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Isolation of Gram –ve proteolytic bacteria
from milk waste and study its sensitivity
against some antibiotics By
Ali Hussein Shabi
Student ID:201111487
B.Sc. student, Biology Department
Faculty of Science, Jazan University
Abstract
Gram –ve bacilli, protease producing was isolated. Sample was collected
from milk wastes; spoiled residue milk. Skim milk nutrient agar media was
used for qualitative screening for protease using streaking method. Because
of partial hydrolysis of milk casein, colonies forming transparent zones were
selected. Purified colonies of selected isolate was streaked on Nutrient agar
slant and stored at 4°C. After being incubated for 24 hrs, a plate containing
milk and agar showed the growth of several colonies. The zone formations
around the bacterial colony indicated the protease positive strain which may
be due to hydrolysis of casein. It was chosen one strain from the plate
showed the highest number of enzyme producers followed by the clear zone.
It was observed that, the production of protease was tolerant up to 45°C. The
potent protease producer was found in G-ve Streptobacilli Bacterium isolate.
This bacterial isolate was resistant to antibiotic cefidime and sensitive to
trimethoprim/sulphamethoxazole and cefaclor.
1
1.Aim of the work
This study is aimed to isolate a bacterial strain can able to secrete useful
products from proteineous waste as milk. The study of their sensitivity
against antibiotics and partial identification was carried out.
2.Introduction
2.1. Thermophilic Bacteria
A thermophile is an organism of extremophile that thrives at relatively high
temperatures, between 45 and 122 °C (113 and 252 °F). Thermophilic
eubacteria are suggested to have been among the earliest bacteria.
Thermophiles are found in various geothermally heated regions of the earth,
such as hot springs and deep sea hydrothermal vents, as well as decaying
plant matter, such as compost. As a prerequisite for their survival,
thermophiles contain enzymes that can function at high temperatures. Some
of these enzymes are used in molecular biology (for example, heat-stable
DNA a polymerases for PCR), and in washing agents (for example
proteases, amylases and lipases). Thermophiles are classified into obligate
and facultative thermophiles: Obligate thermophiles (also called extreme
thermophiles) require such high temperatures for growth, whereas
facultative thermophiles (also called moderate thermophiles) can thrive at
high temperatures, but also at lower temperatures (below 50°C) [1].
2
2.2. The milk waste
Milk and microorganisms have long and interesting associations. Foods as
milk are not only nutritious to consumers, but are also excellent source of
nutrients for microbial growth. Depending upon the microorganisms present,
milk may spoil or preserved by fermentation. Microorganisms can be used
to transform raw milk into fermented delights. Foods as milk also can act as
a reservoir for disease transmission, and thus detection and control of
pathogens and spoilage organisms are important areas of food microbiology
[2].During cold storage after milk collection they dominate the flora, and
their extracellular enzymes, mainly proteases and lipases, contribute to the
spoilage of dairy products. The extracellular enzymes can resist
pasteurization and even ultrahigh temperature processing. Also the
sensitivity of milk’s fat and protein to physical-chemical alterations can also
lead to deterioration, thus, reducing its quality [3].
2.3. Gram –ve bacteria and public health
Gram - negative bacteria are bacteria that do not retain crystal violet dye in
the Gram staining protocol. Compared with gram-positive bacteria, gram-
negative bacteria are more resistant against antibiotics, despite their thinner
peptidoglycan layer, because of their additional, relatively impermeable lipid
membrane. The pathogenic capability of gram-negative bacteria is often
associated with certain components of gram-negative cell envelope, in
particular, the lipopolysaccharide layer. Antibiotic resistance can cause
serious diseases and is an important public health problem [4, 5]. As showed
in the next table are the genera of gram –ve bacteria that contain the most
important human pathogenic bacteria species [6].
