 37C for
24 h. Pathogenic bacteria (Escherichia coli, Bacillus cereus, Lysteria monocytogenes, Salmonella
enterica, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Aeromonas
hydrophilla, Edwardsiella ictaluri, Aeromonas sobria, Plesiomonas shigelloides and Klebsiella
pneumonia were cultivated in brain heart infusion (BHI) broth at 37C for 24 h.

2.2. Bile tolerance

LAB strains were grown at 37°C for 24 h in MRS broth without bile. Approximately 5 μL of the LAB
culture broth was spotted onto MRS agar containing bile salt (oxgall) at concentrations of 0.1%, 0.2%,
0.3%, and 0.4%, respectively. Bacterial growth in bile was determined after incubation at 37°C for 48 h
[6].

2.3. Acid tolerance

LAB strains tested that were resistant to bile salts were subsequently underwent acid tolerance tests. The
LAB grown in 0.5 mL MRS broth at 37C for 24 hour were collected by centrifugation at 5400 rpm for
15 min under room temperature. Cell pellet was washed with 1 mL of phosphate-buffered saline (PBS)
vortex in few seconds and collected by centrifugation at 5400 rpm for 15 min under room temperature.
The pellet cell resuspended with 1 mL sterile PBS with the pH values ranged from 2-2.5 (adjusted using
5 M HCl) to achieve 106 cfu/mL. PBS was prepared by dissolving NaCl (9 g/L), Na2HPO4.2H2O (9 g/L)

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The 9th International Symposium for Sustainable Humanosphere IOP Publishing
IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

and KH2PO4 (1.5 g/L) in distilled water. The tubes were incubated at 37C for 4 hours. The viable cell
was determined by spotted the cell suspension onto Glucose-Yeast Extract-Peptone (GYP) agar
incubated at 37°C for 48 h. Bacterial growth was then observed [6,7].

2.4. Antimicrobial test

Agar spot test method was used for detection of antimicrobial activity. The certain LAB passed previous
test were tested antimicrobial test. Overnight cultures of LAB (5 µL) were spotted on the surface of 20
mL GYP agar plates (containing 1.8% agar) and incubated for 24 hours at 37°C to allow colonies to
develop. Overnight culture of indicator pathogens P. aeruginosa 2%, K. pneumonia 2%, B. cereus 2%,
E. ictaluri 5%, E. coli 5%, A. sobria 5%, L. monocytogenes 5%, A. hydrophilla 5%, S. aureus 5%, and
P. shigelloides 5% were inoculated into 5 mL of soft Mueller Hinton (MH) agar (containing 0.7% agar)
and S. agalactiae 5%, S. enterica 5% inoculated into 5 mL of soft BHI agar (containing 0.7% agar) were
poured over the plate which the LAB was grown, respectively. The plates were incubated at 37°C for
24 hours. At the end of the incubation, inhibition zone diameter surrounding the spotted isolates was
measured [8,9].

2.5. Simulated gastric juice (SGJ) and simulated intestinal juice (SIJ) resistance analysis
SGJ which was prepared by suspending 3 mg/mL pepsin in sterile saline solution (0.85% NaCl, w/v)
was adjusted to pH 2.5. SGJ was inoculated with active LAB cultures (incubated at 37C for 24 h in
MRS Broth at an inoculums concentration of 1% (v/v) and incubated at 37C for 4 h [7].
Pancreatin or SIJ resistance test was prepared by dissolving bile salt (0.3%) and pancreatin (1 mg/mL)
in sterile saline solution adjusted to pH 8.0. SIJ was inoculated with active LAB cultures at an inoculum
size of 1% (v/v) and incubated at 37C for 6 h. The viable cell population was determined before and
after incubation on GYP agar plates by the spread plate method [7].

