OPEN ACCESS Pakistan Journal of Nutrition

ISSN 1680-5194
DOI: 10.3923/pjn.2018.361.367

Research Article
Isolation and Characterization of Lactic Acid Bacteria Proteases
from Bekasam for use as a Beef Tenderizer
1
Afriani, 2Arnim, 3Yetti Marlida and 2Yuherman

1
Department of Animal Production, Faculty of Animal Science, Jambi University, Indonesia
2
Department of Animal Production, Faculty of Animal Science, Andalas University, Padang, Indonesia
3
Department of Animal Nutrition, Faculty of Animal Science, Andalas University, Padang, Indonesia

Abstract
Background and Objective: Proteases are important enzymes and have high economic value due to their wide applications in the food
industry as a meat tenderizer. Protease use in the food industry necessitates an understanding of the capabilities and influencing factors
of these enzymes to accelerate enzymatic reactions. This study aimed to isolate and characterize the proteases of lactic acid bacteria
(LAB) from Bekasam. Methodology: The samples were obtained from the third, fifth, seventh, ninth and eleventh day of fermentation
to isolate the proteolytic LAB. Characterization of proteases includes the incubation time, casein substrate concentration, optimum
temperature and pH, metal ion contents and stability. The LAB with the highest protease activity is identified molecularly and isolated
through 16S rDNA sequencing and phylogenetic analysis based on the Neighbor Joining method. Results: The results showed that the
best isolate was BAF-715 because it had the highest protease activity (18.84 U mLG1) at 40 h of incubation. The optimum activity of this
protease on a casein substrate at 2.5% occurred at an incubation temperature of 40EC at pH 7 and in the presence of Mg2+ and Mn2+
(5 mM) as activators. Based on molecular DNA identification, the BAF-715 isolate is determined to be Pediococcus pentosaceus.
Conclusion: A protease produced by Pediococcus pentosaceus showed the highest proteolytic activity, making it the best protease for
application as a beef tenderizer.

Key words: Lactic acid bacteria, protease, bekasam

Received: September 27, 2017 Accepted: May 04, 2018 Published: July 15, 2018

Citation: Afriani, Arnim, Yetti Marlida and Yuherman, 2018. Isolation and characterization of lactic acid bacteria proteases from bekasam for use as a beef
tenderizer. Pak. J. Nutr., 17: 361-367.

Corresponding Author: Afriani, Department of Animal Production, Faculty of Animal Science, Jambi University, Indonesia

Copyright: © 2018 Afriani 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 author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files.
 Pak. J. Nutr., 17 (8): 361-367, 2018