3
Genus Important species Shape
Bordetella Bordetella pertussis Small coccobacilli
Borrelia Borrelia burgdorferi Spirochete
Brucella
Brucella abortus
Brucella canis Brucella melitensis Brucella suis
Small coccobacilli
Campylobacter Campylobacter jejuni
S-shaped bacilli with single, polar flagellum
Escherichia Escherichia coli Short rods (bacilli)
Francisella Francisella tularensis Small, pleomorphiccoccobacillus
Haemophilus Haemophilus influenzae
Ranging from small coccobacillus to long, slender filaments
Helicobacter Helicobacter pylori
Curved or spiral rods pultiple polar flagella
Legionella Legionella pneumophila
Slender rod in nature, cocobacillary in
laboratory. monotrichious flagella
Leptospira Leptospira interrogans
Long, very slender, flexible, spiral- or corkscrew-shaped rods
Neisseria
Neisseria gonorrhoeae Neisseria meningitidis
Kidney bean-shaped
Pseudomonas Pseudomonas aeruginosa rods
Rickettsia Rickettsia rickettsii Small, rod-like coccobacillary
Salmonella
Salmonella typhi
Salmonella typhimurium Bacilli
Shigella Shigella sonnei rods
Treponema Treponema pallidum
Long, slender, flexible, spiral- or corkscrew-shaped rods
Vibrio Vibrio cholerae
Short, curved, rod-shaped with single polar flagellum
Yersinia Yersinia pestis Small rods
2.4. Proteases
Proteases represent one of the three largest groups of industrial enzymes and
account for about 65% of the total worldwide sale of enzymes. Proteolytic
4
enzymes catalyze the cleavage of peptide bonds in polypeptides and proteins
and resolve racemic mixtures of amino acids [7].
Proteases are physiologically necessary for living organisms, they are found
in a wide diversity sources such as plants, animals, and microorganisms.
Papain , bromelain, and keratinases represent the known proteases of plant
origin. The use of plants as a source of proteases is governed by several
factors such as the availability of cultivated land and the suitability of
climatic conditions for growth. The most familiar proteases of animal origin
are trypsin, chemotrypsin, pepsin, and rennins. Their production depends on
the availability of livestock for slaughter which is governed by political and
agricultural policy. The inability of the plant and animal proteases to meet
current world demands has led to an increased interest in microbial
proteases. Microorganisms represent an excellent source of enzymes owing
to their broad biochemical diversity and their susceptibility to genetic
manipulation. Microbial proteases account for approximately 40% of the
total worldwide enzyme sales. Proteases from microbial sources are
preferred since they possess almost all the characteristics desired for their
biotechnological applications [8].
Proteases can be used in many industrial applications, such as laundry
detergent, leather preparation, meat tenderization, peptide synthesis, food
industry, dehairing process, pharmaceutical industry; as well as in
bioremediation process. Proteases are also used in textile industry for
removing the stiff and dull gum layer of sericine from the raw silk fiber
leading to its brightness and softness. An interesting role in the
decomposition of gelatinous coating of X-ray films from which silver was
recovered [7, 9].
5
3. Materials and methods
3.1. Media
Nutrient agar medium containing 5 g peptone , 3 g beef extract , 5 g NaCl
and 15 g agar agar per liter was prepared [10]. The pH was adjusted to pH
7.0 by NaOH. Skim milk nutrient agar; is nutrient agar supplemented with
5% skim milk. Skim milk was sterilized separately and mixed just before
solidification. Physiological saline solution contained 8.5 g NaCl per liter
[11]. All media, saline solution and tools were sterilized at 115 °C using
autoclave.
3.2. Isolation of bacteria
Samples were collected from milk wastes (Figure 1). One ml of sample was
added to a sterilized test tube containing 9 ml sterilized saline solution.
Serial dilution in physiological saline solution has been done for mixed
samples and each dilution was used to inoculate the nutrient agar medium by
plating method as described by Aftab et al. (2006) [12]. Agar plate medium
containing 25ml solidified medium was inoculated with 100 microliter from
6
each dilution. These plates were incubated at 37°C for 24 h. The plates that
showed considerable single colonies were selected for this purpose.
Figure (1):
Shows the source of isolation; milk waste.
3.3 Determination of viable count
Viable bacterial count (colony forming units, CFU) was carried out as
described earlier [11]. One ml of the bacterial culture was taken and diluted
with presterilized physiological saline solution (under aseptic conditions) to
reach 10-4
dilution. A hundred µl of each dilution was plated on nutrient
7
milk plates, incubated overnight at 37 °C and the number of developed
colonies was processed to obtain the colony forming units per ml culture.
