Isolation of Gram ve proteolytic bacteria from milk waste and …colleges.jazanu.edu.sa/sci/Documents/s12.pdf · Qualitative screening of protease enzyme strain Skim milk nutrient

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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.

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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].

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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].

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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

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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].

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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

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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

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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

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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].

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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,

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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

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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.

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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

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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

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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

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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).

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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.

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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

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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

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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

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