INTRODUCTION eleventh fermentation days of 2 replications and the bacteria

were grown on MRS agar medium. From each sample, five
Bekasam is a traditional salt-fermented product from different colonies were grown on MRS agar medium+CaCO3
fish, which are found in many areas in Indonesia. Many using the pour plate method. The isolate, which formed a clear
microorganisms contain lactic acid bacteria (LAB)1. LAB can zone, was then purified by the streak plate method on the
produce several metabolites; they can also produce same medium.
proteolytic enzymes around cell walls, in cytoplasmic
membranes and in cells2. Protease is an enzyme that breaks Test of proteolytic lactic acid bacteria9: A LAB proteolytic test
the peptide bonds in proteins to produce simpler compounds, was used with an agar skim milk (ASM) medium with 3% skim
such as small peptides and amino acids. and 3% agar composition. The proteolytic isolates were
Protease is a very important enzyme in food and non-food characterized by the formation of a clear zone on the agar
industries. Some uses of proteases in the food industry include skim medium and then, the clear zone was measured. The
reducing turbidity in the beer industry, reducing gluten in the proteolytic index was calculated by measuring the ratio of the
bread industry, agglomerating milk in the cheese industry and diameter of the clear zone/colony diameter (R).
tenderizing meat in the meat processing industry3,4. Proteases
can be easily isolated from various sources of plants, animals Production and isolation of protease enzymes: The
and microbes through fermentation processes 5. production and isolation of protease enzymes are performed
Microbes used as an enzyme source are more by adding one dose of isolate to 50 mL MRS broth and
advantageous than plant and animal sources because incubating the sample in a shaker incubator with an agitation
microbes can grow rapidly on inexpensive substrates and can speed of 150 rpm at 37.5EC for 24 h. The inoculum was then
be easily enhanced by manipulating the growth conditions inoculated on protease production media with a composition
and applying genetic engineering6 and biotechnology7. The of 2% skim milk, 0.5% peptone, 0.1% yeast extract, 2% glucose,
existence of a superior microbe is an important factor in the 0.1% NaCl, 0.008% KH2PO4, 0.01% MgSO4.7H2O and 0.04%
production of enzymes. (NH4)2SO413. The sample was incubated in a shaker incubator
Several types of LAB have proteolytic activity, such as with an agitation speed of 150 rpm at 37.5EC with various
L. plantarum, L. brevis, L. pentosus, Pediococcus acidilactici, production times, namely, 8, 16, 24, 32, 40, 48 and 56 h. The
Pediococcus pentosaceus and Lactobacillus spp8-10. LAB are sample was centrifuged at a rate of 12000 rpm for 15 min at
safe microbes and enzymes produced from LAB can be used 4EC to separate the supernatant and its sediment. The
directly on food. supernatant is a crude extract of the protease and is used for
The ability of proteases to accelerate reactions is testing the protease activity.
influenced by several factors, such as pH, temperature
and metals serving as activators or inhibitors, that Protease activity measurements: Protease activity was
change enzymatic characteristics. Proteases from measured by the method of Bergmeyer et al.14 using casein
Pediococcus acidilactici have an optimum activity at substrate Hammerstein 2% (b/v). A unit of protease activity is
pH 4, an incubation temperature of 28EC and a 2% casein defined as the amount of enzyme that can produce one µmol
concentration11. The protease activity from Lactobacillus of tyrosine product per minute under the measurement
plantarum was highest at 4.43 U mgG1, pH 7, an incubation conditions. Isolates with the highest proteolytic activity were
temperature of 47EC and with a Mg2+ metal activator12. The tested further.
220 proteases from P. pentosaceus stabilized at pH 4-7, an
incubation temperature of 20-40EC and with metal Protease characterization: Characterization of proteases
activators/inhibitors of Ca2+, Zn2+, Mg2+ and Fe3+; the protease includes the effect of the casein substrate concentration on
activity was inhibited by EDTA and sodium dodecyl sulfate10. enzyme activity, which was tested by reacting an enzyme
From the description above, this study was conducted to solution with a casein concentration of 0.5, 1, 1.5, 2, 2.5 and
determine the optimum conditions for the production of 3%.
protease enzymes by LAB. The pH effect on the enzyme activity was tested by using
different buffer treatments. The universal buffers used had pH
MATERIALS AND METHODS values of 3, 4, 5, 6, 7, 8 and 9.
The effect of temperature on enzyme activity was tested
Isolation of lactic acid bacteria: LAB was isolated from on the first incubation carried out at different temperatures,
Bekasam samples on the third, fifth, seventh, ninth and i.e., 30, 40, 50, 60, 70, 80 and 90EC.

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The effect of the metal ions Mg2+, Cu2+, Ca2+, Fe2+, Zn2+ and RESULTS AND DISCUSSION
Mn2+ in sulfate salts with a concentration of 5 mM as an
activator or inhibitor on protease activity was tested using an Isolation of lactic acid bacteria and proteolytic test of
optimum pH buffer and optimum temperature. As a protease-producing bacteria: The isolation process obtained
benchmark, the treatment used buffer without the addition of 50 bacterial colonies. Then, from these 50 colonies, which
metal ions (nonmetal). provided clear zones in the MRS and CaCO3 media, 25 isolates
showed the characteristics of LAB. The formation of clear
Molecular identification of selected LAB isolates: LAB zones indicated that the bacteria produced an excess of
isolates with the highest proteolytic activity were molecularly secondary metabolites (lactic acid) so that an excess of lactic
identified by extracting DNA encoding 16S rRNA. DNA acid was shown by the clear zones around the bacterial
sequencing of the encoded 16S rRNA was performed by 1st colonies.
Base, PT Genetics of Indonesia. An analysis of the sequencing The 25 isolates of LAB were tested for proteolytic activity,
results was performed using the BLASTing sequence of as shown by their ability to form a clear zone on the agar skim
nucleotides from the sequencing results of 16S rRNA with the medium. When viewed from the protease activity of the clear
database available at www.ncbi.nlm.nih.gov. Phylogenetic zone/colony diameter (R) size, the number of isolates
analysis was performed with the Neighbor Joining method exhibiting high protease activity (R>2) was less than that of
using the program Molecular Evolutionary Genetics Analysis isolates with R<2 (Table 1). Three isolates of LAB with a high
(MEGA) version 6.0 with Multiple Sequence Comparison by proteolytic index (R>2) were BAF-514, BAF-715 and BAF-1121
Log Expectation (MUSCLE). (Table 2).