2.6. The physiology and enzymatic properties

LAB isolates were subjected to gram staining, catalase test, growth curve, and growth at various
temperature, pH, and salt concentration. Catalase activity was detected on cells grown on MRS agar by
placing a drop of hydrogen peroxide solution on the cells. Growth at temperatures (5C, 30C, 45C,
and 60C), at pH (3, 4, and 6), and at various concentrations of NaCl (3%, 6.5%, and 10%) were tested
using MRS broth medium. For the growth curve, 1% of overnight LAB culture inoculated into 100 mL
of GYP broth then homogenized with vortex. The growth of LAB was measured every 2 hours for 26
hours using UV-Vis spectrophotometry at  600 nm [10].
The selected LAB strains were checked for presence of enzyme (i.e., amylase, protease, and phytase)
activities according to Kim et al. [11] and Taheri et al. [12] with modifications. To detect the amylase,
protease, and phytase activities, active LAB cultures were spot-inoculated onto relevant medium and
then incubated for 48 h at 37°C. Amylase activity was examined using a GYPA medium (without
glucose) consisted of 1% starch. Protease activity was examined using NA medium consisted of 2%
skim milk and phytase activity was examined using modified MRSA medium (without K 2HPO4)
containing 1% sodium phytate. After incubation, the halo zone surrounding each colony was measured
with a caliper. The size of the halo zones surrounding the colonies gave an approximate indication of
extracellular enzyme level.

3. Result and Discussion

Thirty isolates observed in this study were able to grow in the absence of bile as well as in the presence
of 0.1 and 0.2% bile while only ten isolates grew on 0.3% bile, however two of those isolates (Su-ls22,
Su-ls24) could not tolerate the higher concentration of bile (0.4%) (table 1). Two isolates (Su-ls19, Su-
ls20) were not continued for analysis because have no antimicrobial activity (based on preliminary

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The 9th International Symposium for Sustainable Humanosphere IOP Publishing
IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

antimicrobial tests, data not shown). Buntin et al. [6] suggested that bile salt concentration of 0.3% is
considered as critical and high enough to screen for resistant strains. Therefore, the eight LAB isolates
resistance to 0.3% bile were chosen to do the next analysis. Millete et al. [2] emphasized that bile salt
tolerance is considered one of the most important attributes required by LAB to survive in the duodenum
and the upper small intestine. Their ability to hydrolyze bile salt by bile salt hydrolase enzyme (BSH)
are able to reduce this detergent effect. This particular enzyme decreases bile solubility and weakening
its detergent effect thus minimizing its bactericidal effect on strains.
Among eight 0.3% bile resistance bacterial isolates, all isolates were able to survive in acidic
condition (pH 2.5) and one isolate could not survive in pH 2.0 for four hours (table 2). Tolerance to
acidity (pH 2.0-2.5) is considered as a key functional requirement for probiotics, which enables them to
survive during passage through the gastrointestinal tract [7]. Based on Argyri et al. [13] the pH value
2.5 used in this study for the selection of potential probiotic strains is very selective even though it is
not the most common pH value in the human stomach, therefore it assures the selection of very acid-
tolerant strain. The survival ability of lactobacilli at pH 2.5 was also reported. However, variability in
acidic response was obtained among the tested strains, indicating that resistance to low pH is a strain-
specific property [7].

Table 1. The bile salts resistance of LAB origin tape and tempe observation based on bacterial growth.
Concentration of bile Concentration of bile
No Strain salt (Oxgall) No Strain salt (Oxgall)
0.1% 0.2% 0.3% 0.4% 0.1% 0.2% 0.3% 0.4%
1 L. fermentum Su-ls1 + + - - 16 P. pentosaceus Su-ls16 + + + +
2 W. paramesenteroides Su-ls2 + + - - 17 L. vini Su-ls18 - - - -
3 L. plantarum Su-ls3 + + - - 18 L. mesenteroides Su-ls19 + + + +
4 L. plantarum Su-ls4 + + - - 19 W. paramesenteroides Su-ls20 + + + +
5 L. plantarum Su-ls5 + + - - 20 P. pentosaceus Su-ls21 + + + +
6 W. paramesenteroides Su-ls6 + + - - 21 P. pentosaceus Su-ls22 + + + -
7 E. faecalis Su-ls7 + + - - 22 L. fermentum Su-ls23 + + - -
8 E. faecalis Su-ls8 + + - - 23 P. pentosaceus Su-ls24 + + + -
9 E. faecium Su-ls9 + + - - 24 L. fermentum Su-ls25 + + - -
10 L. plantarum Su-ls10 + + - - 25 L. fermentum Su-ls26 + + - -
11 P. pentosaceus Su-ls11 + + - - 26 L. fermentum Su-ls27 + + - -
12 E. faecium Su-ls12 + + - - 27 L. kunkeei Su-ls28 + + - -
13 P. pentosaceus Su-ls13 + + + + 28 L. plantarum Su-ls29 + + + +
14 P. pentosaceus Su-ls14 + + + + 29 L. plantarum Su-ls30 + + - -
15 E. faecalis Su-ls15 + + + + 30 L. mesenteroides Su-ls31 + + - -
(-): no growth was observed

