PROTEASE ENZYME PRODUCTION FROM FOOD WASTE

 

PROTEASE ENZYME PRODUCTION FROM FOOD WASTE

 

AIM: To isolate protease producing organism and assay the enzyme protease from food waste.

 

MATERIALS REQUIRED: Skim milk agar, Baal media for enzyme production, Centrifuge, azocasein, Tris HCl, Trichloroacetic acid, NaOH, UV-visible spectrophotometer, Bovine serum albumin

 

PROCEDURE:

I. Isolation of protease producing bacterial isolates:

 

1.      Food waste samples were randomly collected from near the house.

2.      One gram of the soil was suspended in 9 mL of sterile distilled water.

3.      After a serial dilution (10¹ to 10⁶) of this suspension with sterile distilled water, 100 mL from each diluted suspension was spread on skim milk agar (SMA) plates (2.0% skim milk, 1.0% glucose, 1.5% agar and pH 9.0) and incubated at 37⁰C for 24-48 h.

4.      Bacteria showing clear zones due to hydrolysis of casein in milk were selected as protease producers.

5.      Based on the ratio of clear zone, screening of protease producer was carried out and then pure cultures were obtained on SMA media.

 

II. Production of crude protease in the shake flask:

1.      A basal media (l.0% glucose, 0.5% peptone, 0.5% yeast extracts, 0.1% K₂HPO₄, 0.01% MgSO₄ and 0.5% Na2CO₃; pH 9.0) was used for alkaline protease production unless noted.

2.      For seed culture, 5 ml of sterile basal media was inoculated with an isolated colony and incubated at 37⁰C and 120 rpm for 20 h.

3.      The seed culture was then transferred to 45 ml production media in a 250 ml conical flask and incubated under the conditions as indicated.

4.      The cell free supernatant was obtained by centrifugation at 8000 rpm for 20 min at 4⁰C and used for determining the protease activity or further study.

 

III. Enzyme assay and protein estimation:

 

1.      The reaction mixture was (500 ml of 1.0% azocasein in 50 mM Tris HCI, pH 9.0 and 1 ml of enzyme solution) incubated for 30 min at 40⁰C.

2.      The reaction was stopped by adding 2.8 ml of 5.0% (w/v) trichloroacetic acid (TCA), and was kept at 4⁰C for 15 min.

3.      The precipitate was removed by centrifugation at 8,000 rpm for 20 min.

4.      A 2 ml of the supernatant was added to 2 ml of 1.0M NaOH and the absorbance was measured at 440 nm using a UV-visible spectrophotometer.

5.      One unit of protease activity was defined as the amount of enzyme that produced an increase in absorbance of 0.01 under the above assay conditions.

6.      For negative control, 5.0% of the TCA solution was added before addition of enzyme preparation. Total protein concentration was determined with Bradford protein assay kit using bovine serum albumin as a standard protein.

ACKNOWLEDGE: We are thankful the authors who have been published this article in open source. Al Hakim, Farhana Rumzum Bhuiyan,Asif Iqbal,Tanvir Hossain Emon,Jahed Ahmed,Abul Kalam Azad. Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes. Heliyon 4 (2018) e00646. doi: 10.1016/j.heliyon.2018. e00646

GREEN SYNTHESIS OF SILVER NANOPARTICLES USING Zingiber officinale RHIZOME EXTRACT AND ITS APPLICATION ON DIABETIC FOOT ULCER

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GREEN SYNTHESIS OF SILVER NANOPARTICLES USING Zingiber officinale RHIZOME EXTRACT AND ITS APPLICATION ON DIABETIC FOOT ULCER

1Nivethitha.M, 2Ramachandran A.M

1Post Graduate and Research Department of Microbiology, Dr. N.G.P. Arts and Science college(Autonomous), Coimbatore, Tamil Nadu, India.

