Isolation and Characterization of Microorganisms for Protease Production from Soil Samples from Kosovo and Testing the Enzymes in Food Industry Application

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 687-693

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Isolation and Characterization of Microorganisms

for Protease Production from Soil Samples from

Kosovo and Testing the Enzymes in Food Industry

Application

1,2

Bahtir Hyseni , Flora Ferati , Fatos Rexhepi , Rifat Morina , Ylberinë Baliu , Shkëlqim Hyseni ,

Aida Rushiti , Sabri Hajdini , Emrah Nikerel

Department of Engineering and Food Technology, University of Mitrovica “Isa Boletini”, Mitrovica, Kosovo

Department of Genetics and Bioengineering, Yeditepe University, Istanbul, Turkey

Current address: Faculty of Education, University of Prizren, “Ukshin Hoti”, Prizren, Kosovo

Received: 10/02/2020

Accepted: 03/04/2020

Published: 20/05/2020

Abstract

Soil samples from different locations in Kosovo were screened for bacteria suitable for high-level production of various proteases. 5

isolates were found and selected to be promising candidates and further characterized by biochemical and morphological assays as well as

6S RNA sequencing and identified as Bacillus spp. The isolates were used for protease production via submerged fermentation. The

produced enzymes were tested for different food industry applications, like meat, beer, milk, and feather degradation. The highest protease

activity achieved was 0.63U mL-1 at 37 C pH 4.7 from strain S10-1 which showed high potential for meat industry application.

Keywords: Bacillus spp.; Enzymes; Fermentation; Industrial biotechnology

meat. Alternative to these, enzymatic treatments provide

Introduction¹

affordable, less energy intensive and faster solution for the meat

processing applications by hydrolyzing myofibrils due to

proteolysis (2, 10).

Enzymes are functional proteins, responsible for governing

biochemical reactions in living cells by e.g. lowering the

activation energy in biochemical reactions (1). These can

selectively and effectively convert several substrates molecules to

products even within complex matrix. Typically produced by

fermentation and extracted from living cells, enzymes are

biodegradable compounds making them also environmentally

benign (2). Enzymes are used in many industries like

pharmaceutical (3), detergent (4), leather (5), and food industry

For the beverage industry, proteases are used typically for

clarification of wine, beer or juices, often in combination with

other enzymes (11, 12). In particular for beer, haze in beer usually

and mainly results from a reaction between beer proteins from

grains (barley or wheat) and polyphenols, responsible for 40-75%

of the beer haziness (13). The concentration of only 2mg L-1 of

protein in beer causes haze formation. Enzymatic protein

hydrolysis is one of the possibilities to enrich the beer with

nutrients and prevent haze formation thereby improving

organoleptic properties. In dairy industry proteases are mainly

used in milk coagulation process by cleavage the peptide bond of

κ-casein Phe105-Met106. Coagulated milk is destined for

different type of cheese making. Lately, the effect of proteases in

cheese ripening process and indication in texture and flavor of the

cheese was reported (14, 15).

(

6). Chief among the commercial enzymes are proteases, which

hydrolyze peptide bonds within proteins. These are widely used,

with over 60% of the enzyme market (7) and growing at a

compound annual growth rate of 5.3% of global demand during

014-2019 (6) and is expected to grow further (8).

Proteases are heavily used in food industry e.g. meat

processing applications, where the focus is on providing tools to

prepare ready-to-use, high-quality, tender meat and meat

products. Meat tenderness is a function of the fibrous nature of

the muscle among them myofibrils are responsible for meat

toughness (9). Conventionally, aging the carcasses, mechanical

Proteases find application in treatment of byproduct from

food industry. Poultry is one of these industries, where wastes are

increasing every day with increment in the demand for poultry

products. Feathers contain 91% keratin, which is an insoluble

structural protein and is found in β-sheets form. Their

tenderization,

elevated-temperature

storage,

electrical

stimulation, calcium chloride injection, are some of the

techniques used by slaughterhouses to increases tenderness of

Corresponding author: Emrah Nikerel, Department of Genetics and Bioengineering, Yeditepe University, Istanbul, Turkey. E-mail:

emrah.nikerel@yeditepe.edu.tr.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 687-693

biodegradation is challenging due to extensive disulfide bonds

and cross-links, hydrogen bonds and hydrophobic interactions

(

16). Conventional methods include incineration, (excessive) use

of alkaline solutions or burying. Proteolytic activity of enzymes

provide environmentally benign solution to feather processing.

