Optimization of protease production by bacillus sp. in fresh water Cyprinus carpio fish

Introduction:

Micro organisms play a major role in both beneficial & non beneficial effect in the environment.Several micro organisms are produced to various types of enzymes such as protease,lipase, cellulose etc.,Protease enzymes plays a major role in cell & enormous applications in biotechnology & industries.protease produced by many microbial organisms such as Bacillus sp. Lactobacillus sp., Entero coccus sp., & fungi Aspergillus flavus etc.,

Proteolytic enzymes (PROTEASE) are produced by a variety of micro organisms during fish sauce fermentation. Microorganisms are important for metabolic process such as sporulation differentiation, maturation of eneymes hormones and the maintanence of cellular protein pool.

In the present study an attempt has been made to isolate the potent protease producer and investigate the effect of physical factors like temperature and pH on protease producing bacteria bacillus sp. was isolated from gastro intestinal tracts of fresh water fish cyprinus carpio.

GIT of fish is filled with a number of beneficial bacteria called probiotics.Probiotics means for life as opposed to the term antibiotics means against life.Fish bodies have a symbiotic relationship with probiotics. They help us to digest food kill harmful micro organisms and keep us functioning properly.

Fish gut bacteria in the digestive tract from aquatic environment through water and food which are populated with bacteria. Being rich the in nutrient, the environment of the digestive tract of fish confers a favourable culture environment for the micro organisms. There are only a few probiotics in the gut as it is highly acidic.The number of these beneficial bacteria increases dramatically in the intestine.

Probiotics also a friendly bacteria \ friendly or healthy first application of probiotics occurred in 1986 to test their ability to increase growth

hydrovionts (organisms that live water.Later probiotics where used to improve water quality and control of beneficial infection. Nowadays, there is documented evidence that probiotics can improve the digestibility of nutrients, increase tolerence to stress and encourage reproduction currently probiotics product are commercially from prepared from various bacterial sp. Such as bacillus sp.

Their use is regulated careful management [includes pH and temperature etc.,].Several microbial strains including Bacillus thuringiensis, fungi, Aspergillus flavous, B.proteolyticus, B.subtillis are reported to produce proteases[Elliah et al.,2002].Microbes represent excellent source of enzymes including protease because of their broad biochemical diversity [God frey & West,1996].In 1980 Aunstrup focused on microbial selection & fermentatation of proteases.

In 1905 Dr. Elie Metchnikoff was the first to describe the positive role played by some bacteria among farmers who consumed pathogen containing milk and that ‘’reliance on gut microbes for food makes it possible to change the flora of our bodies and to replace harmful microbes by beneficial microbes.

However the term probiotic was introduced until 1965 by Lilly and Stillwell as a modification of the original word “Probiotica” was described as an agent has the opposite function of antibiotics.The first use of the term to describe a microbial feed/food supplement was by Parker in 1974.He defined it as ‘’organisms and substances that contribute to industrial microbial balance”.

Fuller expanded that “live microbial supplement that benefits the host [human or animal] by improving the microbial balance of the body” and said that it would be effective in a range of extreme temperatures and salinity variations.

Ward 1985 mainly dealt with the sources of microbial proteases and their possible functions and roles in nature.In 1998 Guarner and Schaafsma

assumed that probiotics are live micro organisms which consumed in adequate amounts,confer health benefits to host. Gatesoupe in 1999 defined them microbial cells administered in a certain way, which reaches the gastrointestinal tract and remain alive with the aim of improving health.In the same year, studies were carried out on the inhibition of pathogens using probiotics, this expanded the definition to live microbial supplements which benefits the host by improving its microbial balance.

Rao et al., 1998 published on excellent review, in terms of molecular biology biochemical and genetic aspects of microbial animal and plant proteases.The bio industrial view microbial alkaline proteases from such sources for the production,characterization and commercial applications have also been reviewed Anwar and Saleemuddin (1998).

Microbes can be easily manipulated genetically to generate new enzymes with altered properties that are desirable for their various applications.Beneficial organisms are response of the body and reducing proliferation of cancer in mammals.Because of this provided at certain concentrations and viability,probiotics favourably effect host health .