2Associate Professor , Department of Microbiology, Dr. N.G.P. Arts and Science college(Autonomous), Coimbatore, Tamil Nadu, India

Abstract: The biosynthesis of metal nanoparticles is a vast research area due to the application of novel technologies. Plant extract are cost effective and eco-friendly for green synthesis of silver nanoparticles. To evaluate the diabetics foot ulcer activity from aqueous silver nitrate through the extract of Zingiber officinale rhizome . Ginger extract act as reducing and capping agent. The formation of silver nano particles are confirmed by UV- Vis spectrophotometer and characterization using Scanning Electron Microscopy, EDAX and Fourier Transform Infra red spectroscopy . SEM images indicated the presence of silver nanoparticles as spherical shape and range of between 62 nm to 92 nm. EDAX shows strong signal from silver atom . FTIR spectra suggest that the biological molecules possibly perform dual function of formation and stabilization of silver nanoparticles in aqueous medium. Minimal inhibitory concentration showed effective activity in 3mm concentration.

Index Terms – Diabetic foot ulcer, Ginger extract, UV- Vis spectrophotometer, SEM-EDAX, FTIR and Minimal inhibitory concentration .

I. INTRODUCTION

Diabetes mellitus is a chronic disorder and a major public health problem. Infections are a common complication in diabetic patients, and they are usually more severe than infections in non-diabetic patients. Foot infections are among the most common complications in diabetic patients and a common case of morbidity and mortality. Twenty- five percent of all diabetics develop severe foot or leg problems and foot infections account for about 20% of all hospital admissions in diabetics (Cunha BA. -2000).

There are a number of predisposing factors, which determine the severity of this complications: 1.According to impaired immunologic response, 2. peripheral neuropathy, 3. peripheral arterial disease. Depending on the control of diabetes, these factors can be present in varying degrees (Lipsky BA, et.al.,-2004). In case of poor control, an infection can develop, spread rapidly and produce significant and irreversible tissue damage, resulting in a high rate of amputations (Caputo GM-1994).

Foot infection in diabetic patients usually begins in a skin ulceration. Although most infections remain superficial, ~25% will spread contiguously from the skin to deeper subcutaneous tissues and / or bone. Up to half of those who have a foot infection will have another within a few years. About 10-30% of diabetic patients with a foot ulcer will eventually progress to an amputation, which may be minor (i.e., foot sparing) or major. Conversely, an infected foot ulcer precedes ~60% of amputations, making infection perhaps the most important proximate cause of this tragic outcome.(Amsterdam,1999).

S. aureus is the most important pathogen in diabetic foot infections, even when it is not the only isolate, it is usually a component of a mixed infection (Breen JD et.al.,-1995). Serious infection in hospitalized patients are often caused by 3-5 bacterial species, including both aerobes and anaerobes (Wheat LJ et al., -1986).

Zingiber officinale, commonly known as ginger, belongs to the Zingiberaceae family that originates in Southeast Asia. Z.officinale is an edible rhizomatic herb that is often used as seasoning throughout the world. The ginger rhizome is elongated, reasonably flattened, and its coloration ranges from golden yellow to bight brown, containing longitudinal grooves (Butt MS and Sultan M., 2011).

Ginger (Zingiber officinale) is a world known spice and has been used for over 2500 years in traditional medicine for the treatment of respiratory diseases(rhinitis, asthma), inflammation (rheumatism), heart disease (hypertension, palpitations, cardiopathies), gastrointestinal tract (diarrhea, constipation, vomiting, poor digestion) and metabolic disease(Diabetes)(Mozaffari KH et al., 2014).

Ginger can also be presented in powdered, crystallized form or in solution, which promotes further changes in its composition (Ali BH et al., 2008). The chemical components of ginger can be volatile or non-volatile, the later being responsible for the characteristic

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smell and taste of ginger. Among the volatile compounds are sesquiterpene hydrocarbons, such as zingiberene (35%), curcumene (18%) and farnesene (10%) (Butt MS et.al., 2011). The non-volatile compounds give the ginger the spicy taste, these being zingerone derivatives, although other other non- volatile substances are also recognized for their pharmacological properties, such as gingerols, shogaols and paradols (Arablou T et al., 2014 and Chen CY et al., 2012). Both gingerols and shogaols are very important phenolic components, since they have pharmacological properties that are beneficial to health (Mozaffari KH et al., 2014).