Ubiquitous in nature, proteases are industrially produced

especially by animals, plants, and microorganisms. In particular,

microorganisms are used due to available cultivation practice in

large quantities in a short time as well as high and stable activity

of the enzyme produced (17,18,19). Among those, bacteria are

preferred organisms, since these typically grow and produce at

higher rates, and have large substrate portfolio (20). Additionally,

bacterial enzymes especially from Bacillus spp. show higher

thermal stability than fungal counterparts (21). Furthermore,

genome of bacteria is typically 2-5Mb, significantly smaller than

the genome of fungi around 8-17Mb (22, 23, 24), rendering

molecular characterization or manipulation easier to carry.

Enzyme production using genetically modified organisms

(GMOs) are also common practice due to available molecular

biology toolbox, possibility of tailor-made products as well as

well-established fermentation practice for known industrial

workhorses, reaching high product titers (25,26). However, the

risk for unpredicted outcome of transformation exists, additional

DNA fragments inserted with the gene of interest or gene

sequences rearrangement and deletion (27, 28). Owing to the

highly regulated market rules for foods with GMO ingredients,

the food industry endeavors to avoid using GMO technology if

wild type strains are available (29). Following that, several studies

have been performed for the isolation of protease production

microorganisms from natural sources in an attempt to get

production strains as well as to exploit natural diversity for

enzymes of various functional, desired properties. Typically,

extreme environments are explored to isolate microorganisms,

including hot spring waters, high altitude areas, arable land,

industrially polluted areas, etc.

Kosovo is located in southeastern Europe, with

predominantly continental climate influenced also by

Mediterranean and Alpine climate. The country possesses diverse

territory surrounded by mountains, arable fields, rich areas with

thermal hot water springs, and mines with high deposit of lead,

zinc, silver, nickel, and cobalt. On top of it, it is the third largest

lignite reserve in Europe. To our knowledge this region is not that

much explored for its microbial ecology, hence the aim of this

research is to screen soil samples from different environments in

Kosovo for bacteria with ability to produce protease

extracellularly, and examine their application in different food

industries.

Figure.1: Map of soil sample points in Kosovo

Single colonies with surrounding clear zone around them

were selected and streaked into fresh agar for further investigation

(30, 31). All chemicals used are purchased from Sigma Aldrich

or Merck. Bacillus subtilis 168 ATCC 23857 is used as reference

microorganism.

2.2 Characterization of Bacillus spp. colonies Isolation and

selection

Morphological and cultural characteristics were used to

identify colonies. Each colony was examined using microscopy

and rod shape cells identified as potential Bacillus spp. The Gram

staining (Beveridge, 2001) (32) and catalase test (Taylor and

Achanzar, 1972 (33) were used to further characterize the

colonies. Semi-quantitative protease activity of each colony was

measured in duplicate data, where colonies were inoculated with

sterile needles on 5% skimmed milk agar. The radius of bacterial

colony and radius of halo formed after 24h were measured using

image processing software GIMP 2.10.12. The area of the halo

around the culture was used to calculate protease index (Eq. 1).

퐴푇퐻

푃퐼 = _퐴_퐶

(1)

where PI is the protease index, ATH is the area of total halo, AC

the area of the bacterial colony. Colonies with large ATH and

small AC are considered to be candidate host for further

characterization using liquid culture.

Materials and methods

.1 Isolation and selection

Soil samples from different locations corresponding to

different environments (high altitude, heavy metal contaminated

area, hot spring water, food industry zones) in Kosovo (Fig. 1)

were collected and stored at +4 C until further screening was

performed. During the sampling, all used equipment were

sterilized beforehand.

To isolate the microorganisms, one gram of soil sample was

mixed with 9mL sterile 0.9% saline solution and incubated at

.3 Cultivation process and determination of protease activity

A single colony from an agar plate is grown for 18hr in 10mL

nutrient broth in a 50mL falcon tube to be used as inoculum. The

-1

final fermentation is carried in YPD medium with salt g L :

2 4 2 4

peptone, 10; glucose, 5; yeast extract, 5.5; KH PO , 1; K HPO

O, 3.7; MgSO .7H O, 0.1; (34, 35). To assess the effect of

50rpm for 15min at room temperature. Out of this mixture,

temperature, two different incubation temperatures (30 and 37 C)

are used while the pH was kept at 7; and to assess the effect of

00μL is further diluted and spread on the skim milk agar plates.