Amplification of DNA encoding 16S rRNA with PCR Protease enzyme activity: A qualitative test of protease
(polymerase chain reaction): The amplification reaction activity showed that the BAF-514, BAF-715 and BAF-1121
of the DNA sample was carried out in 0.2 mL PCR tubes. isolates could produce extracellular protease by forming a
To each PCR reaction tube was added 0.25 µL RBC large clear zone around the cell colony. The larger clear zone
Taq (5 U mLG1), 10×5 µL Taq buffer (containing Mg2+), that was formed indicated that the microbes were highly
4 µL total of 2.5 mM dNTP and universal primer 63F capable of changing the substrate that was contained in the
(5'-CAGGCCTAACACATGCAAGTC-3') and universal primer medium18.
1387R (5 'GGGCGGWGTGTACAAGGC-3') at 1.25 µL (20 pmol) The proteolytic index of isolates BAF-514, BAF-715
and 1.25 µL (20 pmol), respectively; 2.5 µL (100 ng) of genomic and BAF-1121 was 2.06, 2.29 and 2.02, respectively. The
extract and ddH2O up to 50 µL were also added. PCR protease activity of the BAF715 isolate in the skim milk
amplification was performed using a PTC 100 PCR instrument agar medium was demonstrated by the presence of clear
(MJ Research, Inc.) at an initial denaturation temperature of zones around the colony (Fig. 1). The results reported
95EC for 5 min, followed by 30 cycles of annealing at 94EC for by Wikandari et al.9 indicated that the LAB with a
30 sec, as well as an extension at 50EC for 1 min, 72EC for proteolytic index >2 were L. plantarum N2352,
2 min and a final stage of 72EC for 5 min. The PCR products L. plantarum B1765, L. plantarum T2565, L. plantarum
were stored at 4EC for further examination using 1% agarose
electrophoresis in 1×TAE, 100 V for 30 min15. Table 1: Number of isolates of lactic acid bacteria and proteolytic lactic acid
bacteria from Bekasam
Isolates Total isolates
Analysis of the DNA sequence of the genes encoding 16S
Acid-forming bacteria 50
rRNA: The DNA sequencing of the genes encoding 16S rRNA Lactic acid bacteria (LAB) 25
was performed by using 1st Base, PT, Indonesian Genetics. The LAB proteolytic 17
LAB proteolytic R>2 3
sequencing analysis was performed by BLAST to obtain the
LAB proteolytic R<2 14
nucleotide sequence from the 16S rRNA sequencing results LAB non-proteolytic 8
with the available database at www.ncbi.nlm.nih.gov. Then, a
multiple alignment was performed by using the Clustal W Table 2: Chosen isolates of lactic acid bacteria and the proteolytic index
Isolates Proteolytic index
Program. Furthermore, visualization of kinship was performed
BAF-514 2.06
using a phylogenetic tree combination via the Neighbor BAF-715 2.29
Joining method16 with the MEGA 6.0 program17. BAF-1121 2.02

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 Pak. J. Nutr., 17 (8): 361-367, 2018

25

Protease activity (U mLG )

1
20

15

10

0
3 4 5 6 7 8 9
pH

Fig. 4: Effect of pH on the protease activity of lactic acid

bacteria BAF-715

The results for the protease activity as a function of

incubation time (Fig. 2) showed that the optimum activity of
the BAF-514 and BAF-1121 isolate proteases at 48 h of
incubation time was 11.32 and 9.65 U mLG1, respectively. This
Fig. 1: Clear zone as a qualitative indicator of LAB proteases result indicates that the proteases were produced at a
stationary phase, namely, the peak cell growth phase.
Meanwhile, the isolate protease activity of BAF-715 at 40 h of
Protease activity (U mLG )

20 BK-5.1.4
1

BK-7.1.5
15 BK-11.2.1 incubation time was 18,844 U mLG1, revealing that the
protease was produced along with the cell growth before
10
reaching the stationary phase. The value of this
5
protease activity was higher than that of Putranto19. The
0 Lactobacillus acidophilus bacteria were at the highest activity
0 8 16 24 32 40 48 56
when the incubation time was 18 hours (0.752 U mgG1).
Incubation time (h)
Because isolate BAF-715 showed the highest proteolytic
Fig. 2: Activity of isolate enzymes BAF-514, BAF-715 and activity, this isolate was selected for further research.
BAF-1121 for different incubation times
Characterization of the crude extract protease of the
25
selected LAB isolate (BAF-715)
Protease activity (U mLG )
1