Nanotechnology can be termed as the synthesis, characterization, exploration and application of nano-sized (1-100nm) materials for the development of science. It deals with the materials whose structure exhibit significantly novel and improved physical, chemical and biological properties. Nanotechnology holds a trusting future for the design and development of many types of novel products that are used in early detection , treatment, prevention of various diseases in food technology. It also plays a role in pharmaceutical by using green synthesis from plant extracts and microorganism favou.6rs numerous benefits to ecofriendliness application (Thanaa.I.Shalaby et al.,2015)

The upcoming applications in various fields like optical sensors, in spectrally coating for absorption of solar energy (G.Haripriyaa et al., 2014), bioremediation of radioactive wastes, functional electrical coating, synthesis of enzyme electrodes, in medicine such as delivery of antigen for vaccination, gene delivery for treatment or prevention of genetic disorders, in medical field as a bactericidal and a therapeutic agent. Silver Nanoparticles have wide reactivity due to large surface to volume ratio and plays a crucial role in inhibiting bacterial growth, It is a well known that silver is very toxic to the microorganism (Chandan singh et al., 2011).

Hence nanosized organic and inorganic particles are finding increased attention in medical application due to their amenability to biological functionalization. Byased on enhanced effectiveness, the new age drugs nanoparticles of polymers, metals or ceramics, which can combact conditions like cancer and fight human pathogens like bacteria (Govindaraju et al.,2010).

Silver is a non- toxic, safe, inorganic antibacterial agent being used for centuries and capable for killing microorganism that cause disease. It has a significant potential for a wide range of biological applications such as antibacterial agent for antibiotic resistant bacteria, preventing factors, healimg wounds and anti- inflammatory. Silver ions (Ag+) and its compounds are highly toxic to microorganism exhibiting strong biocidal effects on many bacterial species but have a low toxicity towards animal cells.

Biosynthetic methods have been investigated as an alternative to chemical and physical one. These methods can be divided into two categories depending on the place where the nanoparticles or nanostructures are created as many microorganism can provide inorganic materials either intra or extracellularly. For example, the organism isolated from silver mine materials is able to reduce Ag+ ions and accumulates silver nanoparticles. The metabolic activity of microorganisms can lead to precipitation of nanaoparticles in external environment of a cell ( Sadowski et al., 2008). Among noble metal nanomaterials , silver nanoparticles have received considerable attention due to their attractive physicochemical properties. The surface plasmon resonance and large effective scattering cross section of individual nanoparticles make them ideal candidates for molecular labelling (Schultz et al., 2000, Elchiguerra et al., 2005).

Nanoparticle characterization is necessary to establish understanding and control of nanoparticle synthesis and application .Characterization is done by using a variety of techniques, mainly drawn from material science .Common techniques are Electron microscopy (TEM ,SEM) , Atomic force microscopy (AFM), Dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS) and UV spectroscopy (Das et al ., 2009).SEM shows macroscopic aggregates composed of nanosized silver particles. Obviously these particles have only a limited use as biocidal materials in liquid systems because of their low colloidal stability (Sondi and Sondi ,2004).

Silver nanoparticles are attractive as these are nontoxic to human body at low concentration and having broad spectrum antimicrobial nature. Silver nanoparticles inhibit the bacterial growth at very low concentrations than antibiotics as of no side effects are reported (Dharnendra and Behari., 2009).

Major benefits of the project was effective AgNPs against Diabetic Foot Ulcer by biosynthesis process. The biosynthesis process helps to avoid many chemicals. These process doesn't contain any chemical and which are not harmful. At 30mM concentration gives effective activity against DFU. Overall its an cost effective and eco-friendly work.