The plates were incubated at 37 C for 24h.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 687-693

pH, the fermentations are carried at three different pH (5, 7 and

difference is noted as hydrolysed protein (13). The same

experiment is repeated with reference enzyme (Protease from

Aspergillus oryzae Sigma cat Nr: P6110), where 5mL of enzyme

is used instead of crude enzyme, keeping all other conditions

same.

) while the temperature was kept at 37 C. The cultures are

sampled 24h for growth (measured by optical density at 600nm

OD600) and protease activity. Protease activity was measured by

casein method (36), with 0.65% casein (w/v) in potassium

phosphate buffer at pH 7 or sodium acetate buffer at pH 5. The

assay conditions were set to 10 min at 37 C and terminated using

2.5.3 Milk-clotting activity

0% trichloroacetic acid. The tyrosine released is determined

The milk-clotting activity was determined according to the

method of Iwasaki (42): 0.1mL of the crude enzyme was mixed

using Folin’s phenol reagent (36). One unit of protease activity

was defined as the amount of enzyme liberating 1μmol of tyrosine

per minute under defined assay conditions.

well with 1mL of skim milk solution (10g skim milk powder in

100mL of 0.01M CaCl

solution) and incubated at 37 C for 1h

and 30min. Each 10 min tubes were checked for clotting

formation. Obtained coagulation time is used for calculating milk

clotting units, where one unit of milk clotting activity was defined

as the amount of enzyme capable of coagulating 1mL of milk for

40 min, Eq. (2).

.4 16S rRNA sequencing and analysing

Sequencing ribosomal RNA (rRNA) 16S is well-accepted

method to identify bacteria, yielding species-specific signature

37, 38). Bacterial DNA from the liquid culture was extracted

(

using commercial kit (high pure PCR template preparation kit,

Roche) and was used as a template for PCR amplification of 16S

rRNA sequence. PCR reaction consist of: 10X Taq buffer

푀퐶푈

푚퐿

2400푠

(2)

(푇ꢀ푚푒(푠)∗푣표푙푢푚푒표푓푡ℎ푒 푒푛푧푦푚푒 ꢀ푛 푚퐿)

Thermo-scientific (100mM Tris-HCl, 500mM KCl, 0.8% (v v )

Nonidet P40), 25mM MgCl , dNTP 0.4mM, universal primers

7F (5’-AGAGTTTGATCATGGCTCAG-3’) and 1492R (5’-

GGATACCTTGTTACGACTT-3’) each 0.4µM, Taq polymerase

.5.4 Enzymatic treatment of feathers

The effect of the culture on feather degradation from isolated

-1

.05U μL and the DNA template 100ng. The regime of the

colonies was examined. The method was based on the work of

Kaya and Nikerel (43) with small changes. In a 50mL of 48h

grown culture, 1.2g of washed and autoclaved feather was added

and the mixture is incubated at 150rpm, 37 C for 12h. After 12h

content was filtrated using filter paper, dried and the difference in

the dry feather considered as degraded by the enzyme.

temperature used was, first denaturing at 95 C for 3min, followed

by 31 cycles of denaturation at 95 C for 0.30min, annealing at

o o

5 C for 1min, extension at 72 C for 1min, and a final extension

o o

step at 72 C for 10 min, then storage temperature at 4 C (39). The

amplified 16S sequence was purified using the Macherey-Nagel

PCR purification kit and sequenced. The forward and reverse

sequences were assembled using BioEdit version 7.0.5.3. The

resulting sequences were BLAST’ed against GeneBank Database

for 16S ribosomal RNA sequences (Bacteria and Archaea).

3 Result and discussion

To isolate and identify Bacillus spp. capable of producing

extracellularly protease, 102 soil samples from 34 different areas

in Kosovo (Fig. 1) were screened. Out of initial sample pool, 66

single colonies were selected based on formation of clear halo in

skim milk agar. The colonies were further identified as potential

Bacillus spp. based on the form and color of colonies in nutrient

agar, as well as based on Gram staining and catalase test as

morphological tests. All identifications were compared with the

reference strain B. subtilis 168. From the 66 colonies only 29

selected colonies resulted Gram-positive rod shape

microorganisms and out of which, 28 colonies were catalase-

positive (44). Remaining colonies were subjected to semi-

quantitative protease activity measurement in skim milk agar, to

select best extracellular protease producers.