For many years,studies focused on micro organisms characteristic from intestinal microbiota and mainly restricted to gram positive lactic acid bacteria particularly genera Bifido bacterium,Lacto bacillus and Streptococcus.Generally proteases are produced from micro organisms are partially inducible in nature and under the most culture conditions,Bacillus sp. Produce extra cellular proteases.

Protease synthesis is also affected by rapidly metabolizable nitrogen sources such as amino acid in the medium.Physical and chemical factors like pH,temperature,inoculums density,incubation time also affected the amount of protease production.So optimized and maintained this physical and chemical factors.

Several studies have reported that protein and peptides are necessary for effective protease production.Secades et al., 2001 reported that the

production of a calcium induced metallo protease by the fish pathogen Flavo bacterium psychrophillum.

The enzymes was detected only the strains was cultivated in the presence of calcium.Several alkaline proteases by the genius bacillus have been purified and characterized [Ferraro et al.,(1996) singh et al.,(2001)] Antibiotics are widely distributed in the nature where they play an impartant role in regulating the microbial populations of soil, water, sewage and compost.

The genus bacillus contains several industrially important species and commercial production of enzymes derivatives from the bacillus sp(beg and gupta,2003).These strains are extracellular protease producers and cultivated under extreme temperature and Ph conditions to gives rise to products that are turn,stable in a wide range of harsh environment Florikoshi et al., (1996).

The genus bacillus are gram (positive),aerobic,rod shaped spore forming bacteria are produced antibiotics as secondary metabolites.Most studies have reported that the different strains of Bacillus sp. and Lactic acid bacteria found in fish gut and fish products.Because probiotics bacteria are present in fish gut.Probiotics may provide an alternative way to reduce the use of antibiotics and applied to human and animals.

I have studied that to isolate Bacillus sp. Strain are produced protease enzymes from freshwater Cyprinus carpio fish gut.Among these B.strains are one of the most important producers of extra cellular and commercially viable proteases among various micro organisms.Some bacillus strains secrete significantly large amounts of extra cellular proteases with good stability at high temperature and ph values.

Currently large proportions of the commercially available extra cellular proteases are derived from B.strains Gupta et al., 2002.However, few reports exist on proteolytic enzymes from Pseudomonas sp. Jang et al., 1996 reported alkaline serine protease from an alkaline resistant P.sp.

Protease producing P.aeruginosa K-187 was used for deprotenization of shrimp and shell wastes (Wangard and Chio in 1998).In contrast to terrestrial animals,gastro intestinal microbiota of aquatic sp. Is particularly depends on the external environment due to the flow of water passing through the digestive tract.

Thus the majority of bacteria are transient in the intestine,due to constant intake of water and food together with micro organisms present in them.Although in the gastro intestinal tract (GIT) of aquatic animals have been reported potentially pathogenic bacteria such as Salmonella,Listeria, E.coli ,Probiotic bacteria other micro organisms have also been identified.

These include gram(+) bacteria such as Bacillus,Carnobacterium,enterococcus & several sp. Of Lactobacillus.Gram(-) bacteria such as Vibrio & Pseudomonas as well as certain yeast, fungi & algae of the genera Debaryomyces etc., Beneficial micro organisms with beneficial effects for the host able to persists in the digestive tract because of its tolerance to acid & bile salt.

Moreover a B.subtilis strain combained with hydrolytic enzymes to produce biogen was used to supply the feed of Oreochromis niloticus,obtaining significant increases in productivity.Probiotic micro organisms have the ability to release chemical substances with bactericidal or bacteriostatic effect on pathogenic bacteria that are in the intestine of the host, thus constituting a barrier against the proliferation opportunistic pathogens.

Proteases are commercially used to pharmateutical industry, microbial industry & biochemical industry.Probiotics are added to fish cultures they adhere to the intestinal mucosa of them developing & exercising their multiple benefits. According to Soccol et al., the global market for probiotic ingredients, supplements & food reached US S 15,900 million in 2008 & is projected to increase as to US S 19,600 in 2013 representing an annual growth rate of 4.3%.