3.4. Qualitative screening of protease enzyme strain
Skim milk nutrient agar media was used for qualitative screening for
protease using streaking method at 30, 37 and 45°C. It used to detect
protease-producing isolates [13]. Colonies forming transparent zones,
because of partial hydrolysis of milk casein, were selected. Purified cultures
of selected isolates were streaked on Nutrient agar slants and stored at 4°C.
3.5. Demonstration of bacterial shape and Gram staining type
Preparation of a bacterial smear. Ten μl of sterile water in the center of a clean glass slide was placed. A pure
protease-producing bacterium was mixed with water drop on slide. The
water-bacteria mixture was spreaded over an area of about 1 inch square
then allowed to air dry. The slide was hold with forceps and heated on the
hot plate for several minutes [14].
Staining
The smear was covered with a few drops of crystal violet and left for 1
minute. The slide was washed carefully over the top with distilled water
until no large amounts of color wash off. The smear was covered with
Gram’s iodine and left for 1 minute. The smear was decolorized with 95%
ethyl alcohol (Decolorizer) then the slide was washed carefully over the top
with distilled water from a wash bottle until no large amounts of color wash
off. Immediately the slide was rinsed with distilled water. The smear was
8
covered with safranin and left for 1 minute followed with gently rinsed with
distilled water then blot dried with a paper towel. The specimen was
observed under the microscope to differentiate is it Gram positive or
negative bacterium and also to know the shape of the isolate [14].
3.6. Effect of temperature
Bacterial cells was activated by growing them overnight on milk agar plate
at 37 °C. Single colony was streaked on 3 plates at 30, 37 and 45°C for
24hrs.
3.7. Disc diffusion sensitivity
Using an asceptic technique, a single colony of the isolate was picked using
a sterile loop. The nutrient agar was streaked .The plate was allowed to dry
for approximately 5 minutes. A filter-paper disk contained the antibiotics;
trimethoprim/sulphamethoxazole, cefaclor and cefidime separately were
dispensed onto the plate. Using a flame-sterilized forceps, each disc was
gently pressed to the agar to ensure that the disc is attached to the agar.
Plates should be incubated overnight at an incubation temperature of 37°C
[15].
9
4. Results and discussion
Enzymes are organic catalysts involved in vital biochemical reactions and
play a major role in a variety of industrial processes. Microbial enzymes
were discovered and used in industries as early as 20th century. Since then,
Their use in technical and industrial processes has increased tremendously.
Microbial enzymes are superior to any other sources for commercial
applications. Among hydrolytic enzymes, microbial proteases are a group of
most important and extensively studied enzymes. Proteases are highly stable
and have been found to be more suitable for a wide range of applications
owing to their high activity and stability in extreme physiological conditions
such as high pH, temperature and inhibitory compounds such as detergents.
Microbial proteases belong to acid, neutral or alkaline based on their pH
optimum for activity and the active sites. Most of the commercially used
proteases are produced by Bacillus, Pseudomonas, Clostridium, Proteus
species and also fungi. Among these, Bacilli is the major protease producer
with application in pharmaceuticals, food, leather processing, medicine,
10
molecular biology, tannery, detergent, metal recovery and peptide[16].
During the recent years, efforts have been directed to explore the means to
reduce the protease production cost through improving the yield, and the use
of either cost free or low cost feed stocks [17].
The largest structures in the fluid portion of the milk are "casein micelles":
aggregates of several thousand protein molecules with superficial
resemblance to a surfactant micelle, bonded with the help of nanometer-
scale particles of calcium phosphate [18].So, milk fermentation considers
one from the sources of the proteolytic enzymes; where during cold storage
after milk collection they dominate the flora, and their extracellular
enzymes, mainly proteases and lipases, contribute to the spoilage of dairy
products [3].
