Bioscience Horizons Volume 11 2018 10,1093 / biohorizons / hzy004

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Artikel Penelitian

Isolasi, identifikasi fi kation dan karakterisasi bakteri asam

laktat yang memproduksi enzim dari makanan fermentasi
tradisional
Jason Chen Kok Ho * dan Lim Yin Sze

Sekolah Biosciences, Fakultas Sains, Universitas NottinghamMalaysia Kampus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia.

* Penulis yang sesuai: Taman Rasa Sayang, 47.301 Petaling Jaya, Selangor, Malaysia. E-mail: jasonchen808@gmail.com

Pengawas: Dr Lim Yin Sze, School of Biosciences, Fakultas Sains, Universitas NottinghamMalaysia Kampus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia

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proses industrialisasi rentan untuk menghasilkan berbagai bentuk limbah yang dapat dimanfaatkan untuk menghasilkan silase. limbah ini dapat diobati
dengan menggunakan bakteri asam laktat (BAL), yang diketahui potensial produsen enzim. Tujuh strain LAB diisolasi dari makanan fermentasi tradisional, ' tapai
pulut '' tempe '' tempoyak ' dan ' fu yu ' dan disaring untuk amilase, selulase dan produksi protease dalam rangka untuk memilih untuk strain yang berpotensi dapat
digunakan industri dalam produksi silase. Semua tujuh LAB isolat dipamerkan produksi protease tinggi, dan dua dari mereka juga dipamerkan amilase tinggi
dan produksi selulase. Kedua isolat menunjukkan amilase dan selulase produksi yang tinggi dipilih dan identifikasi fi ed sebagai

Pediococcus acidilactici FY2 dan durans Enterococcus FY3 melalui pro biokimia fi ling (API 50 CHL) dan 16 s rDNA sequencing.
E. durans ditemukan memiliki amilase tertinggi ( V max: 5.51 μ mol / mL / menit; K m: 0,300 g / 100mL) dan selulase ( V max: 3.50
μ mol / mL / menit; K m: 0,006 g / 100mL) produksi, sementara menunjukkan produksi protease yang kuat ( V max: 0,51 μ mol / mL / menit;
K m: - 0,287 g / 100mL). P. acidilactici ditemukan memiliki amilase yang kuat ( V max: 4.43 μ mol / mL / menit; K m: 0,433 g / 100mL) dan selulase ( V max: 2,66 μ mol / mL / menit; K
m: 0,002 g / 100mL) produksi sementara menunjukkan produksi protease tertinggi ( V max: 2.14
μ mol / mL / menit; K m: - 0,348 g / 100mL). Hasil ini menunjukkan bahwa E. durans adalah kandidat yang lebih baik untuk aplikasi industri di masa depan sebagai keseluruhan memiliki

kecepatan reaksi enzim lebih tinggi bila dibandingkan dengan P. acidilactici. Penelitian lebih lanjut harus dilakukan untuk con fi rm K m Nilai untuk produksi protease, untuk memurnikan dan

mengkarakterisasi semua tiga enzim yang diproduksi dan untuk mengoptimalkan kondisi pertumbuhan E. durans.

Kata kunci: bakteri asam laktat, amilase, selulase, protease, Pediococcus acidilactici, Enterococcus durans

Disampaikan pada 7 Agustus 2017; keputusan editorial pada 2 Mei 2018

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pengantar
Dalam era modern ini, proses industrialisasi tumbuh pada tingkat yang
mengkhawatirkan. Penemuan terus menerus dan penemuan teknologi state-of-the-art
terus mendorong dunia industri berkembang dengan cepat. Dengan demikian,
memprioritaskan penggunaan sumber daya yang ada adalah penting untuk
memastikan produksi yang optimal
dan mencegah pemborosan. Berbagai proses industri pertanian dan konsumsi
pangan umum menghasilkan sejumlah besar limbah, dan beberapa di antaranya
dapat dimanfaatkan dalam produksi silase sebagai limbah yang biasanya kaya
akan gula dan protein ( Show dan Guo 2012 ). Makanan tandon yang diawetkan
tanaman hijauan yang umumnya difermentasi oleh bakteri asam laktat (BAL) dalam
kondisi anaerob dan biasanya kosong dari yang tidak diinginkan

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© The Author (s) 2018. Diterbitkan oleh Oxford University Press.

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 Research article Bioscience Horizons • Volume 11 2018
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mikroorganisme karena produksi asam laktat oleh LAB ( Ni et al., 2015b ). menjadi 10 mL MRS broth, diikuti dengan inkubasi pada 37 ° C hingga 72 jam
Dengan demikian, para ilmuwan mencari sumber mikroba alternatif untuk fi nd sebelum subkultur berikutnya.
strain yang lebih baik untuk memproses silase, seperti LAB adalah produsen
Dalam hal tidak ada pertumbuhan, 0.2mL dari saham gliserol diinokulasi di 10
terkenal enzim ekstraseluler ( Patel, Shah dan Prajapati, 2013 ; Tosungnoen,
mL MRS broth dan diinkubasi pada 37 ° C selama 24 jam. Kemudian, beruntun
Chookietwattana dan Dararat 2014 ; Ni et al., 2015a ). LAB dengan amilase
empat poin dilakukan pada MRS agar untuk memastikan tidak ada kontaminasi
dan aktivitas selulase untuk mendegradasi gula kompleks seperti pati dan
terjadi. Sebuah koloni murni tunggal dipilih dan diinokulasi di 10 mL MRS broth
selulosa yang banyak dicari, karena hal ini akan mengurangi biaya substrat
dan diinkubasi pada 37 ° C selama 24 jam, diikuti oleh subkultur lain di 10 mL
pretreatment ( Shibata
MRS kaldu dan inkubasi pada 37 ° C selama 24 jam, membentuk aktif LAB
24-h-tua.
et al., 2007 ; Yitbarek dan Tamir 2014 ).