Further studies of the works such as toxicity checking by MTT Assay, animal studies and formulation of product as ointment or spray will be performed later.

II. MATERIALS AND METHODS

2.1 Collection of Sample:

The Diabetic Foot Ulcer sample was collected from a local private hospital by providing a nutrient broth and swab for taking a swab from the wound and then inoculate it into the nutrient broth. The broth was incubated for 24hrs at 37°C. Then serial dilution were made, from that some colonies are inoculated in a nutrient agar for isolation of pure culture. Those cultured are subcultured , identified and used for this study.

2.2 Identification of Microorganism:

The culture was processed for identification such as : Gram staining, motility, Biochemical Test (IMVIC, oxidase, TSI, urease) and enrichment media are used (EMB, Mac conkey, Mannitol Salt Agar, Blood Agar).

2.3Preparation of Ginger Extract:

Ginger (Zingiber officinale) rhizome extract was used in the study. The materials were collected from farmers market in Coimbatore. Then rinsed with sterile distilled water to remove any associated debris. The extract was prepared by taking 10gm thoroughly washed ginger rhizome, taking out the upper part by the knife, chopped into the fine pieces and converting it into paste using mortar and pestle followed by addition of 40ml of MilliQ water, boiling for 10 mins then filtered by Whatman filter paper , centrifuged at 5000rpm for 15mins and collection of suspension , which was further used as ginger rhizome broth for the experiment(Chandan singh et al.,2011).

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2.4 Bio-Synthesis of Silver Nanoparticles:

For the reduction of Ag+ ions 5ml of ginger rhizome broth was added to 50ml of 1mM Silver Nitrate solution, 10ml for 3mM and 15ml for 5mM silver nitrate solution. The ginger broth was slowly and drop by drop added to the silver nitrate solution, which kept in a magnetic stirrer in 40°C at 500 rpm for 1.30 hours in dark.

2.5 UV- Visible Spectrophotometer Analysis:

Bioreduction of Ag+ in aqueous solution was monitored by UV-Vis spectroscopy. UV- Visible spectrophotometer analyses of silver nanoparticles synthesized were recorded as a function of time at room temperature using SHIMADZU UV-2600 spectrophotometer, operated between 800 to 400nm wavelength ranges at a resolution of 1nm.

2.6 FTIR Analysis: ( Fourier Transform Infrared Spectroscopy)

After the completion of Ag+ ions by the ginger rhizome extract, 10ml solution of silver nanoparticle was centrifuged at 4000 rpm for 5 mins and these process to make nanoparticle free from proteins or other bioorganic compounds present in the solution. There after 5ml of purified suspension was given for analysis in SHIMADZU -FTIR for % Transmittance at 4cm-¹ resolution.

2.7 Scanning Electron Microscopy - EDAX:

The powdered silver nanoparticle were characterized using SEM-EDAX. The biosynthesized nanoparticles were lyophilized to make powder form then given for analysis.

SEM analysis was done to study the size and the elements present in the sample. The energy dispersive analysis of X-ray (EDAX) was also done along with the SEM analysis.

2.8 Anti-Bacterial Activity:

The Diabetic Foot Ulcer sample were serial diluted and plated on nutrient agar medium and various bacterial colonies were isolated and those organisms are: Staphylococcus aureus, Proteus sp, Klebsiella sp, Pseudomonas sp, Escherichia coli and Bacillus sp.

Well Diffusion method was used for checking anti-bacterial activity. Sterile Muller Hinton Agar plates were prepared. These plates were swabbed with the respective clinical isolates. Using a sterile cork borer, wells were cut on the Muller Hinton Agar plates. The biosynthesized liquid silver nanoparticles of ginger was then added, and in the remaining wells silver nitrate solution and ginger rhizome broth also added in the respective wells. The plates were the incubated at 37°C for 24 hours. Control plates with silver nitrate solution and culture broth were kept for incubation along with the test plates. After incubation the zones were examined and the zone diameter was measured. For identifying Minimal Inhibitory Concentration (MIC) various value such as 1mM, 3mM and 5mM of silver nanoparticles are added.