Table 2. The acid resistance of selected bile-resistant LAB based on bacterial growth.
pH 2.5 pH 2.0
No. Strain
0h 4h 0h 4h
1 P. pentosaceus Su-ls13 + + + +
2 P. pentosaceus Su-ls14 + + + -
3 E. faecalis Su-ls15 + + + +
4 P. pentosaceus Su-ls16 + + + +
5 P. pentosaceus Su-ls21 + + + +
6 P. pentosaceus Su-ls22 + + + +
7 P. pentosaceus Su-ls24 + + + +
8 L. plantarum Su-ls29 + + + +
(-): no growth was observed

Acid and bile tolerance properties are two fundamental properties that indicate the ability of probiotic
microorganism to survive the passage though the upper gastrointestinal tract, particularly acidic
condition in the stomach and the presence of bile in the small intestine [6]. Those properties are
important for selection of the probiotic candidate, since the probiotic strains have to possess the ability
to overcome the extremely low pH and the detergent effect of bile salts for leading to the site of action
in a viable physiological state [10]. Thus, only eight tested LAB strains with acid and bile tolerance are

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The 9th International Symposium for Sustainable Humanosphere IOP Publishing
IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

suggested to survive in the gastrointestinal tract. Its reflected the phenomenon of bacterial strain-
dependent in response to bile and acid tolerance [14].
Tolerance to stomach and intestinal conditions is an important trait for probiotic bacteria in terms of
their performance to survive, grow, and exert action in the gut [14]. After 4 hours exposure to SGJ at
pH 2.5, this study showed slightly decreased population of LAB compared to initial number of
population but still survived well (table 3, figure 1A). Concerning on the survivability in the SIJ at pH
8.0, all tested isolates were survived well after exposure to SIJ for 6 h (table 3, figure 1B) suggesting a
potential recuperation of the initial levels during the passage of the small intestine. However, the
susceptibility or resistance of probiotic cultures to bile is species dependent as well as strain specific as
mentioned [2].

Table 3. Viable count of selected LAB in simulated gastric and intestinal juice test.
SGJ (log cfu/mL) SIJ (log cfu/mL)
No. Strain
0h 4h 0h 4h
1 P. pentosaceus Su-ls13 8.48 4.86 8,30 8,13
2 P. pentosaceus Su-ls14 8.51 6.09 8,69 8,54
3 E. faecalis Su-ls15 8.24 4.85 7,39 8,58
4 P. pentosaceus Su-ls16 7.88 4.85 7,91 7,84
5 P. pentosaceus Su-ls21 8.31 4.77 7,50 7,23
6 P. pentosaceus Su-ls22 8.07 4,39 7,50 7,55
7 P. pentosaceus Su-ls24 8.00 4,17 8,12 8,06
8 L. plantarum Su-ls29 9.07 5,34 9,00 8,93

10 8.48 8.51 8.24 8.31

9.07 10 8.38.13
8.69
8.54
8.58
8.12
9 8.93
7.88 8.07 8 7.91
LOG CFU/ML

7.39 7.84 7.57.23 7.57.55 8.06

LOG CFU/ML

8
6.09
5.34
4.86 4.85 4.85 4.77
4.39 4.17
6
5
4
2

0H 0 0
0H
Su-ls13
Su-ls14
Su-ls15
Su-ls16
Su-ls21
Su-ls22
Su-ls24
Su-ls29

4H 6H

A B STRAIN
STRAIN
Figure 1. Survival of LAB strains in simulated gastric (A) and intestinal juice (B).