LAB adalah kelompok Gram-positif bakteri yang menghasilkan asam laktat selama
Dua hari sebelum assay, 0.2mL dari isolat LAB aktif diinokulasi di 10 mL
fermentasi dan penting untuk pembuatan makanan, terutama di susu, sayuran dan
MRS broth pada 37 ° C selama 24 jam dan disubkultur sekali lagi untuk
industri daging ( Konings et al., 2000 ; González et al., 2010 ). Berbagai LAB umumnya
membentuk LAB 24-h-lama aktif yang siap digunakan.
dianggap aman dan telah digunakan untuk waktu yang sangat lama sebagai kultur
starter untuk menghasilkan makanan fermentasi melalui cara tradisional, karena
mereka dapat menghasilkan asam laktat dan bakteriosin yang bertindak sebagai
Persiapan enzim kasar
pengawet alami dan dengan demikian dapat memperpanjang umur simpan silase ( Holzapfel,
Geisen dan Schillinger 1995 ; Enzim mentah yang diproduksi oleh isolat LAB yang disiapkan baru sebelum
digunakan. Sebanyak 2% ( v / v) LAB 24-h-lama (rata-rata OD 600nm = 0,713)
Perez, Zendo dan Sonomoto 2014 ). Oleh karena itu, LAB harus berlimpah ditemukan diinokulasi di 10 mL MRS broth dan diinkubasi pada 37 ° C selama 24 jam.
dalam makanan fermentasi. Supernatan bebas sel (CFS) mengandung enzim kasar dikumpulkan dengan
sentrifugasi dari kultur bakteri di 10 000 g selama 10 menit pada 4 ° C dan
Dalam studi saat ini, tradisional makanan seperti beras ketan yang
disimpan dalam es sampai digunakan.
difermentasi fermentasi ' tapai pulut ', tempe ' tempe ', fermentasi durian
bumbu ' tempoyak ' dan fermentasi dadih kedelai ' fu yu ' diselidiki. Tujuan
dari penelitian ini adalah untuk mengisolasi dan layar bagi produsen LAB
Skrining untuk produsen enzim
diduga untuk enzim amilase, selulase dan protease dari makanan
fermentasi tradisional untuk aplikasi industri di masa depan. Para aktivitas amilase assay
produsen enzim diduga berada identifikasi fi ed menggunakan teknik
pengujian ini diadaptasi dari metode Zhang dan Zeng (2007) untuk
biokimia dan molekuler, dan kinetika enzim ditentukan.
mengevaluasi aktivitas amilase enzim kasar dari LAB isolat. Dalam pengujian
ini, 1 kali lipat, 2 kali lipat dan 10 kali lipat CFS diencerkan digunakan sesuai
untuk mendapatkan pembacaan absorbansi kurang dari 2,5. Pati (1 g / 100mL;
Bendosen) digunakan sebagai substrat dan α- amilase (16 unit / mg padat;
Sigma-Aldrich) digunakan sebagai kontrol positif. Campuran reaksi disiapkan
material dan metode
sesuai dengan Tabel 1 . Campuran reaksi diinkubasi pada 37 ° C selama 30 menit,

Isolasi LAB dari makanan fermentasi dan reaksi enzimatik dihentikan dengan menggunakan rendaman es. Sebanyak
0.3ml dari 3,5-dinitrosalicyclic acid (DNS) ditambahkan ke dalam campuran
Empat jenis makanan fermentasi lokal, fermentasi beras ketan ' tapai pulut '(
reaksi dan campuran dipanaskan selama 5 menit dalam 90 ° C air mandi. Reaksi
TAP), tempe ' tempe '
kemudian berhenti menggunakan bath es. Kemudian, 0.2mL dari campuran
(TEM), fermentasi durian bumbu ' tempoyak '( TYK) dan fermentasi dadih
reaksi dipipet ke piring 96-baik dan diukur untuk absorbansi pada panjang
kedelai ' fu yu '( TA) yang dibeli dari toko kelontong di Selangor, Malaysia.
gelombang 550 nm menggunakan Fluostar OPTIMAmicroplate pembaca.
Sebanyak 10 g sampel makanan dicampur dengan 90ml air pepton buffered
dan diinkubasi pada suhu kamar selama 30 menit. Sebuah 10-kali lipat
pengenceran seri dilakukan dari 10 - 1 10 - 7. Volume 0.1ml sampel diencerkan
tersebar di de Man, ROGOSA dan Sharpe (MRS) agar piring dan diinkubasi
pada 37 ° C selama 48 jam. MRS agar adalah media selektif untuk Sebuah kurva standar maltosa dihasilkan untuk mengukur jumlah gula
pertumbuhan LAB. Setelah inkubasi, koloni dengan morfologi yang berbeda pereduksi dalam campuran reaksi. Satu unit amilase de fi ned sebagai
dipilih secara acak, Gramstained dan diperiksa untuk morfologi sel di bawah aktivitas enzimatik yang membebaskan salah satu mikrogram maltosa per
mikroskop. menit per mililiter CFS. The kecepatan awal ( V 0) enzim mentah untuk
membebaskan satu mikromol dari maltosa per menit per mililiter CFS
ditentukan.
Pemeliharaan dan persiapan isolat bakteri
aktivitas selulase assay

pengujian ini diadaptasi dari metode Wood and Bhat (1988) to evaluate
Setiap isolat LAB diawetkan dalam 20% ( v / v) gliserol dan disimpan dalam - 20 ° the cellulase activity of crude enzymes from LAB isolates. In this assay, 1
C freezer sampai digunakan. Awalnya, isolat terus aktif melalui subkultur oleh g/100mL carboxymethyl cellulose (CMC; Sigma-Aldrich) was dissolved in
pipetting 0.2mL budaya aktif 0.05M

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Table 1. Reaction mixtures for amylase, cellulase and protease activity assay

Reagents Mixture volume (mL)

Amylase Cellulase Protease

Blank − ve + ve Sample Blank − ve Sample Blank − ve Sample

control control control control

CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250

α- Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000

1 g/100mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000

0.4M Tris-HCl 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000
buffer, pH 9

1 g/100mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000

0.6 g/100mL 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250
Casein

5 g/100mL 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500
Tricloroacetic acid

Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000

glycine/NaOH buffer (pH 9) and used as a substrate. The reaction
mixtures were prepared according to Table 1 . The reaction mixtures were
incubated at 37 ° C for 30min, and the enzymatic reaction was halted by
using an ice bath. A total of 0.36mL of DNS were added to the reaction
mixture and the mixture was heated for 5min in a 90 ° C water bath. The
reaction was then stopped using an ice bath. Then,
0.2mL of the reaction mixture was pipetted into a 96-well plate and was
measured for absorbance at 550 nm wavelength using Fluostar OPTIMA
microplate reader.
A glucose standard curve was generated to quantify the amount of
reducing sugar in the reaction mixture. One cellulase unit was de fi ned as
the enzymatic activity that liberates one microgram of glucose per minute
per millilitre CFS. The
V 0 of the crude enzyme to liberate one micromole of glucose per minute
per millilitre CFS was determined. Positive control was not available due
to limitation of resources in the laboratory.
Protease activity assay
This assay was adapted from the method by Kanekar et al.
(2002) to evaluate the protease activity of crude enzymes from LAB
isolates. In this assay, 0.6 g/100mL casein (SigmaAldrich) was dissolved
in 0.4M Tris-HCl buffer (pH 9) and used as a substrate. The reaction
mixtures were prepared according to Table 1 . The reaction mixtures were
incubated at 37 ° C for 30min, and the enzymatic reaction was terminated
by adding 0.5mL of 5 g/100mL tricloroacetic acid.
Then, 0.25mL of the reaction mixture was mixed with
0.25mL of Bradford ’ s reagent in a 96-well plate and was allowed to react
for 5min at room temperature.
A tryptophan standard curve was generated to quantify the amount of
protein in the reaction mixture. One protease unit was de fi ned as the
enzymatic activity that liberates one microgram of tryptophan per minute
per millilitre CFS. The
V 0 of the crude enzyme to liberate one micromole of tryptophan per minute
per millilitre CFS was determined. Positive control was not available due to
limitation of resources in the laboratory.
Identi fi cation via carbohydrate metabolism tests using API®
50 CHL kit
Before testing the LAB for carbohydrate fermentation, the two isolates were
subjected to a catalase test. A sterile loop was used to place a small
amount of LAB growth onto the base of a Petri dish, then one drop of
hydrogen peroxide was added and the Petri dish was immediately covered
with a lid. The development of bubbles (effervescence) indicates a positive
result.
The selected LAB isolated were then tested for their carbohydrate
fermentation pattern using the API ® 50 CHL system kit (BioMérieux). The
identi fi cation procedure was conducted in accordance to the manufacturer ’ s
instructions. Active 24-h-old LAB isolates were added into the API 50 CHL
basal medium and were fi lled into wells on the API 50 CHL test

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strips. The strips were then incubated at 37 ° C for 48 h. The strips were
visually examined at 24 and 48 h, and the indication of a positive or negative
result was determined from the colour change from a scale of 1 to 5, from
purple (1) to green (3) to yellow (5) at the 48-h mark, whereby a value of ≥ 3
Statistical
was considered positive. The results were then cross-referenced to the API ® databases
using APIweb ™.
DNA extraction, polymerase chain reaction ampli fi cation
and DNA sequencing of 16s rDNA gene
DNA extraction was carried out using Wizard ® Genomic DNA Puri fi cation
Kit (Promega) on 24-h-old LAB isolates. Polymerase chain reaction (PCR)
was carried out in total volumes of 20 μ L containing 5 U/ μ L DreamTaq
polymerase, 10 × DreamTaq Buffer, 2mM deoxyribonucleotide phosphate,
10mM U8 Forward primer (5 ′- AGA GTT TGA TCC TGG CTC AG-3 ′), 10mMA total of 12 LAB were isolated from the four fermented foods, two isolates
1492 reverse primer (5 ′- CGG TTA CCT TGT TAC GAC TT-3 ′), sterile
distilled water and 5 ng/ μ L LAB DNA samples. DNA ampli fi cation was
performed for 30 cycles and the PCR cycle was set as the initial
denaturation at 95 ° C for 4min, annealing at 65 ° C for 1min, elongation at
72 ° C for 1min, and followed by another 29 cycles of denaturation at 95 ° C TEM2, TYK2, TYK3, FY2 and FY3 were chosen for preliminary screening
for 1min, annealing at 65 ° C for 1min and elongation at 72 ° C for 1min. of enzyme producers. This loss of viability for the fi ve isolates could be
A total of 20 μ L of the ampli fi ed PCR products and a negative control
were electrophoresed on 1 g/100mL agarose gel containing SYBR ® Safe
and 1 × Tris-acetate – EDTA buffer (TAE) at 75V for 40min. The gel was
visualized using Gel Doc ™ EZ Gel Documentation System (Bio-Rad). The
remaining ampli fi ed PCR products were then sent to First BASE
Laboratories Sdn. Bhd. for sequencing using the U8F and 1492R primers
under the DNA Sequencing Service + Plus (SS1201) service. The DNA
sequence obtained was then compared with sequences in the GenBank
database using BLAST by the National Center for Biotechnology
Information (NCBI).
Determination of the enzyme kinetics by using Hanes – Woolf
plot
The Michaelis – Menten constant ( K m) and maximal velocity ( V max) of the crude
enzymes were calculated using the Hanes –
Woolf plot. The V 0 of the enzyme reaction was determined according to
methods described in Sections 3.4.1 – 3.4.3. In this assay, a range of
substrate concentrations were used for amylase (ranging from 0 to 20
g/100mL starch), cellulase (ranging from 0 to 1.3 g/100mL CMC) and
protease (ranging from 0 to 1.5 g/100mL casein). The Hanes – Woolf graph
was then plotted to determine the K m and V max values of the targeted crude
enzymes.
A graph of [S]/ V 0 ( g min/100 μ mol) against substrate concentration, [S]
(g/100mL) was plotted. As the Hanes – Woolf equation is ‘[ S]/ V 0 = ( 1/ V max)([ S])
The seven LAB isolates tested were able to produce amylase, cellulase and
+ K m/ V max ’, the V max was calculated by taking the reciprocal value of the
gradient of the
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Bioscience Horizons • Volume 11 2018
graph. K m is equal to the negative of the x- intercept, thus was calculated
by taking the negative of the [S] value when [S]/ V 0 = 0.
analysis
Statistical analyses were carried out using IBM SPSS Statistics
23. All experimental data were obtained in triplicates and were analysed
using the analysis of variance (ANOVA) test followed by Duncan ’ s post hoc
test at P ≤ 0.05. Correlation between amylase and cellulase assay was
analysed using Pearson ’ s correlation coef fi cient test.
Results and discussion
Isolation of LAB from fermented foods
from ‘ tapai pulut ’ ( TAP1 – 2), four isolates from ‘ tempeh ’ ( TEM1 – 4), three
isolates from ‘ tempoyak ’
(TYK1 – 3) and three isolates from ‘ fu yu ’ ( FY1 – 3). However, only 7 out of
12 were still viable after repeated subcultures, hence only TAP2, TEM1,
due to two factors, (i) the selective media (MRS) used was not providing
the nutrients needed and (ii) the changes in growth environment.
This fi nding was similar to those reported by Birollo, Reinheimer and
Vinderola (2000) showing that MRS may show a lower viable count of
bacteria and there are other suitable alternatives to grow LAB. Other than
that, the other LAB isolates may be in the unique form of viable but
nonculturable state, whereby they cannot be cultured on routine
microbiological media but are still viable ( Fakruddin, Mannan and
Andrews, 2013 ). Thus, the LAB isolates were most likely unable to
acclimatize and grow continuously in MRS broth. This may be due to
unfavourable environmental factors such as starvation, non-optimal pH or
others ( Pinto, Santos and Chambel, 2015 ).
Morphological and physiological
characterization of LAB
The seven LAB isolates tested were Gram-positive, and two isolates were
rod-shaped and the remaining fi ve were coccusshaped. FY2 had showed a
tetracoccus cell arrangement, TEM2 and TYK3 showed diplococcus cell
arrangement, while TEM1 and FY3 had no distinct coccus arrangements.
The two rod-shaped isolates, TAP2 and TYK2 tended to form short chains.
A summary of the morphology of the LAB isolates can be seen in Table 2 .
Screening for enzyme producers
protease enzymes at different strengths. The enzyme activity of the LAB
isolates was determined via the
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Table 2. Colony and cell morphology of LAB isolates