III. RESULT AND DISCUSSION

3.1 Isolation and Identification of bacterial pathogens from Diabetic Foot Ulcer:

The samples were collected and the isolates predominantly showed 7 bacterial colonies which are subcultured and identified based on morphological and biochemical analysis. The isolated bacterial species were examined by gram staining, some isolates showed gram negative rods and few were gram positive cocci in shape. In the study , the most common isolates were Escherechia coli, Klebsiella sp, Pseudomonas sp, Staphyloccus aureuc, Streptococcus pyogenes, Proteus sp and Bacillus sp.

3.2 Ginger Rhizome Silver Nanoparticle:

Ginger broth and 1mM, 3mM and 5mM of Silver nitrate solution were mixed in order to reduce the silver nitrate to Ag+ ions. The process of silver reduction was taken place in magnetic stirrer at 40°C at 500 rpm. The reduction was confirmed by Colour change and UVVisible spectrophotometer , then which are characterized by SEM-EDAX and FTIR.

• Colour Change: The ginger broth was pale yellow in colour before the addition of AgNO3. Then colour changed into golden brown on completion of the reaction with Ag+ ions for 1.30 hours. This change in colour is primarily due to the surface plasma resonance of silver nanoparticles. In the case of silver nitrate solution alone, no change in colour was observed. The intensity of the colour increased with time. At the end of the reaction golden or brown colour was observed in the reaction mixture. Reduction of silver ions was reflected in colour change of the ginger broth from pale yellow to golden brown colour. The appearance of the brown colour indicates the silver nanoparticles formation (Nithya and Raghunathan, 2009). The periodical analysis in colour of the ginger with silver nitrate as control was observed and results were recorded.

• UV-Spectrophotometer Absorbance: Ginger Rhizome capped AgNPs showed absorbance peak with maximum at 435nm wavelength indicated the reduction of Silver nitrate into silver nanoparticles.(fig.1)

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Figure 1: UV-Vis absorption spectra of biologically synthesized AgNPs using ginger rhizome .

1.925

1.500

1.000

0.500

0.000

-0.174

351.38 400.00 500.00 600.00 700.00 800.00

nm.

The (figure 1)reveals the UV Vis absorption peak at 435nm wavelength.

• SEM -EDAX Analysis:The SEM Analysis enables the high- resolution imaging of single nanoparticles (NPs) with sizes well below 10nm (V-D Hodoroaba et al.,2016). The SEM Analysis of Ginger rhizome silver nano compounds were observed. The micrograph of nanoparticles obtained, which showed that silver nanoparticles are spherical shape well distributed without aggregations in solution with an average range between 62nm-92nm.EDAX spectrum recorded in the spot- profile mode from one of the densely populated silver nanoparticles regions on the surface of film. Strong signals from the silver atoms in the nanoparticles are observed (Varshney et al., 2009).EDAX study showed strong signals from silver and from oxide. These are likely to be due to X-Ray emission from some plant compounds (fig 2 and 3) .

The SEM image reveals the size if AgNPs ranges between 62nm-92nm.(fig.2)

EDAX Analysis

SEMSEMSEM ANALYSIS ANALYSIS ANALYSIS ANALYSIS

FigureFigure FigureFigure 2: SEMSEMSEM ImageImageImage of AgNPs of AgNPsof AgNPs of AgNPsof AgNPs biosynthesized usingbiosynthesized using biosynthesized using biosynthesized usingbiosynthesized usingbiosynthesized usingbiosynthesized usingbiosynthesized using biosynthesized usingbiosynthesized usingbiosynthesized usingbiosynthesized usingbiosynthesized usingbiosynthesized using biosynthesized using Ginger GingerGinger Ginger rhizomerhizomerhizome rhizomerhizomerhizome extractextractextractextractextractextractextract

1

Abs.

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Figure 3: EDAX signals of biosynthesized AgNPs

The EDAX study showed the strong signal towards Ag=ions(fig.3).