In recent years, proteases from the gut of fishes received much attention Chi et al., 2007. Protease production is influenced by some physical factors such as aeration,inoculums size, Ph, temperature, incubation time &biological factors such as the genetic nature of the organism, which are influencing the metabolic & biochemical behaviour of the microbial strain.

No defined medium has been established for the best production of any metabolite due to the genetic diversity present in different microbial sources because each organisms (or) strains to have its own special conditions for maximum product yield.Therefore, it is essential to have a detailed investigation on the newly isolated microbial strain to understand the production pattern during different environment conditions and optimization trend to achieve the maximum production of enzyme (Ellaiah et al., 2002). Infact bacteria isolated from digestive tract of fish gut have shown enormous enzymes such as proteases, celluloses, trypsin & lipases.

OBJECTIVES:

To isolate and screen the protease producing bacterial strains from the gut of fresh water fish Cyprinus carpio.

To perform the biochemical test for identification of protease positive bacteria by using Bergeys Determinative Bacteriology.

To optimize the identified bacterial strains with varing parameters such as carbon sources, organic nitrogen sources, sodiumchloride, Ph, temperature, metal ions, inoculum, incubation time through the submerged fermentation (SF) process.

Cyprinus carpio

Materials and methods:

Collection of samples:

The experimental fish Cyprinus carpio was collected from the Kadachanendhal in Madurai.The number of incidental organisms was reduced by washing fish skin with 70% ethanol.

Gut microbial analysis:

The ventral surface of the fish was opened with sterile scissor. After dissecting the fish 1g of gut was taken out and homogenized with 0.1mM of phosphate buffer solution was added and ground well under aseptic

conditions used as astock.This stock solution was serially diluted and spreaded on the agar plate.

The method is used to isolate pure culture and also for estimating the total viable colonies (TVC).Number of bacterial population was expressed as number of colony forming units (CFU) per ml of samples.

Mi crospical identification bacteria:

Gram’s staining:

Most of the bacteria can be differentiated by their grams reaction due to differences in their cell wall structure. The technique was discovered by Hans Christian Gram.

The organisms which stained dark purple with crystal violet (primary stain) and not decolourized by 95% ethanol are gram positive and those organisms which after being stained with crystal violet lost their colour treated with alcohol and stained red with saffranin (counter stain) are referred as gran negative.

(ii) Motility test

Bacterial motility was identified by hanging drop method. Bacterial motility is characterized by the presence of flagella.

Biochemical tests:

I Catalase test

This test is used to determine ability of an organism to produce the catalase enzyme. During aerobic respiration, the organism produce H2O2 and super oxide and the accumulation of these substances are toxic to the organisms. Hence it produces catalase and degrades H2O2 into oxygen and water.

II Oxidase test

This test is used to identify the organisms, which produce the enzyme oxidase forms the paart of electron transport system, possessed by some bacteria. This enzyme oxidizes the oxidase disc, which contain the reagent N-N Tetramethy1 Paraphenylene Diamine Dihydro Chloride to a deep purple colour.

III Triple sugar iron test

Triple sugar iron agar (TSI) is used to determine whether a Gram-negative rod uses glucose, lactose or sucrose fermentative and form hydrogen sulfide (H2S). TSI contains 10 parts lactose, 10 parts sucrose, 1 part glucose and peptone. Phenol red and ferrous sulphate serve as the indicators of acidification and H2S formation respectively. When fermentive organisms use glucose, the entire medium becomes acidic (yellow) within 8 to 12 hours. The gut remains acidic after the recommended 18 to 24 hours of incubation period because of the presence of organic acids, which results from the fermentation of glucose under aerobic conditions in the gut of the tube. The slant however, reverts to the alkaline (red) state because of oxidation of the fermentation products under aerobi conditions of the slant. The changes, is a result of the formation of CO2 and H2O and the oxudation of peptones in the medium of alkaline amines. In addition to glucose, lactose and sucrose, the large amout of fermentation products formed on the slant will neutralize the alkaline amines and render the slant acidic (yellow), provided the reaction is read in 18 – 24 hours. Reaction in TSI should not be read beyond 24 hours of incubation, because aerobic oxidation of the fermentation product from lactose and sucrose does proceed and the slant will eventually revert to the alkaline state. The formation of CO2 and H2 9hydrogen gas) is indicated by the presence of bubbles of cracks in the agar or by separation of the agar from the slides or bottom of tube.