LAB isolates Colony morphology Gram stain Shape

TAP2 Circular, milky Positive Short chain rod

TEM1 Circular, milky Positive Coccus

TEM2 Circular, milky Positive Diplococcus

TYK2 Circular, white Positive Short chain rod

TYK3 Circular, white Positive Diplococcus

FY2 Circular, milky Positive Tetracoccus

FY3 Circular, white, pinprick sized Positive Coccus

Table 3. Amylase, cellulase and protease activity of LAB isolates

LAB Amylase activity Amylase reaction Cellulase activity Cellulase reaction Protease activity Protease reaction
isolates (mg/ mL/min) velocity, V 0 ( μ mol/ (mg/ mL/min) velocity, V 0 ( μ mol/ (mg/ mL/min) velocity, V 0 ( μ mol/
mL/min) mL/min) mL/min)

TAP2 0.725 ± 0.053 d 2.118 ± 0.155 d 0.406 ± 0.012 e 2.252 ± 0.064 e 1.888 ± 0.347 a 9.245 ± 1.700 a

TEM1 0.962 ± 0.029 b 2.809 ± 0.085 b 0.569 ± 0.014 c 3.158 ± 0.080 c 2.058 ± 0.399 a 10.076 ± 1.956 a

TEM2 0.572 ± 0.009 e 1.672 ± 0.026 e 0.401 ± 0.012 e 2.229 ± 0.064 e 2.162 ± 0.471 a 10.585 ± 2.304 a

TYK2 0.803 ± 0.032 c 2.347 ± 0.093 c 0.484 ± 0.019 d 2.688 ± 0.105 d 2.079 ± 0.164 a 10.180 ± 0.804 a

TYK3 0.184 ± 0.008 f 0.538 ± 0.022 f 0.083 ± 0.007 f 0.460 ± 0.039 f 2.035 ± 0.271 a 9.963 ± 1.328 a

FY2 1.027 ± 0.071 b 3.000 ± 0.208 b 0.655 ± 0.013 b 3.638 ± 0.072 b 2.150 ± 0.427 a 10.529 ± 2.093 a

FY3 1.224 ± 0.007 a 3.575 ± 0.021 a 0.816 ± 0.007 a 4.528 ± 0.040 a 2.307 ± 0.044 a 11.295 ± 0.216 a

Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are signi fi cantly different at P ≤ 0.05.

liberation of a speci fi c substrate based on the designated enzyme assay are 2-folds higher than amylase and cellulase activities under the assay
depicted in Table 3 . conditions. However, no signi fi cant difference in protease activity was
observed between isolates. This fi nding was in accordance with the study by Shin
The amylase activity of isolate FY3 was ranked highest, followed by
et al. ( 2008) , where LAB from the genera Pediococcus and Enterococcus displayed
FY2 and TEM1 (Table 3 ). This fi nding was interesting as these three
high amylase and cellulase activities but did not exhibit any signi fi cant
isolates were not isolated from
difference of protease activity compared to other bacteria.
carbohydrate-rich food sources ( ‘ fu yu ’ and ‘ tempeh ’), whereas isolates from
carbohydrate-rich food sources ( ‘ tapai ’ and ‘ tempoyak ’) such as TAP2, TYK2
and TYK3 did not show high level of amylase activity.
Therefore, the two top producers for amylase and cellulase isolated from ‘ fu
yu ’— FY2 and FY3 were selected for further identi fi cation and enzyme kinetic
Similarly, the cellulase activity of FY3 was also ranked highest, followed by
assay.
FY2 and TEM1 (Table 3 ). Pearson ’ s coef fi cient test showed that there was a
signi fi cant positive correlation ( r 2 =
+ 0.979) between amylase and cellulase activity in all LAB isolates. This fi nding
suggests that amylase and cellulase production could be correlated. However, Bacterial identi fi cation via carbohydrate metabolism
to the author ’ s knowledge, no relevant published literature was found. tests using API® 50 CHL kit
FY2 and FY3 which displayed the highest amylase and cellulase activities
All LAB isolates were good producers of proteases as seen in Table 3 . were identi fi ed by using the API 50 CHL system kit to test for their
The protease activity of all isolates was at least carbohydrate fermentation patterns