• FTIR:FTIR Analysis was used for the characterization of the extract and the resulting nanoparticles. FTIR measurements were carried out to identify the biomolecules for capping and efficient stabilization of metal nanoparticles synthesized by plant root extract (M. Kurian et al., 2016).FTIR measurement were performed to identify the potential biomolecules in the ginger rhizome responsible for the reduction and then for stability to the bio-reduced AgNPs. The peak (fig.4) was centered at 1645.28cm-¹ which indicates the presence of alkanes (C=C) in the residual solution. The band at 3547cm-¹ and 3653cm-¹ (fig.4) indicates OH stretching in alcohols. From the analysis of FTIR spectra, we conclude that the presence of these functional groups proves the plant extract act as capping and stabilizing agent for silver nanoparticles.

Figure 4: FTIR Spectrum of bio-synthesized silver nano particle using ginger rhizome

3.3 Anti-Bacterial Activity:

The silver nanoparticles obtained from ginger rhizome were tested for antibacterial activity against clinical isolates of DFU (Staphylococcus aureus, E. coli, Proteus sp., Pseudomonas sp., Klebsiella sp., Bacillus sp., Streptococcus pyogenes). 20mm zone of inhibition was observed in 3mM concentration ( tab.1) of this formulation reveals most effective against S. aureus.

TheTheThe spectrum imagespectrum image spectrum imagespectrum imagespectrum imagespectrum image spectrum imagespectrum imagespectrum imagespectrum imagespectrum imagespectrum image impliesimpliesimpliesimpliesimpliesimpliesimplies thethethe presence presencepresencepresencepresencepresencepresence of functionalfunctionalfunctional functionalfunctionalfunctionalfunctionalfunctionalfunctional groups groupsgroups in thein thein thein thein thein the AgNPs.AgNPs. AgNPs.AgNPs.

TheTheThe spectrum imagespectrum imagespectrum imagespectrum imagespectrum imagespectrum imagespectrum imagespectrum imagespectrum imagespectrum image spectrum image spectrum image implies impliesimpliesimplies implies thethethe presencepresencepresencepresencepresencepresencepresencepresence of functionalfunctionalfunctionalfunctionalfunctionalfunctional functional groups groups groupsgroups in the in thein thein thein the AgNPs. AgNPs.AgNPs.AgNPs.

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3.4 Minimal Inhibitory Concentration (MIC):

The MIC of silver nanoparticles from ginger was showed inhibition zone( tab.1) for 1mM (Staphylococcus aureus- 10mm, E.coli- 12mm and Klebsiella sp- 8mm).

For 3mM AgNPs (Staphylococcus aureus-20mm, E.coli-18mm,Pseudomonas sp17mm, Klebsiella sp-13mm and Bacillus sp- 16mm), zone of inhibition was observed.

For 5mM of AgNPs (Strephylococcus aureus-14mm, E.coli-13mm,Proteus sp12mm, Pseudomonas sp-10mm,Klebsiella sp-13mm,Bacillus sp-14mm and Streptococcus pyogenes-15mm).AgNPs most effective against all the clinical isolates at 3mM concentration when compared to other different concentration, so the MIC of ginger rhizome silver nanoparticles is 3mM concentration.

Table 1. Antibacterial Activity of biosynthesized AgNPs towards DFU pathogens

S.No

Test Organism

Zone of Inhibition (Diameter in mm)

1mM of AgNPs

3mM of AgNPs

5mMof AgNPs

1.

Staphylococus aureus

10

20

14

2.

Escherechia coli

12

18

13

3.

Klebsiella sp

8

13

13

4.

Bacillus sp

-

16

14

5.

Pseudomonas sp

-

17

10

6.

Streptococcus pyogenes

-

-

15

7.

Proteus sp

-

-

12

The table implies the Triplicates mean values of each organism in all concentration. Average value for Staphylococcus aureus- 15 -, E.coli 14, Klebsiella sp- 11, Bacillus sp- 10, Pseudomonas sp- 9, Strephylococcus pyogenes- 5 and Proteus sp- 4.