iv.Sugar fermentation:

This test is used to determine the ability of the organisms to degrade and ferment sugar with the production of an acid gas.Fermentation is an aerobic metabolic process in which the final electron acceptor is an organic

substrate instead of oxygen.The most commonly produced end product is lactic acid even though many other substances are also produced as the result of different fermentation pathways.Most organisms obtain their energy through a series of orderly and integrated enzymatic reactions leading to biooxidation of the substrate.The medium contains nutrient broth sugar and the pH indicator nutrient broth supports the growth of organisms.The sugars enhance the fermenting capability of the organisms and the pH indicator phenol red at neutral pH and yellow at acidic Ph.Following incubation the fermented carbohydrates produce acidic Ph causing changing the colour to yellow.

V. Indole production test.

The test is used to determine the ability of an organism to split tryptophan to form the compound indole.

Vi. Urea hydrolysis.

The test is used to determine the ability of an organism to produce the enzyme urease, which hydrolyzes urea. Hydrolusis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium and the pH shift is detected by the colour change of phenol red from light orange at pH 6.8 to magenta at pH 8.1.

Vii.Casein hydrolysis.

Casein, the major milk protein is a macro,olecule of aminoacids linked by peptide bonds. A molecule of this magnitude is incapable of permeating cell membranes. The protein should undergo stepwise degradation into peptones, polypeptones. Dipeptones and finally to aminoacids for cellular nutrition. This is mediated by cellular exoenzymes called proteases and the process is called as peptidization or proteolysis.

Viii. Starch hydrolysis.

Starch is a linear polymer of glucose molecule linked together by glycoisidic bonds. Starch as such cannot be transported into the cell fdor energy production, because of its high molecular weight. To assimilate starch for energy and catabolic reactions, it must be degraded into basic glucose units by starch hydrolyzing enzymes. These enzymes are secreted by the microorganisms into the medium, which degrade starch primarily to glucose.The resulting low molecular weight soluble glucose molecules are now able to pass into the cell for energy production via, glycolysis.

Serial dilution preparation:

The serial dilution of the sample was done for inoculation of the sample into plates and enumerate the number of bacterial cells per mg of sample.This was done by serially diluting the sample with sterile distilled water. To this 1ml of stock solution was mixed with 9ml of distilled water in a test tube and subsequently serially diluted samples were plated onto the surface of zobell marine agar medium.

Bacteria and protease activity:

The bacteria used in this study were isolated (B.sp.) as a potent proteolytic bacterium from the gut of Cyprinus carpio.About 9ml of distilled water was sterilized.To this 1ml of stock solution was added the following dilution were prepared ie 10-1,10-2, 10-3,10-4 from each dilution 0.1 sample was added to the petriplate with nutrient agar medium. The plates were incubated at 37C for 24 hrs. After incubation the number of colonies formed were counted.In the present study the colonies were reported at 10-3 dillution were taken for further enumeration.

Preparation of nutrient agar slant:

Nutrient agar was aseptically prepared and poured into test tubes kept in slanting position for solidification.After solidification single colony was isolated out and streaked on the agar slants.Then the agar slants were stored at 4C in refrigerator condition.

Preparation of inoculums:

The protease producing bacterial strains were subcultured from the pure culture for further experimental analysis.The cells were taken from the agar slant.The cells were added into sterile distilled water and resulted cell suspension at 10% level and it was transferred to 250ml of Erlenmeyer flasks.This flask containing 45ml of sterile medium with beef extract (0.3%), peptone(0.5%), NaCl(0.5%) and glucose(0.5%) at Ph for 7 for 24 hrs.The culture was then incubated for 2 days by reciprocal shaking(120rpm) at 35C.The cells were then harvested by centrifugation at 10,000 rpm for 15 mins and the supernatant was used fof further protease assay method.