The results of spot-on-lawn test performed in this study showed that all isolates presented a wide
range of inhibitory effects. All lactobacilli inhibited the growth of Escherichia coli, Bacillus cereus,
Lysteria monocytogenes, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus,
Edwardsiella ictaluri, Aeromonas sobria, Plesiomonas shigelloides, Klebsiella pneumonia, Aeromonas
hydrophilla, Streptococcus agalactiae and the strongest antimicrobial effect was shown by L. plantarum
SU-LS29 while antimicrobial effect of other lactobacilli were varies (table 4, figure 2).The inhibitory
properties of LABs are commonly formed by producing some substances such as organic acids (lactic,
acetic, propionic acids), carbon dioxide, hydrogen peroxide, diacetyl, low molecular weight
antimicrobial substances, and bacteriocins. Bacteriocins may enhance the survival of LAB in complex
ecological systems. It is more important with respect to probiotics may inhibit growth pathogenic
microorganisms by secreted products, and not merely an effect of acidic pH [6,8]. Besides the various
essential characteristics, the probiotic should exhibit health benefits with functional properties.
Probiotics should present their antimicrobial actions particularly to the pathogens in the GI system [15].
Probiotics affect the host animal by improving its intestinal balance and mode of action is related to the
competition for attachment sites (competitive exclusion). The probiotics attach to the intestinal mucosa

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IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

and block the attachment of pathogenic bacteria by forming a physical barrier [16] and control intestinal
pathogens by production of antibacterial compounds [17].

Su-ls13 Su-ls14
Su-ls21 Su-ls22

TE GM

Su-ls15 Su-ls16 Su-ls24 Su-ls29

Figure 2. Antimicrobial activity of selected LAB against Bacillus cereus, TE: Tetracycline 30 µg, GM:
Gentamicin 10 µg.

Table 4. Diameter of inhibition (mm) of selected LAB against pathogenic bacteria.

Inhibition zone (mm)
Strain
EC BC LM SE PA SA SAg AH EI PS KP AS
P. pentosaceus
13.0 20.0 16.5 16.0 7.5 15.0 15.0 15.0 20.0 19.00 6.0 20.0
Su-ls13
P. pentosaceus
13.0 19.5 18.0 17.0 8.0 16.0 15.0 16.5 20.0 17.00 7.0 20.0
Su-ls14
E. faecalis Su-ls15 10.0 13.0 14.0 10.0 5.0 10.0 10.0 10.0 15.5 14.00 6.0 15.5
P. pentosaceus
13.0 19.0 19.0 17.0 9.0 16.0 15.0 18.5 21.0 19.00 7.0 21.0
Su-ls16
P. pentosaceus
14.0 22.5 18.0 15.0 9.0 19.0 14.0 16.0 21.0 14.00 8.0 21.0
Su-ls21
P. pentosaceus
14.0 24.0 19.5 18.0 11.0 20.0 17.0 19.0 22.0 16.00 8.0 22.0
Su-ls22
P. pentosaceus
14.0 22.5 19.5 17.0 10.0 19.5 16.0 17.0 21.0 17.50 7.5 21.0
Su-ls24
L. plantarum Su-
19.0 25.0 24.0 24.0 14.0 26.0 22.0 26.0 26.0 22.00 14.0 26.0
ls29
Tetracycline 30
17.0 25.0 22.0 19.0 0 15.0 11.0 22.0 15.0 11.00 18.0 15.0
µg
Gentamicin 10 µg 11.0 21.0 19.0 13.0 12.0 15.0 16.0 15.0 16.0 14.00 10.0 16.0
EC: Escherichia coli, BC: Bacillus cereus, LM: Lysteria monocytogenes, SE: Salmonella enterica, PA: Pseudomonas
aeruginosa, SA: Staphylococcus aureus, EI: Edwardsiella ictaluri, AS: Aeromonas sobria, PS: Plesiomonas shigelloides,
KP: Klebsiella pneumonia, AH: Aeromonas hydrophilla, SAg: Streptococcus agalactiae