SUMMARY AND CONCLUSION:

The present study was made to isolates the bacterial populations from Diabetic Foot Ulcer. And the clinical isolates are identified as Staphylococcus aureus, E. coli, Proteus sp., Pseudomonas sp., Klebsiella sp., Bacillus sp., Streptococcus pyogenes.

Silver had huge antimicrobial activity, and it is a non-toxic to humans. So Silver Nanoparticles are formed in this study. Ginger Rhizome had many benefits and it also had more physiochemical properties, even it shows antibacterial activity and cost effective.

Here, Ginger Rhizome broth was prepared by adding silver nitrate solution of various concentration to form Silver Nanoparticle. Where ginger rhizome act as a capping and stabilizing agent. Magnetic Stirrer method was used in 40°C at 500 rpm for 1.30 hours.

Silver Nanoparticles were formed , which are confirmed by different characterization such as colour change, shows pale yellow to golden yellow colour. Then UV- spectrophotometer was used to prove that silver Nanoparticles were formed by absorbance peak at 435nm.

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SEM performed to show the size of the silver nanoparticles which ranges from 62nm- 92nm at 20X magnification. EDAX study, showed a strong signal from silver that indicates the presence of Ag+ ions.

FTIR measurement indicates, the presence of functional group such as alkanes (C=C) and the presence of alcohol whereas some plant residues. These shows the ginger rhizome act as a capping and stabilizing agent for Silver Nanoparticles.

Antibacterial assay was performed by Well diffusion method. To find MIC at three different concentration such as 1mM, 3mM,and 5mM are plated for the isolated 7 clinical isolates. It shows a highly effective zone of inhibition for Staphylococcus aureus of 20mm. Finally the effective MIC is 3mM concentration. The minimal concentration of 3mM able to inhibit the causative agent of Diabetic Foot Ulcer. Further, study of toxicity checking and animal studies for the product formation will be performed later.

IV. ACKNOWLEDGMENT

The author thanks DST-FIST laboratory, DBT Star Scheme, chairman and managing Trustee of Dr. N.G.P Arts and Science College, Coimbatore, Tamil Nadu, for providing necessary facilities for carrying out this research work. Communication number: DrNGPASC 2020-2021 BS 087. Author extended her thanks to PSG Institute of Advanced studies for their help and support for characterization of nanoparticles.

V. Conflicts of Interest

The authors have no conflicts of interest to publish this research article in this journal

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27) Sondi I, Sondi BS. Silver Nanoparticles as antimicrobial agent: A Case study on E.coli as a model for gram negative bacteria.J Coll Inter Sci 2004, 275:177-182.

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29) Cunha BA. Antibiotics Selection for Diabetic foot infections: a review.J Foot Ankle Surgery. 2000; 39(4): 253-7.

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Methylene Blue Reduction Time for Milk

 

Determination of Methylene Blue Reduction Time for Milk (MBRT Test)

 

Principle:

This test is based on the principle that methyl blue (an oxidation-reduction dye or indicator) which is blue in its oxidized state, is reduced to colorless compound (leuco form) as a result of the metabolic activities of bacteria in milk. When a solution of dye is added, the organisms present in milk consume the dissolved oxygen and lower O-R potential to a level where methyl blue or similar indicators are reduced or decolorized.

 Reagent Required: Methylene Blue Reduction Dye

Procedure:

1. The samples of milk are mixed thoroughly. If the milk is in a bottle/ pocket, it shall be inverted at least 25 times to mix the fat uniformly with the milk.

2. Take 10 ml of milk into a test tube and add 1 ml of standard methylene blue solution.

3. Invert the test tube to mix the milk and methylene blue solution.

4. Place the test tube in a thermostatically maintained water bath at 37⁰C and note down the time of incubation.

5. Observe the test tubes after 30 min for decolourization reduction of dye. If there is no decolourization the tubes are inverted once and transferred to the water bath for further incubation.