Isolation and screening of protease producing bacteria
Isolated bacterial strains were screened for protease producing ability on
skim milk agar. After being incubated for 24 hrs, a plate containing milk and
agar showed the growth of several colonies. The zone formation around the
bacterial colony indicated the protease positive strain which may be due to
hydrolysis of casein. Hence the strains were identified as a protease producer
and it was taken for further experimental studies. Figure 2 shows the total
bacterial strains isolated from milk waste incubated overnight on 37°C as
described in the materials and methods. The count reached to 5.4x105
CFU/ml. When some resulted colonies were screened on nutrient agar
supplemented with skim milk; it was found that one strain showed the
highest number of enzyme producers the clear zone (Figure 3). To study the
effect of temperature as mentioned in the materials and methods; it was
11
observed that; when incubated over night at 37°C was ˃ or = at 30°C. It was
tolerant at 45°C (Figure 4).
Fig 2: Represents plating of the milk waste on Nutrient milk agar with 10-3
dilution with count 5.4x105 CFU/.
Fig (3): Represents Qualitative screening for protease by different colonies
isolated from milk waste incubated at 37 °C.
12
Fig (4):Represents Qualitative screening for protease by the isolates from
milk waste and incubated at A)37,B) 30 and C)45 °C respectively ; using
skim milk nutrient agar medium.
A B
C
13
The potent protease producer was found in G-ve Bacterium bacilli singles
and aggregates in chains known as streptobacilli isolate (Figure 5).
Fig. (5): Shows that; the protease bacterium isolate from spoiled milk is
gram -ve bacilli.
A
14
The G-ve bacilli isolate gave was resistant against some antibiotics and
sensitive against some others as mentioned in the materials and
methods.(Figure 8).
Fig. (8): Shows that; the effect of different antibiotics on the G-ve bacilli
bacterium isolated from spoiled milk, where A) cefaclor, B)
trimethoprim/sulphamethoxazole, and C) Cefidime.
A
B
C
15
5- Conclusion
1-When milk kept out the refrigerator, some pathogenic Gram –ve bacteria
can spoiled it.
2-In this case, the isolated bacteria secrets extra-enzymes as lipase and
protease to lyses the casein.
3-Due to protease represents 65 % from the enzymes importance and sale
worldwide and for easing of getting it from microorganisms, we can
use this enzyme after extraction and purification in many industries as
laundry detergent, leather preparation, meat tenderization, peptide
synthesis, food industry, dehairing process, pharmaceutical industry;
as well as in bioremediation process.
6. References
1. Takai T. (2008): Cell proliferation at 122°C and isotopically heavy
CH4 production by a hyperthermophilic methanogen under high-
pressure cultivation. PNAS.105 (31):10949–51.
2. Goff, H. D., & Griffiths, M. W. (2006): Major advances in fresh
milk and milk products. Journal of Dairy Science, 89, 1163-1173.
3. Bali,O.S., Imène F., Rouaa L., Hamadi A.& Ayadi,M.A.(2013):
Study of Proteolytic and Lipolytic Activities of Pseudomonas spp.I
solated From Pasteurized Milk in Tunisia. Journal of Agricultural
Science, 5(7).
16
4. Baron S, Salton MRJ, Kim KS (1996). "Structure". In Baron S et
al.. Baron's Medical Microbiology(4th ed.). Univ of Texas Medical
Branch. ISBN 0-9631172-1-1. PMID 21413343
5. http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/
Antibiotic_resistant_bacteria
6. http://en.wikipedia.org/wiki/Pathogenic_bacteria
7. Anwar, A., and M. Saleemuddin (1998): Alkaline proteases: A
review. Bioresource Technology. 64: 175 – 183.
8. Rao, M.B., A.M. Tanksale, M.S. Ghatge, and V.V. Desh pande
(1998): Molecular and biotechnological aspects of microbial
proteases. Microbiology and Molecular Biology Reviews. 62 (3): 597
– 635.
9. El-Shafei H, Abdel-Aziz M, Ghaly M, Abdalla A (2010):
Optimizing some factors affecting alkaline protease production by a
marine bacterium Streptomyces albidoflavus. Proceeding of fifth
scientific environmental conference, Zagazig University,125 – 142
10. Ganesh, A., S., Swarnalatha, S., Gayathri, N., Nagesh, and G.,
Sekaran (2008): Characterization of an alkaline active – thiol
forming extracellular serine keratinase by the newly isolated Bacillus
pumilus. Journal of Applied Microbiology 104:411–419
11. Pelczar, M.J., and E.C. Chan (1977): Laboratory Exercises in
Microbiology, 4th edition, McGraw Hill, Inc.