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Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit

No. Substrates LAB isolate code No. Substrates LAB isolate code

FY2 FY3 FY2 FY3

0 Control − − 25 Esculin ferric citrate + +

1 Glycerol − − 26 Salicin − +

2 Erythritol − − 27 D- Cellobiose + +

3 D- Arabinose − − 28 D- Maltose − +

4 L- Arabinose − − 29 D- Lactose (bovine origin) − +

5 D- Ribose + + 30 D- Mellibiose − +

6 D- Xylose + − 31 D- Saccharose (sucrose) − +

7 L- Xylose − − 32 D- Trehalose − −

8 D- Adonitol − − 33 Inulin − −

9 Methyl- β D- xylopyranoside − − 34 D- Melezitose − −

10 D- Galactose − + 35 D- Raf fi nose − +

11 D- Glucose + + 36 Amidon (starch) − −

12 D- Fructose + + 37 Glycogen − −

13 D- Mannose + + 38 Xylitol − −

14 L- Sorbose − − 39 Gentiobiose ± +

15 L- Rhamnose − + 40 D- Turanose − −

16 Dulcitol − − 41 D- Lyxose − −

17 Inositol − − 42 D- Tagatose − −

18 D- Mannitol − − 43 D- Fucose − −

19 D- Sorbitol − − 44 L- Fucose − −

20 Methyl- α D- Mannopyranoside − − 45 D- Arabitol − −

21 Methyl- α D- Glucopyranoside − − 46 L- Arabitol − −

22 N-Acetylglucosamine + + 47 Potassium gluconate − −

23 Amygdalin − + 48 Potassium 2-ketogluconate − −

24 Arbutin − + 49 Potassium 5-ketogluconate − −

( −) negative, (+) positive, ( ±) undetermined.

(Table 4 ). A catalase test was performed to complement the API 50 CHL biochemical pro fi le of P. acidilactici in the APIweb ® system as compared to
test, where both isolates were found to be catalase-negative. the API 50 CHL results of FY2. FY2 exhibited the inability to utilize L- arabinose,
D- galactose, L- rhamnose, salicin, D- trehalose and D- tagatose, whereas P.
acidilactici in the APIweb ® system had the following biochemical pro fi le: ( L- arabinose
Based on the API 50 CHL results in Table 4 , FY2 was ini- 100%), ( D- galactose 100%), ( L- rhamnose 75%), (salicin 75%), ( D- trehalose
tially identi fi ed with a low % ID value of 34.2% as P. acidilactici. The % ID
75%) and ( D- tagatose 100%), whereby the percentage represents the
for FY2 meant that 34.2% of LAB with this speci fi c biochemical pro fi le was likelihood of that particular LAB strain to have the ability to ferment that
found to be P. acidilactici. sugar.
The low % ID was due to the discrepancies between the

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Figure 1. 16 s rDNA sequence of FY2 ( A) and FY3 ( B).

On the other hand, based on Table 4 , FY3 was initially
identi fi ed as Pediococcus pentosaceus with a higher % ID value of 80.9%
but also displayed biochemical discrepancies such as the inability to utilize L-
arabinose, D- trehalose and
D- tagatose as compared to the APIweb ® system ’ s biochemical pro fi le for P.
pentosaceus ( L- arabinose 100%), ( D- trehalose 99%) and ( D- tagatose 99%).
Bacterial identi fi cation via 16s rDNA gene sequencing
analysis
Since the API carbohydrate fermentation test was unable to con fi rm the
identity for FY2, both isolates were then subjected to 16 s rDNA gene
sequencing analysis for further con fi rmation. Based on the gene sequences
(Fig. 1 A) and BLAST