Protease assay (Syner and Moghissi, 1972);

The assay system consists of following ingredients such as 1.25 ml Tris buffer(pH 7.2), 0.5 ml of 1% aqueous casein solution and 0.25 ml culture supernatant. Appropriate controls were also maintained. The mixture was incubated for 30 min at 30c. Then 3 ml of 5% TCA was added to this mixture and placed at 4c for 10 min to form precipitate. Then it was centrifuged at 5000 rpm for 15 min. Form this, 0.5 ml of supernatant was taken, to this 2.5 ml of 0.5M sodium carbonate was added, mixed well and incubated for 20 min. Then it was added width 0.5 ml of folin phenol reagent and the absorbance was read at 660 nm using UV-V is Spectrophotometer (TECOMP 8500). The amout of protease produced was estimated and expressed in microgram of tyrosine released under standard assay conditions.U/ml = O.D of the sample -----------------------× concentration of the standard O.D of the standard OPTIMIZATION OF MEDIA COMPONENTS:

Effect of environmental conditions on protease production:

Various process parameters that influence the protease production by Bacillus sp was evaluated by carrying out fermentations in the basal media. The pH of the basal media was adjust to 7 under various temperatures ( ) and kept in a on orbital shaker at 120 rpm.

The effect of pH of media on protease production by Bacillus sp was determined during fermentation in medium with different pH ( ) The cell free supernatant was obtained by centrifugation and it was used for the determination of protease production. The enzyme assay was carried out after 48h of incubation at 35c.

Effect of nutritional parameters on protease production.

The protease production by the selected bacterial strains was optimized by supplementing the different organic nitrogen sources in basic production medium. The protease production has bee assayed from culture media supplemented with casein, peptone, beef extract, urea, yeast extradct, soy meal and skim milk powder. They were tested individually at the concentration of 0.5% in the basal medium inoculatd with 2% of 24h culture of test organisms.

The impact of inorganic nitrogen souces on protease production was studied by using various inorganic nitrogen sources viz., ammonium sulphate, ammonium chloride, sodium nitrate, ammoniuk nitrate and potassium nitrate. They were tested individually at 0.5% concentration in the basal medium inoculated with 2% of 24h culture of test organisms.

For the selection of suitable carbon source for protease production: galactose, sucrose, arabinose, fructose, maltose, raffinose, starch, glucose and dextrin were supplemented individually at 0.5% concentration in the basal medium inoculated with 2% of 24h culture of test organisms.

1.Effect of carbon sources on protease production:

To asses the effect of different carbon sources on amylase production was determined by using varios carbon source such as galactose, sucrose, fructose, maltose, starch, glucose, and lactose were supplemented individually at 0.5% concentration in the optimized basal meium inoculated with 2% of 24h broth culture.

2.Effect of nitrogen sources on protease production:

The effect of organic nitrogen sources was studied by using various nitrogen sources such as soya meal, beef extract, peptone, urea, yeast extract, skim milk powder, casein, sodium nitrate, ammonium sulphate. The effect was studied after 48hrs of incubation.

3.Effect of NaCl on protease production:

The effect of NaCl on protease production was studied by supplying various concentration of NaCl to the production media. The experiment was carried out individually at 0.5 – 5% NaCl in the optimized basal medium inoculated with 2% of 4hr culture broth.

4.Effect of pH on protease production:

To asses the effect of Ph on protease production by the experimental microorganisms was determined by using different ph. The assay was carried out individually at various ph such as 5, 6, 7, 8, 9,10.

5.Effect of temperature on protease production:

The effect of temperature on protease production was studied by incubating the enzyme and substrate solution at various temperatures such as 25 35 45 55c. The assay was carried out individually at these temperatures after 48hrs of incubations.

6.Effect of incubation time on protease production:

The effect of incubation time on protease production was studied by the optimized production medium with different hrs of incubation. The

experiment was carried out individually at various hrs of incubation such as 24hr, 48hr, 72hr, and 96hr and then enzyme assay was performed.

RESULTS:

Isolation of proteolytic bacterial strains:

A total of 4 out of 16 bacterial isolates/ strains possessed proteolytic activity. Among the four bacterial isolates, only one bacterium was found to be higher protease production.The isolate was identified as Bacillus sp.to produced protease enzyme by using skim milk agar plate.