.5 Application of produced enzymes in the food industry

The enzymes are tested in various food industry applications,

ranging from meat to beer processing to feather meal preparation.

The details are explained in this section.

.5.1 Breakdown of meat myofibrils

Myofibril extraction was performed with fresh cow muscle

tissue by using extracting buffer (100 mM KCl, 20 mM potassium

phosphate (pH 7.0), 1 mM EDTA, and 1 mM sodium azide) at

C based on the method described by of OLSON & al. (40).

.95mL of extracted myofibril at pH 5.5 (approximately the pH

of the red meat) was aliquoted in a 15mL falcon tube and assayed

Based on semi quantitative protease index results, five

colonies were selected (Fig. 2). Protease index takes into account

both the biomass formed and the proteolytic activity. S7-1 has

small colonies, yet large halo resulting highest protease index, and

despite S21-1 has about the same halo size, it also has largest

biomass colony, therefore lowest protease index. Interestingly,

protease index for S7-1 was 22 fold higher than the index of B.

subtilis 168. These 5 colonies were further used for enzyme

production by submerged fermentation, during which total DNA

is isolated for final identification by sequencing genomic region

encoding 16S rRNA.

with 50µL crude enzyme extracts. The hydrolysis takes place in

an orbital shaker 150rpm at 37 C for 1h and terminated with 2mL

of 15% trichloroacetic acid. The mixture was cooled at room

temperature for 15minute and centrifuged at 1000 xG for 15min.

Finally the optical density of the supernatant was determined at

80nm (41). The same experiment is repeated with reference

enzyme (Protease from Aspergillus oryzae Sigma cat Nr: P6110),

where 50µL of enzyme is used instead of crude enzyme, keeping

all other conditions same.

.5.2 Beer haze treatment

5mL of hazy beer was treated with 5mL of crude enzyme (or

3.1 Identification by 16S sequencing

distilled water for blank), incubated at 25 C for 18h. Pre-weighted

DNA samples were isolated and genomic regions encoding

16S rRNA were sequenced. Results sequences were BLAST’ed

against 16S ribosomal RNA sequences (Bacteria and Archaea)

filter paper 0.45µm were used to filtrate the treated beer. After

two hours of drying at 65 C filter paper was re-weighed and the

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 687-693

database in NCBI (Table 1), with all 0.0 e-value and percent

identity ranging between 88-99%.

at an altitude of around 1800m. Soil from this region exceed the

limits for heavy metal like Pb, Cd, and Zn as reported by

Kelmendi (48). Under mesophilic conditions (pH 7, 37 C) the

highest activities were observed with strains S37-2, and S6-4 with

.54 and 0.3U mL-1, respectively (Fig. 3d). Neutral proteases

produced by Bacillus spp. are important due to generation of less

bitterness hydrolysates from proteins. This is significant a major

factor for food industry applications in particular for beverage

industry where enzyme application significantly effects

organoleptic characteristics.

Table 1: BLAST results of the assembled sequences with NCBI

6S ribosomal RNA sequences (Bacteria and Archaea) database

S. name Closest match e-value (%) Identity

S6-4

S7-1

S10-1

S21-1

S37-2

B. pumilus ATCC 7061

B. albus MCCC 1A02146

B. cereus ATCC 14579

Bacillus albus strain BPB24 16S 0.0

B. mobilis MCCC 1A05942 0.0

0.0

97.92

89.29

88.23

97.9

99.02

The organism S37-2 is isolated from walnut plantation soil in

Rahove in north part of Kosovo and S6-4 is isolated from highly

mineralized hot spring water at Kllokoti with 3.6g L-1 minerals

reported by Kosovo Environmental Protection Agency

(2010)(49). Lastly, contrary to our expectations and several

reports in literature on alkaline protease production by Bacillus

spp., isolated strains failed to grow and produce well at pH 9 and

7 C (Fig. 3. f).

.3 Myofibril hydrolysis

To mimic meat processing application, the crude extract from

culture supernatants at two different time points were used for

myofibril hydrolysis and compared with the reference protease.