20 Effect of the casein substrate concentration: The effect

15
of the casein substrate concentration on the protease
activity is shown in Fig. 3. The highest protease activity
10
was obtained at a concentration of 2.5% at 88.4 U mLG1.
5 Meanwhile, the protease activity decreased when the
concentration of the casein substrate was 3%. Comparing the
0
0.5 1.0 1.5 2.0 2.5 3.0 results of Oke and Onilude11 study using P. acidilactici and
Casein the results of Akinkugbe and Onilude20 study using
Lactobacillus acidophilus, the highest protease activity was
Fig. 3: Effect of the casein substrate concentration on the achieved at a casein concentration of 2%.
protease activity of the BAF-715 isolate
Effect of pH: The pH activity profile of an enzyme describes
B1465, L. pentosus B2555 and Pediococcus pentosaceus the pH at the time that the proton should be donated or
B1666. Interestingly, research by Yusmarini et al.8 proved received on the catalytic side of the enzyme to be at the
that LAB from the genus Lactobacillus R.1.3.2 and desired ionization level. Figure 4 shows that the isolate
R.11.1.2 has a proteolytic zone of 1.725 and 1.650 cm, protease activity of LAB has an optimum pH of 7 with an
respectively. enzyme activity of 19.89 U mLG1. Changes in alkaline or acidic

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 Pak. J. Nutr., 17 (8): 361-367, 2018

35
pH conditions result in a decrease in enzyme activity. It is
estimated that the enzyme activation change is caused by 30

Protease activity (U mLG )

1
ionization of the enzyme s ionic group, on the active side or on 25

the other side, thus indirectly affecting the active side. The 20

ionic group plays a role in maintaining the active side 15

conformation for binding the substrate and converting it into 10

the product. These enzyme activity results were higher than 5

that reported in the research of Putranto19, in which 0

30 40 50 60 70 80 90
Lactobacillus acidophilus bacteria had an optimum pH of 6 Temperature (°C)
with an enzyme activity of 13.5 units mgG1; the protease
product of P. acidilactici had an optimum pH of 4.0. The Fig. 5: Effect of temperature on the protease activity of lactic
research results by Oke and Onilude11 using the protease from acid bacteria BAF-715
Lactobacillus plantarum had an optimum pH of 7, which was
45
in agreement with the research results by Sulthoniyah et al.12.
40

Protease activity (U mLG )

Ionization can also be experienced by the substrate or

1
35
substrate-enzyme complex, which also affects enzyme 30

activity21. 25
20
15
Effect of temperature: Temperature is a vital factor affecting 10
protease activity. According to Baehaki and Budiman22, in 5

general, each enzyme has a maximum activity at a certain 0

Non metal Cu Ca Fe Mg Mn Zn
temperature, meaning that the enzyme activity will Type of metal ion (5 mM)
increase as the temperature increases until the optimum
temperature is reached. The increase in temperature affects Fig. 6: Metal ion effect on the protease activity of lactic acid
the substrate conformation change so that the substrate s bacteria BAF-715
active side encounters barriers while entering the enzyme's
active site, causing a decrease in enzyme activity. In Fig. 5, 39.39 U mLG1, respectively. While with Cu2+, Ca2+, Fe2+ and Zn2+,
the optimum activity at 40EC was 29.19 U mLG1 and the the protease activity values were 11.54, 10.03, 12.19 and
enzyme activity decreased as the temperature increased. At a 12.21 U mLG1, respectively, showing a reduction compared
temperature of 30EC, there was an increase in protease with the activity of the treatment without adding metal ions
activity. At 50-90EC, a decrease in protease activation was in which the activity was only 12.67 U mLG1, as shown in
observed. The stability of an enzyme depends on several Fig. 6. Cofactor bonds or enzyme inhibitors may alter the
things, namely, hydrogen bonding, hydrophobic bonding, enzyme's ability to bind to the substrate and alter the
ionic interactions and disulfide bridges. The stability of the enzyme's catalytic capacity because the enzyme structure
enzyme in response to temperature can be maintained in the presence of an inhibitor or cofactor undergoes
when the protein s amino acids form a certain conformation physical and chemical changes such that the activity
that is resistant to the effects of denaturation; in general, changes24. The research results by Xu et al.10 demonstrated
proteins are denatured at high temperatures23. Compared that P. pentosaceus had 220 proteases, the metal activator
with the Lactobacillus plantarum protease, the optimum was the Ca2+ ion, the inhibitors were Zn2+, Mg2+ and Fe3+
temperature is 47EC with an activity of 9.76 U mLG1; and the activity was inhibited by the metal-chelating EDTA
the research results by Sulthoniyah et al.12 on the and sodium dodecyl sulfate. Regarding the protease of
optimum activity of the proteases of Streptococcus lactis Lactobacillus plantarum, the metal activator was Mg2+ with
and Lactococcus lactis revealed an optimum temperature of an enzyme activity of 7.5 UI mgG1, as shown in the
28EC, in agreement with the research results by Akinkugbe research results by Sulthoniyah et al.12. According to
and Onilude20. Suhartono24, the presence of cofactors or inhibitors may alter
the enzyme's ability to bind the substrate and may alter the
Effect of metal ions: The results showed that the metal enzyme's catalytic capacity because the enzyme structure
ions that act as activators are Mg2+ and Mn2+ ions at a undergoes physical and chemical changes such that the
concentration of 5 mM with an activity of 23.16 and activity changes.