Identification of protease positive colony:

Based on the morphological, physiological, and biochemical characteristics the suspected colony was identified as Bacillus sp. by the following standard keys of Bergeys Manual of Determinative Bacteriology and the isolated bacterial strain was screened for protease producing on skimmilk agar. The zone formation around the bacterial colony indicated the protease positive strain PCSIR EA-3. Hence the strain was identified as an protease producer and it was taken for further experimental studies.

Effect of carbon sources:

The effect of carbon sources on protease production by Bacillus licheniformis after 48hr of incubation period indicated that it was maximum in lactose supplemented medium.

Effect of organic nitrogen sources

Among the tested nitrogen sources the maximum amount of protease production was registered in peptoneadded medium after 48hr of incubation. But the minimum amount of protease production was observed in tryptone.

Effect of various concentration of NaCl:

The effect of NaCl on protease production by Bacillus sp. Inferred that, It was high in 2% concentration . The lowest amount of enzyme production is 4% concentration.

Effect of pH :

The effect of pH on protease production revealed that it was maximum at 7.0 and followed by 6,8 and 5. The minimum range of protease production was obtained in pH 10.

ᵒ

Effect of temperature:

The effect of protease production revealed that it was maximum at 40ᵒC. The least amount of protease production was recorted at 25ᵒC for

Bacillus sp

Effect of incubation time:

Figure shows incubation interval on the protease production and the results indicated that the 4hrs of incubation was suitable for the candidate

bacterium. A gradual reduction of protease yield was registered with increase in incubation period.

Discussion:

The microenvironment of bacteria associated with the gastrointestinal tract of an animal influences the host in many ways,including the metabolism of nutrients. Given the importance in digestion and health, the composition, diversity and morphological characteristics of the gut microflora in many

species of marine and fresh water fishes and invertebrates have been extensively researched.

The optimization for growth and protease production increased proteolytic activity and was in development of started culture for acceleration the fermentation process. The protease produced from various microorganisms. The optimum pH was 7.0 for the production of high amount of protease enzyme. At this pH condition the bacillus isolates produced 18mMol/ml and showed high CFU when compared with other pH ranges.similer resuls have been reported by Sugita et al.,. Alkaline proteases mostly have their isoelectric points near to their ph optimum in the range of 8 to 11 was studied by Gupta et al.,

Rahman et al.,found that enzyme synthesis and energy metabolism of bacteria was controlled by temperature. Moreover, temperature significantly the synthesis and secretion of bacterial extracellular protease enzyme. Hence, our study was focused to optimize temperature for the efficient production enzyme. It was found that optimum temperature of 35ᵒC was effective condition to produce high amount of protease enzyme when compared with other. The temperature was found to influence extracellular enzyme secretion; possibly by changing the physical properties of cell membrane. Elsafey et al., reported the same findings in production , purification and characterization of proteases enzyme from bacillus subtilis.

From an application point of view, it should be taken into consideration whether protease activity or microbial cell is needed for development and acceleration of fish sauce fermentation process. Probiotics were used to improve water quality and control of bacterial infections and it also can improve the digestibility of nutrients, increase tolerance to stress, and encourage reproduction. Kozasa made the first emprical application of probiotics in aquaculture considering the benefits exerted by the use of probiotics on humans and poultry.

The aim of this study by the use of Bacillus licheniformis were produced to enormous amount of protease produced in various ways

(pH,temperature etc). The optimum pH of protease production is 8-12 and this pH protease enzymes were produced. The optimum temperature is 35-40ᵒC in Bacillus sp was best to produce the protease production. High or low pH and temperature to produce low amount of protease production. So optimum pH and temperature should be maintained.

Further studies have stressed probiotics ability to stimulate appetite, improve absorption of nutrients, and strengthen the host immune system. Although in the gastrointestinal tract have been reported potentially pathogenic bacteria such as Salmonella, Listeria, E.coli, probiotic bacteria and other microorganisms have also been identified.

The biological diversity of marine and estuarine species provides a wide array of enzymes with unique properties. In recent years, from the gut of fishes received much attention (Chi et al. , 2007). It contributes to the development of highly added applications or products by using the enzyme-aided digestion of proteins from different sources including those from marine animals. Moreover, proteases have a wide variety of industrial applications such as detergents, leather processing, and silver recovery, medical purposes, food processing, feeds, chemical industry, and waste treatment (Kumar and Tagaki 19999). In the aquaculture industry, proteases are the needful enzyme for the preparation of high-quality functional feeds through bioconversion of low-cost feed materials because the worldwide sustainability of the aquaculture industry depends on the availability of low-cost, high-quality feeds. However, very scare information is available in the area of gut bacterial enzymes especially proteases and its role in the intestine.