The determination of the growth phase was important to form the growth curve in order to analyze
the speed of which the bacteria reached the exponential phase and the bacteria generation time [18].The
fast growing characteristics of LAB and their metabolic activity have been the key in most applications
including food production, agricultural industry, and probiotics [19]. The isolates’ growth was measured
to determine biomass in the growth phases of each bacterial isolate. The growth curves formed in the
observation period of 26 hours showed that isolates had varied patterns. Based on the initial growth
phase or lag phase during the counting of colony numbers, it was showed that the probiotic candidates
Su-ls13, Su-ls14, Su-ls16, Su-ls21, Su-ls22, Su-ls24 and Su-ls29 could reach high mass in their
exponential phase compared to Su-ls15 isolate (figure 3A).

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IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

2,5

Su-ls29

2 Su-ls16
Su
Su--ls24
ls22
Su
Su--ls14
ls13
Su-ls21

1,5

Su-ls15
1

Protease Phytase
0,5

0
0 5 10 15 20 25 30
Time (h)

A. B.
Figure 3. Growth curve (A) and enzyme produced (B) of selected LAB.
Table 5. Physiological tests and enzymatic characteristic of probiotic candidate.
Cata NaCl (%) Temperature C Enzyme (clear zone/LAB colony in mm)
No. Strain Gram
lase 3 6.5 10 5 30 45 60 Protease Amylase Cellulase Phytase
1 P. pentosaceus Su-ls13 + - + + - - + + - 9/5 - - 15/5
2 P. pentosaceus Su-ls14 + - + + - - + + - 9/5 - - 13/5
3 E. faecalis Su-ls15 + - + - - - + + - 11/5 - - 7/5
4 P. pentosaceus Su-ls16 + - + + - - + + - 9/5 - - 16/5
5 P. pentosaceus Su-ls21 + + + + - - + + - 9/5 - - 14/5
6 P. pentosaceus Su-ls22 + + + + - - + + - 12/9 10/10 - 12/8
7 P. pentosaceus Su-ls24 + + + + - - + + - - 10/10 - 13/8
8 L. plantarum Su-ls29 + - + + - - + + - - - - 22/5

The probiotic candidates were characterized by classical methods. All the isolates were Gram
positive and three isolates have positive catalase (Su-ls21, Su-ls22, Su-ls24). Based on physiological
tests, the isolates showed good growth at 37°C and 45°C whereas no growth was observed at 5°C,
indicating their mesophilic character. The lactobacilli were able to tolerate salt concentration of 6.5%,
but were unable to grow at 10% of salt, (table 5). In addition as mentioned [10], higher temperature
degrees and high salt concentrations might be faced during industrial processing. The sodium chloride
concentration and temperature play an important role in cell growth.
The size of the halo zones surrounding the colonies gave an approximate indication of extracellular
enzyme levels. All bacterial isolates displayed different protease, amylase, and phytase activities (figure
3B). These results were similar to those reported by Kim et al. [11]. These exogenous enzymes will help
the host’s endogenous enzymes in hydrolyzing nutrients such as breaking down long chains found in
carbohydrates and protein. Breaking down complex molecules into simpler molecules will make the
process of digesting and absorbing digestive tract easier [18]. Its production of dietary enzymes will
promote animal growth [11]. Probiotics are able to produce several exogenous enzymes to digest feed
may increasing the nutritional value food/feed.

4. Conclusions
The six isolates of P. pentosaceus (Su-ls13, Su-ls14, Su-ls16, Su-ls21, Su-ls22, Su-ls24), E. faecalis Su-
ls15, and L. plantarum Su-ls29 origin from tempe and tape were considered as potential candidates of
probiotics. Those isolates demonstrate high tolerance to 0.3% bile salt and acid (pH 2.5), simulated
gastric juice (SGJ) and simulated intestinal juice (SIJ) and inhibited 12 pathogenic bacteria, while some
strains producing dietary enzyme.

Acknowledgment
Authors thank to Research Center for Biology - LIPI for supporting the research funding.

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IOP Conf. Series: Earth and Environmental Science 572 (2020) 012026 doi:10.1088/1755-1315/572/1/012026

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