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 Research article
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results for FY2, 16 s rDNA gene sequencing analysis showed a similarity
of 94%, 94% and 92% with P. acidilactici,
P. pentosaceus and P. stilesil, respectively. However, the query cover for P.
acidilactici was at 97%, whereas P. pentosaceus was at 94%. Thus, it was
highly suspected that FY2 was indeed P. acidilactici due to a higher similarity
in the biochemical test compared to P. pentosaceus. Besides that, the
morphology of the LAB to form tetracoccus also supported the evidence of
FY2 to be under the genera Pediococcus.
Based on the gene sequences (Fig. 1 B) and BLAST results for
FY3, a similarity of 95%, 95% and 94% with Enterococcus durans, E. faecium and
E. mundtii was obtained respectively. The query cover for E. durans and E.
faecium was the same at 80%, thus the biochemical properties were examined
to assist in identifying the species. However, the APIweb ™ database did not
show LAB from the genera Enterococcus, hence other literature were used as a
reference. According to Bergey ’ s manual of Determinative Bacteriology, FY3
showed discrepancies for both Enterococcus LAB such as raf fi nose (+) and
mellibiose (+) ( Holt, 2000 ). However, based on Bergey ’ s manual FY3 was
more likely to be E. durans due to its inability to utilize glycerol ( −) and D- mannitol contain a basic pool of chromosomal genes that is responsible for starch
( −), as compared to E. faecium.
The discrepancies between the biochemical and molecular identi fi cation
techniques might be due to the fact that principles of both techniques are
fundamentally different. API 50 CHL is used to classify LAB based on their
phenotypical properties by comparing the fermentation pattern of 49
different types of carbohydrate with other bacteria that are registered in the
APIweb ™ database. One of the limitations of this is that the APIweb ™ database
has been found to be lacking in biochemical pro fi les of certain LAB ( Boyd et
al., 2005 ). Besides that, phenotypical characterization has been known to
have poor reproducibility and that the whole information potential of the
LAB genome is not always expressed, as gene expression is directly
related to environmental conditions such as the growth conditions in the
laboratory ( Mohania
et al., 2008 ).
On the other hand, 16 s rDNA sequencing relies on the ampli fi cation
and sequencing of the highly conserved 16 s ribosomal RNA gene, which
strain that was isolated from the gut of the phytophagous insect Oxya
is akin to a unique biosignature for any organism ( Isenbarger et al., 2008 ). Janda
and Abbott (2007) reported that 16 s rDNA sequencing is an alternative to
provide identi fi cation of unknown bacteria with unrecognized biochemical
pro fi les or a low likelihood. This sequencing technique can provide
information about the genus and species of most unknown bacteria, with
high levels of genus identi fi cation (>90%) and moderate levels of species
identi fi-
cation (65 – 83%; Janda and Abbott, 2007 ). A study by Ba g˘ der et al. ( 2014) (2015)
compared
, whereby P. acidilactici which was isolated from biomass piles of Eucalyptus
the results of the API 50 CHL test with 16 s rRNA results and found that
the API test did not give reliable identi fi cation results, with only 71 out of
152 tested isolates were in agreement. Another study by Moraes et al. ( 2013)
reported the possibility for high reliability rates in the API 50 CHL to diverge
greatly from 16 s rDNA results, supporting the identi fi cation of FY3 which
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Bioscience Horizons • Volume 11 2018
had a high API 50 CHL % ID for P. pentosaceus ( 80.9%) but was identi fi ed
as E. durans ( 95%) through BLAST. However, one of the limitations of
using 16 s rDNA sequencing is that LAB in the genera Enterococcus are dif fi
cult to differentiate due to the highly conserved nature of 16 s rDNA ( Moraes
et al., 2013 ). Thus, the API 50 CHL biochemical test results were used as
supporting evidence to support the main identi fi-
cation technique, 16 s rDNA sequencing to help discern the LAB isolates
based on their genome and carbohydrate metabolism patterns.
The fi nding of the amylase producers — FY2 and FY3 from the species of
P. acidilactici and E. durans were in accordance to a report by Velikova et
al. ( 2016) in which their targeted strains P. acidilactici and E. durans were
also found to have highest extracellular and intracellular amylase activities,
respectively. However, this fi nding was in contrast with a study by Musikasang
et al. ( 2009) , as bacterium from the genera Pediococcus and E. durans that
were isolated from chicken ’ s gastrointestinal tract did not exhibit the ability
to digest starch. According to Velikova et al. ( 2016) , amylaseproducing LAB
hydrolysis. However, only the strains that are forced to survive in starchy
environment are able to display these genetically determined properties.
Hence, the gastrointestinal tract of chicken might not have an ideal
environment for these LAB to grow. These fi ndings suggest that LAB
isolated from different sources such as plants or animals may exhibit
different enzymatic properties.
According to Bergey ’ s manual, P. acidilactici can grow at higher
temperatures of up to 50 ° C, whereas E. durans can grow at 45 ° C. This may
be bene fi cial as the two LAB could possibly produce thermostable amylases
which are of special interest in the industrial fi eld, especially for starch
sacchari fi-
cation ( Saxena et al., 2007 ). Hence, further tests should be carried out to
evaluate the ability of FY2 and FY3 to produce potential thermostable
amylases.
On the other hand, the cellulase-producing potential of FY3 ( E. durans) was
similar to the fi nding by Shil et al. ( 2014)
where they fi rst discovered cellulase activity from an E. durans
velox. However, this was in contrast to a study by
Mazzucotelli et al. ( 2013) , as the E. durans isolated from cheese whey did
not exhibit the ability to degrade cellulose. These fi ndings further support
the previous suggestion that LAB isolated from various sources may exhibit
different enzymatic properties. Besides that, FY2 also exhibited high
cellulase activity, similar to a study reported by Ventorino et al.
camaldulensis was found to have high levels of azo-carboxymethylcellulase
activity.
The protease-producing potential of FY2 and FY3 was contrasted with
a study by Tuncer (2009) , whereby E. durans
isolated from Turkish tulum cheese showed varied levels of protease
activity, ranging from low to high based on the strain observed, and even
more variation among different species.
 Bioscience Horizons • Volume 11 2018 Research article
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Besides that, Moslehishad et al. ( 2013) reported that P. acidilactici PTCC1424

from the Iranian Research Organization for Science and Technology
exhibited moderate protease activity in supernatant form. However, Moslehishad
et al.
(2013) also reported that different LAB had varying levels of protease
activity based on the culture conditions, such as in CFS form, anaerobic
conditions and enriched CO 2 conditions, whereby anaerobic conditions
showed more favourable protease activity. Although the protease activity
was about 2-fold higher than the amylase and cellulase activities, these

fi ndings suggest that LAB protease activity could be even higher, if the
assay were to be optimized.

Determination of the enzyme kinetics by using Hanes – Woolf

plot
According to Berg, Tymoczko and Stryer (2002) , enzymes are important to
enhance the rate of biochemical reactions, thus the kinetic description of
their activity is required to understand about the enzyme ’ s function. Thus, a
Figure 2. Hanes – Woolf plot for starch concentration, [S]/ V 0 against [S].
graph of [S]/ V 0
against [S] was plotted to calculate V max and K m. V max is the maximum reaction
velocity achieved by an enzyme and can be used to indicate the maximum
capacity of an enzyme to carry out enzymatic reactions, provided suf fi cient
substrates are available. K m is equal to [S] at which reaction velocity is equal
to half of V max and is independent of enzyme and substrate concentrations. K
m shows the required [S] needed for signi fi cant catalysis to occur and
determines the viability of the enzyme for industrial use.

K m and V max of LAB isolates FY2 and FY3 were calculated by plotting graphs
as seen in Figs 2 – 4 . For Fig. 4 , the graph not extrapolated the past y- axis due
to the nature of the graph.