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 Pak. J. Nutr., 17 (8): 361-367, 2018

Pediococcus pentosaceus E24-160

89/48/30
P. pentosaceus E24-168
89/66/32
P. pentosaceus V12-27
100/100/90
P. pentosaceus V3-100
BK 715
49/97/93 P. parvulus S-182
P. inopinatus DSM 20285
84/92/89 L. graminis LMG 9825
Lactobacillus kefiri NBRC 15888

0.005

Fig. 7: Phylogenetic trees of isolate BAF-175 based on the 16S rRNA gene with maximum likelihood (ML), neighbor joining (NJ)
and maximum parsimony (MP) analysis
The 0.005 scale shows the evolutionary distance at the branch length, whereas the number on the branch shows the bootstrap value 1000 (ML/NJ/MP)

Molecular identification of isolate BAF-715: Genotypic that it is Pediococcus pentosaceus. A protease produced by
characterization of the LAB isolate was performed based on Pediococcus pentosaceus from Bekasam showed a high
DNA sequencing of the gene encoding 16S rRNA to determine proteolytic activity, making it the best protease for application
the genus and species. The DNA encoding the 16S rRNA can as a beef tenderizer.
be used as a molecular marker for the definition of a species
because these molecules exist in every organism and they SIGNIFICANCE STATEMENTS
perform identical functions in all organisms. DNA from
isolate BAF-715 was amplified using 63F and 1387R primers. This study discovers the optimum conditions for
These primers do not form a duplex structure with an the production of the protease enzyme by Pediococcus
enzyme-recognizable 5 edge 5'-3exonuclease and no pentosaceus bacteria; the findings can be beneficial for the
nucleotides are cut off at the 5' end to affect the primary food industry to improve meat tenderizers. This study will help
annealing temperature25. researchers uncover the ability of Pediococcus pentosaceus
The DNA sequence analysis of the 16S rRNA BAF-715 to produce a protease enzyme that can be explored by other
isolate obtained sequences with a length of 1200 bp. The researchers. Thus, a new theory on the optimum conditions for
sequencing of the 16S rRNA BAF715 isolates compared Pediococcus pentosaceus to produce the protease enzyme
with sequences in the GenBank NCBI database using the may be developed.
BLAST algorithm. The BLAST result for isolate BAF-715 showed
99% homology/similarity to Pediococcus pentosaceus (KP ACKNOWLEDGMENTS
18922.8.1) (Fig. 7). The classification of isolate BAF-715 is as
follows: Kingdom: Bacteria; Division: Firmicutes; Class: Bacilli; This study was funded and supported by the Directorate
Order: Lactobacillales; Family: Lactobacillaceae; Genus: General of Higher Education of the Ministry of Research,
Pediococcus; Species: Pediococcus pentosaceus. Technology and Higher Education through the Hibah Doktor
scheme 2017, No: 48/UN21.17/PP/2017. We are very grateful
CONCLUSION to the Minister of Research, Technology and Higher Education
for allowing us to conduct this study.
Isolate BAF-715 has a higher protease activity than that of
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