12. Aftab, S.; Ahmed, S., Saeed, S. and Razoo, S.A. (2006): Screening,
isolation and characterization of alkaline protease producing bacteria.
Pak. J. Biol. Sci. 9:2122-2126.
17
13. Hanaa A. El-Shafei1, Mohamed S. Abdel-Aziz1, Mohamed F.
Ghaly and Ahmed A. H. Abdalla(2010): Optimizing some factors
affecting alkaline protease production by a marine bacterium
Streptomyces albidoflavus. Proceeding of fifth scientific
environmental conference, Zagazic Uni., 125 - 142
14. Maryland University. (2000): The Gram Stain. Pathogenic
microbiology.
http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/Gr
amStain.htm
15. Cheesbrough M.(2000): District laboratory practice in tropical
countries, part 2Cambridge,university press
16. Vijayalakshmi S., Venkat Kumar S. and Thankamani V (2013):
Optimization and Cultural Characterization of Bacillus RV.B2.90
producing Alkalophilic Thermophilic Protease. Research Journal of
Biotechnology. Vol. 8 (5):37-43
17. Kuberan, T., S. Sangaralingam, and V.Thirumalaiarasu (2010):
Isolation and optimization of Protease producing Bacteria from
Halophilic soil. J. B iosci. Res., 2010. Vol. 1(3):163-174
18. Fox, P. F. (1995): Advanced Dairy Chemistry, Lactose, Water, Salts
and Vitamins. 2nd ed. Chapman and Hall: New York,vol.3
18
المستخلص العربى
مخلف الحليبم عزل ساللة بكتيرية منتجة النزيم البروتييز و مقاومة لبعض المضادات الحيوية من ت
لمدة يوم خارج الثالجة لنمو البكتيريا ومن ثم عزلها امن مخلف الحليب بعد تركه ةعين أخذتم وقد
جار اآل و الحليب المقشودب الوسط الغذائى المدعماستخدم قد و . على اطباق وسط مغذى االجار
وقد تم . باق االجارزراعة البكتيريا بطريقة التخطيط على اطباستخدام طريقة يزلبروتيالفحص
ساللة أختيرتقد .كازين الحليبلتشكيل مناطق شفافة، وذلك بسبب التحلل ذات اختيار المستعمرات
بعد أفضل ساللة بكتيرية منتجة لالنزيمتم حفظ . من المخلف نزيملال انتاجواحدة أظهرت أكبر
73 بالمقارنة بدرجة مئوية 73 ان درجة الحرارة المثلى هىلوحظ . درجة مئوية 4 تنقيتها فى
بصبغ السالله .درجة مئوية 44لكن يستطيع الميكروب المعزول أن يتحمل حتى درجة مئوية
و . الناتجة النزيم البروتييز بصبغة كريستال فايلوت وجد انها عصوية الشكل و سالبة صبغة جرام
السيفيدين و حساسة لبعضها مثلمثل قد وجد ان السالة المعزولة مقاومة لبعض المضادات الحيوية
trimethoprim/sulphamethoxazole , cefaclor .إنتاج يمكن االستفادة من المخلف ب عامة
، واألحماض الذائبة اتبروتينالة مثل صناعة يدشجع قيام صناعات عدو من ثم ي زييالبروت انزيم
معالجة األدوية، ودباغة الجلود، صناعة ,مساحيق الغسيلوصناعة األلبان، و تطرية اللحوماألمينية
.إلى مركبات مفيدة المخلفات و تحويلها
19
المملكة العربية السعودية
كلية العلوم -جامعة جازان
قسم االحياء
منتجة النزيم سالبة صبغة الجرام و عزل ساللة بكتيرية
ض لبع دراسة حساسيتها و مخلف الحليبمن البروتييز
المضادات الحيوية
اعداد الطالب
على حسين شعبى:رقم جامعى
781111102
اشراف
خالد السيد الجيار/د
قسم األحياء -أستاذ مساعد
جامعة جازان –كلية العلوم
20
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