Based on Fig. 2 , the amylase V 0 of FY3 is generally higher than FY2, as its V max
value was slightly higher (5.51 – 4.43 μ mol/ mL/min) and K m value was lower
(0.299 – 0.433 g/100mL) than FY2. This result suggests that both LAB isolates
are not very ef fi-
cient at degrading starch, as a high substrate concentration (average 4.97
g/100mL) would be required for its enzyme to achieve maximum capability.
However, due to the lack of amylase-producing LAB, the two isolates may
still be of industrial use during fermentation processes ( Fossi and Tavea, Figure 3. Hanes – Woolf plot for CMC concentration, [S]/ V 0 against [S].
2013 ). The cellulase V 0 of FY3 was also higher than FY2 as seen in Fig. 3 .
FY3 had a higher V max value (3.50 – 2.66 μ mol/mL/ min) but also had a higher K
m value (0.006 – 0.002 g/100mL) than FY2. Hence, these results suggest that
FY3 is a better cellulase producer than FY2 and the cellulase produced by
Interestingly, FY2 had a higher V max value than FY3 (2.14 – 0.51 μ mol/mL/min)

both isolates are ef fi cient at degrading carboxymethylcellulose due to their but also a higher negative K m
( − 0.348 to − 0.287 g/100mL) in terms of protease activity as seen in Fig. 4 .
low K m value.
This negative value is due to a drastic reduction in protease reaction activity
at casein concentrations higher than 1.2 g/100mL, causing the FY3 graph
to have an R 2

Based on both amylase and cellulase assay, FY3 would be a better
option to be studied as an industrial enzyme producer as it can achieve
higher enzymatic activity when substrate concentration is saturated. FY3
would have a lower requirement of substrate concentration to achieve
optimum enzymatic activity for amylase while also having a low requirement
of substrate concentration for cellulase.
value of 0.27. Koka and Weimer (2000) had a similar observation, whereby
Pseudomonas fl uorescens was observed to have a sudden reduction in
protease activity at casein concentrations greater than 100mmol. This was
suspected to be caused by either casein micelle formation or
self-aggregration, which would reduce the number of available bonds for
hydrolysis ( Koka and Weimer, 2000 ). In terms of protease,