Moreover, fish extracts readily spoil thereby proving to be an excellent source of nutrients for bacterial growth. Particularly enzymatic hydrolysate from fish waste extract (Jassim et a;. 1988) and autolysis of fish viscera (Clausen et al. 1985) have been proved as an excellent substrate for bacterial growth. Das and Tripathi (1991) observed that optimum protease activity between pH 7.6 and 8.4 in both the fingerling and adult grass carp and among

fingerlings the activity increased proportionately with higher amount of protein in diet upto a limit.

The results of the present study indicated that the micro organisms isolated from fish digestive tract are capable of producing proteolytic enzyme. In the present study the total viable count was ranged from 72× 104 CFU per gram gut of cyprinus carpio. From this study, it was concluded that the microorganisms have a beneficial effect in the digestive process of fish investigations have suggested that micro organisms have a beneficial effect in the digestive process of fish (Rigo et al., 1995) but it rarely applied in aquaculture.

The main strategy in the use of probiotics is to isolate intestinal bacteria with favourable properties form marine animals of the same species (Gildberg et al., 1997). In commercial aquaculture beneficial bacteria could be introduced by incorporating them into artificial fish diets. In aquaculture, most of the experiments conducted with utilization of probiotics for the alternative methods of disease control. In animal husbandry, probiotics are used to increase nutrient utilization and growth rate. Such experiments are not very common in aquaculture. The enzyme producing microorganisms isolated from the fish digestive tracts can be beneficially used as a probiotics while formulating the diet for fish especially in the larval stages.

In consistence with this present study, bacteria such as Bacillus sp. (Prakasham et al., 2006) and Bacillus clausii (Joo et al., 2003) were also reported that higher protease production in maltose supplied medium. The experimentation on the effect of sodium chloride showed that these bacterial strains could utilize the salt range between 0.5% to 4% sodium chloride supplied medium. Shanmuga Priya et al., (2008) reported that the protease production by marine bacterium Roseobacter sp. absolutely require 3% NaCl.

Metal ions and trace elements are often required by bacterium for its growth and physiological activities. Some bacteria and fungi showed maximum enzyme production with metal ions such as ca, mg and inhibited by the addition of EDTA (Sumantha et al.(2005).pH effect indicated that the

protease production by B.flexus and Pseudomonas sp.was maximum at pH 7.0. The effect of inoculum concentration on protease production indicated that the highest protease production at 5% inoculum added in this medium.

In this present study 72× 10 4 CFU/ml at 35-40c temperature effective for bacillus sp.but at 40ᵒC bacterial culture was produced to low amount of protease enzyme 20mMol/ml and the isolates was the capable of growing in the pH range of 6.0-10.0 with maximum growth at 7.0. The production of protease substantially decreased above and below the the optimum pH 7.0. At pH 7.0 the protease production increased and leveled off around 18mMol/ml. So optimum p H and temperature was produce maximum amount of protease production.

SUMMARY

Based on the morphological, physiological and biochemical characteristics the suspected colony was identified as Bacillus licheniformis by the following standard keys of Bergeys Manual of Determinative Bacteriology and the isolated bacterial strain was screened for protease producing ability on skimmilk agar. The zone formation around the bacterial colony indicated the protease positive strain. Hence the strain was identified as an amylase producer and it was taken for further experimental studies.

The effect of carbon sources on protease production by bacillus licheniformis after 48h of incubation period indicated that, it was maximum in lactose supplemented medium.

Among the tested nitrogen sources, the maximum amount of protease production was registered in peptone added medium.

Among the tested incubation time results indicated that the 48h of incubation was suitable for the candidate bacterium.

The effect of ph on protease production revealed that it was maximum at 7.0 and minimum at 10.

The effect of temperature on protease production revealed that it was maximum at 40c and minimum at 25c.

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