Among the tested cultures, sample at the 48th hour from S10-1

culture exhibited the highest myofibril hydrolysis, nearly 10% of

the commercial enzyme, which is considered to be promising as

crude extract ought to be further purified before final use (Fig.

4a). The sample at 24th hour from S7-1 exhibited lowest

myofibril hydrolysis, possibly due to low biomass level.

Figure 2: a) Selected colonies in 5% skim milk agar for extracellular

protease expression, b) Protease index of each colony calculated by using

the area of colony and the total halo formed. The protease index for S7-1

is much higher than the rest and is shown with a different axis

.4 Hazy beer treatment results

Here, the supernatant from five different cultivations, at two

.2 Enzyme production and activity

Selected species are cultivated at different pH and

different times (24 and 48h) were used as crude extracts for

treatment of beer haziness (Fig. 4b). The total amount of

proteinaceous material in untreated beer used for experiment was

estimated to be between 346 to 525 mg L-1, in agreement with

literature (13). Sample from cultivation of strain S37-2 at 48h

showed the highest beer clarification action, with 212 mg/L haze

protein degradation (nearly 61% decrease in residue) compared

with the result of commercial enzyme with 335mg L-1 haze

protein degradation.

Temperatures. Overall, the highest growth and activity was

obtained from S10-1 grown at pH4.7 at 37 C with the highest

protease activity 0.63U mL-1 Fig. 3b). Protease activity at low pH

is interesting for food industry applications, where acid proteases

are always in high demand (45). Albeit being uncommon,

production of acid proteases by Bacillus spp. is reported (46),

even though Bacillus spp. are commonly known for neutral and

alkaline protease production (47). S10-1 was isolated from

agriculture soil used as source of forage grass from Bajgora region

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 687-693

Figure 3: a) growth and b) activity at pH 4.7 and 37 C; c) growth and d) activity at pH 7 and 37 C; e) growth and f) activity at pH 9 and 37 C; g) growth and

h) activity at pH 7 and 30 C

.5 Milk clotting results

Milk clotting assay was performed with supernatant from

processing, while protease from S37-2 performed better in haze

treatment in beer. Strains S7-1 and S21-1 showed moderate milk

clotting activity, while S6-4 showed promising results in feather

treatment. In conclusion these five strains have a potential for

industrial application.

isolated strains, crude enzyme form cultures S7-1 and S21-1

coagulated the milk. However, the time of coagulation was 1h

0min calculated in milk clotting unit 4.44MCU mL-1. While, for

supernatant from B. subtilis culture took only 45minutes to

coagulate the milk or 8.89MCU mL-1 calculated in milk clotting

unit.

Aknowledgments

This research was funded by Ministry of Education, Science

and Technology of Kosovo based on the project “Isolation of

Bacillus bacteria from soil sample for production of protease from

Food Industry residues” with project number 2-3970-3 and was

realized as cooperation between University of Mitrovica “Isa

Boletini” and Yeditepe University. We thank Petrit Idrizi, Bardha

Hyseni, Valdrin Ramadani and Diellza Zejnullahu at University

of Mitrovica “Isa Boletini” for their help in the practical work and

to Filiz Erçelik from Yeditepe University for helping in molecular

characterization.

.6 Enzymatically feather degradation

In our research we assessed the ability of 48h grown cultures

form five isolated strains for degradation of feathers. Results

showed that S6-4 biodegraded the highest amount of feather

above 41% (Fig. 4c). This shows a good possibility for this culture

to find application in feather biodegradation from poultry

industry.

Conclusion

Five different strains were isolated from different region soils

Ethical issue

in Kosovo and identified as a good production host of protease

enzyme. All isolated strains were characterized as Bacillus sp.

strain by using both morphological and molecular techniques.

Interestingly, there is no single strain that produces best protease

for all applications, rather all five strains are promising for

different applications. S10-1 has shown good activity for meat

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

(avoidance of guest authorship), dual submission, manipulation

of figures, competing interests and compliance with policies on

research ethics. Authors adhere to publication requirements that

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 687-693

submitted work is original and has not been published elsewhere

in any language.

Authors’ contribution

All authors of this study have a complete contribution for data

collection, data analyses and manuscript writing

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