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Figure 4. Hanes – Woolf plot for casein concentration, [S]/ V 0 against [S].
FY2 would be a more suitable choice compared to FY3 due to its higher V max value
albeit it has a higher K m, as the difference between K m values is smaller than
the difference between
V max values of the two isolates.
Conclusion
This study has demonstrated that traditional fermented foods, namely, ‘ tapai
pulut ’, ‘ tempeh ’, ‘ tempoyak ’ and ‘ fu yu ’ are the potential resources for
isolating LAB enzyme producers. All seven LAB isolates tested are strong
producers of protease and two of them (FY2 and FY3) also exhibited the
highest amylase and cellulase production. FY2 and FY3 were identi-
fi ed as P. acidilactici and E. durans based on biochemical pro-
fi ling and 16 s rDNA sequencing. Furthermore, FY3 had a higher overall V 0 than
FY2, hence FY3 is a better candidate for future industrial application.
However, further studies are essential to enhance the understanding on
these producers and the enzymes produced. Future studies should be
carried out to con fi rm the K m value for protease production, to purify and
characterize all the three enzymes produced and to optimize the growth
conditions of FY2 and FY3 for future application in silage treatment.
Author Biography
Jason Chen graduated from the University of Nottingham (Malaysia
Campus) in the year 2017 with a First Class Honours BSc in
Biotechnology. This research article was submitted in partial ful fi lment of
the requirements for Jason ’ s degree in his fi nal year. Having an interest in
microorganisms and food, Jason is hoping to pursue a career related to
the food and microorganism industry. J.K.H., C. designed the study,
carried out the research and wrote the paper. He has
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Bioscience Horizons • Volume 11 2018
primary responsibility for the paper. Y.S., L. contributed in designing the
initial project scope and improving the quality of the paper.
Acknowledgements
I would like to express my gratitude to all those who provided me the
chance to complete this report. Special thanks should be given to Dr Lim
Yin Sze, my fi nal year project supervisor, for her professional guidance,
patience and useful critiques for this project. My grateful thanks are
extended to the School of Biosciences, University of Nottingham Malaysia
Campus, for providing the opportunity to carry out this project. I would like
to extend my thanks to the technicians for their help in providing
resources. Finally, I wish to thank my family and friends for their support
throughout this project.
References
Ba ğ der, E. S., Tokatl ı, M., Dursun, D. et al. (2014) Phenotypic and genotypic identi fi cation of
lactic acid bacteria isolated from traditional pickles of the Çubuk region in Turkey, Folia
Microbiologica, 60 (3), 241 – 251.
Berg, J. M., Tymoczko, J. L. and Stryer, L. (2002) Biochemistry, 5th ed.,
W.H. Freeman, New York.
Birollo, G. A., Reinheimer, J. A. and Vinderola, C. G. (2000) Viability of lac-
tic acid micro fl ora in different types of yoghurt, Food Research International, 33 (9), 799 – 805.
Boyd, M. A., Antonio, M. A. D. and Hillier, S. L. (2005) Comparison of API
50 CH strips to whole-chromosomal DNA probes for identi fi cation of Lactobacillus Species,
Journal of Clinical Microbiology, 43 (10), 5309 – 5311.
Fakruddin, M., Mannan, K. S. B. and Andrews, S. (2013) Viable but non-
culturable bacteria: food safety and public health perspective, ISRN Microbiology, 2013, 1 – 6.
Fossi, B. T. and Tavea, F. (2013). Application of amylolytic Lactobacillus
fermentum 04BBA19 in fermentation for simultaneous production of thermostable
alpha-amylase and lactic acid. Lactic Acid Bacteria, R & D for Food, Health and Livestock
Purposes, accessed at: https://
www.intechopen.com/books/lactic-acid-bacteria-r-d-for-food-healthand-livestock-purposes/application-of-amylolytic-la
(29 March 2017).
González, L., Sacristán, N., Arenas, R. et al. (2010) Enzymatic activity of
lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish
cheese, Food Microbiology, 27 (5), 592 – 597.
Holt, J. G. (2000) Bergey ’ s Manual of Determinative Bacteriology, 9th ed.,
Lippincott Williams & Wilkins, Philadelphia.
Holzapfel, W. H., Geisen, R. and Schillinger, U. (1995) Biological preserva-
tion of foods with reference to protective cultures, bacteriocins and food-grade enzymes, International
Journal of Food Microbiology, 24 (3), 343 – 362.
 Bioscience Horizons • Volume 11 2018
.................................................................................................................................................................
Isenbarger, T. A., Carr, C. E., Johnson, S. S. et al. (2008) The most con-
served genome segments for life detection on Earth and other planets, Origins of Life and
Evolution of Biospheres, 38 (6), 517 – 533.
Janda, J. M. and Abbott, S. L. (2007) 16S rRNA gene sequencing for bac-
terial identi fi cation in the diagnostic laboratory: pluses, perils, and pitfalls, Journal of
Clinical Microbiology, 45 (9), 2761 – 2764.
Kanekar, P. P., Nilegaonkar, S. S., Sarnaik, S. S. et al. (2002) Optimization
of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India, Bioresource
Technology, 85 (1), 87 – 93.
Koka, R. and Weimer, B. C. (2000) Isolation and characterization of a
protease from Pseudomonas fl uorescens RO98, Journal of Applied Microbiology, 89 (2),
280 – 288.
Konings, W. N., Kok, J., Kuipers, O. P. et al. (2000) Lactic acid bacteria:
the bugs of the new millennium, Current Opinion in Microbiology,
3 (3), 276 – 282.
Mazzucotelli, C. A., Ponce, A. G., Kotlar, C. E. et al. (2013) Isolation
and characterization of bacterial strains with a hydrolytic pro fi le with potential use in
bioconversion of agroindustial by-products and waste, Food Science and Technology
(Campinas), 33 (2), 295 – 303.
Mohania, D., Nagpal, R., Kumar, M. et al. (2008) Molecular approaches
for identi fi cation and characterization of lactic acid bacteria,
Journal of Digestive Diseases, 9 (4), 190 – 198.
Moraes, P. M., Perin, L. M., Júnior, A. S. et al. (2013) Comparison of
phenotypic and molecular tests to identify lactic acid bacteria,
Brazilian Journal of Microbiology, 44 (1), 109 – 112.
Moslehishad, M., Mirdamadi, S., Ehsani, M. R. et al. (2013) The proteo-
lytic activity of selected lactic acid bacteria in fermenting cow ’ s and camel ’ s milk and the
resultant sensory characteristics of the products, International Journal of Dairy
Technology, 66 (2), 279 – 285.
Musikasang, H., Tani, A., H-kittikun, A. et al. (2009) Probiotic potential of
lactic acid bacteria isolated from chicken gastrointestinal digestive tract, World Journal of
Microbiology and Biotechnology, 25 (8), 1337 – 1345.
Ni, K., Wang, Y., Cai, Y. et al. (2015b) Natural lactic acid bacteria popula-
tion and silage fermentation of whole-crop wheat, AsianAustralasian Journal of Animal
Sciences, 28 (8), 1123 – 1132.
Ni, K., Wang, Y., Li, D. et al. (2015a) Characterization, identi fi cation and
application of lactic acid bacteria isolated from forage paddy rice silage , PLoS One, 10 (3),
e0121967.
Patel, A., Shah, N. and Prajapati, J. B. (2013) Biosynthesis of vitamins
and enzymes in fermented foods by lactic acid bacteria and related genera — a promising
approach, Croatian Journal of Food Science and Technology, 5 (2), 85 – 91.
.................................................................................................................................................................
Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzy004/5038454 by guest
on 28 June 2018
Research article
Perez, R. H., Zendo, T. and Sonomoto, K. (2014) Novel bacteriocins from
lactic acid bacteria (LAB): various structures and applications,
Microbial Cell Factories, 13 (Suppl 1), S3.
Pinto, D., Santos, M. A. and Chambel, L. (2015) Thirty years of viable but
nonculturable state research: unsolved molecular mechanisms,
Critical Reviews in Microbiology, 41 (1), 61 – 76.
Saxena, R. K., Dutt, K., Agarwal, L. et al. (2007) A highly thermostable
and alkaline amylase from a Bacillus sp. PN5, Bioresource Technology, 98 (2), 260 – 265.
Shibata, K., Flores, D. M., Kobayashi, G. et al. (2007) Direct L- lactic acid
fermentation with sago starch by a novel amylolytic lactic acid bacterium, Enterococcus
faecium, Enzyme and Microbial Technology,
41 (1-2), 149 – 155.
Shil, R. K., Mojumder, S., Sadida, F. F. et al. (2014) Isolation and identi fi-
cation of cellulolytic bacteria from the gut of three phytophagus insect species , Brazilian
Archives of Biology and Technology, 57 (6), 927 – 932.
Shin, M. S., Han, S. K., Ji, A. R. et al. (2008) Isolation and characterization
of bacteriocin-producing bacteria from the gastrointestinal tract of broiler chickens for
probiotic use, Journal of Applied Microbiology,
105 (6), 2203 – 2212.
Show, K. Y. and Guo, X. (2012) Industrial Waste, 1st ed., InTech, Rijeka.
Tosungnoen, S., Chookietwattana, K. and Dararat, S. (2014) Lactic acid
production from repeated-batch and simultaneous sacchari fi cation and fermentation of
cassava starch wastewater by amylolytic
Lactobacillus Plantarum MSUL 702, APCBEE Procedia, 8, 204 – 209.
Tuncer, Y. (2009) Some technological properties of phenotypically
identi fi ed enterococci strains isolated from Turkish tulum cheese,
African Journal of Biotechnology, 8 (24), 7008 – 7016.
Velikova, P., Stoyanov, A., Blagoeva, G. et al. (2016) Starch utilization
routes in lactic acid bacteria: new insight by gene expression assay,
Starch/Stärke, 68 (9-10), 953 – 960.
Ventorino, V., Aliberti, A., Faraco, V. et al. (2015) Exploring the micro-
biota dynamics related to vegetable biomasses degradation and study of
lignocellulose-degrading bacteria for industrial biotechnological application, Scienti fi c
Reports, 5, 8161.
Wood, T. M. and Bhat, K. M. (1988) Methods for measuring cellulase
activities, Methods in Enzymology, 160, 87 – 112.
Yitbarek, M. B. and Tamir, B. (2014) Silage additives: review, Open
Journal of Applied Sciences, 4 (5), 258 – 274.
Zhang, J. W. and Zeng, R. Y. (2007) Psychrotrophic amylolytic bacteria from
deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases, World
Journal of Microbiology and Biotechnology, 23 (11), 1551 – 1557.
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