6. After 30 min, continue to observe for the reduction of dye at an interval of every one-hour.

7. The milk shall be regarded as decolorized, when the entire column of milk is completely decolorized or is decolorized up to 5 mm of the surface.

MBRT Test result and interpretations:

 

MBRT TEST RESULTS	MBR Time (Hrs)	Milk Quality
	5 hrs and above	Very Good
	3 – 4 hrs	Good
	1 – 2 hrs	Fair
	Half an hour	Poor

 

Grading of milk in MBR test

Quality of milk	Reduction time	Approx. keeping quality/ml	Approx. bacterial count per ml
Good	5 hour or more	40 hrs	Below 5,00,000
Fair	2 to 5  hours	30 hrs	5,00,000 to 40,00,000
Bad	20 mins to 2 hrs	10 hrs	40,00,000 to 2,00,00,000
Very bad	20 mins or less	Less than 10 hrs	Above 2,00,00,000

 

Isolation of drug resistant mutants using UV – Gradient plate and Replica plating

 

 Isolation of drug resistant mutants using UV – Gradient plate and Replica plating

AIM:   To become familiar with gradient plate method for isolating antibiotic resistant mutants and to perform replica plating procedure for isolating and identifying Auxotrophic mutants.

Principle

 Mutations often inactivate a biosynthetic pathway of the microorganism, and frequently make a microorganism unable to grow on a medium lacking an adequate supply of the pathway’s end product. Based on this principle microorganism are classified as Prototrophic and Auxotrophic. Prototrophic organisms (wild type) have the same nutritional requirements as that of their ancestors. They need only inorganic salts, an organic energy source such as sugar, fat, protein and water to survive and grow. That is, the Prototroph’s need only "Minimal medium" for their growth and survival. Auxotrophic mutants are unable to grow without one or more essential nutrients. Auxotrophs are mutant for particular nutrient synthesis pathway enzymes. Such an error is known as an inborn error of metabolism, whether it occurs in a bacterium or a eukaryote. An auxotroph can be grown only on an enriched medium that provides the particular nutrient that the mutant cannot metabolize on its own.

Gradient Plate Technique: 

            The gradient plate consists of two wedges like layers of media: a bottom layer of plain nutrient agar and top layer of antibiotic with nutrient agar. The antibiotic in the top layer, diffuse into the bottom layer producing a gradient of antibiotic concentration from low to high.  A gradient plate is made by using Streptomycin in the medium. E. coli, which is normally sensitive to Streptomycin, will be spread over the surface of the plate and incubated for 24 to 72 hours. After incubation colonies will appear on both the gradients. The colonies develop in the high concentration are resistant to the action of Streptomycin, and are considered as Streptomycin resistant mutants. For isolation of antibiotic resistant of gram negative enteric bacteria, the antibiotics commonly used are Rifampicin, Streptomycin, and Erythromycin etc.

 

 

 

Replica Plating Method:

The technique was developed by Joshua and Esther Lederberg in 1952 for providing the direct evidence for the existence of pre-existing mutations. This technique isolates both nutritional mutants and antibiotic resistant mutants. Their actual experiment concerned with replicating master plates of sensitive cells to two or more plates containing streptomycin. Replica plating allows the observation of microbes under a series of growth conditions. The bacteria are grown in an environment that is not selective for given mutation.  This technique is used to transfer the members of each colony to a selective environment. A simple velveteen covered colony transfer device is used to transfer the colonies in nutrient agar medium supplemented with or without a particular antibiotic or nutrient.  The fibers of velvet act as fine inoculating needles, picking up the bacterial cells from the surface of this master plate. The velvet with its attached microbes is then touched to the surface of a sterile agar plate, inoculating it. In this manner, microbes can be repeatedly stamped onto media of differing composition. By comparing the presence of colonies following incubation we can indirectly determine the mutant colonies by their absence in the selective environment. A colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains. The microbes that do not grow on the minimal medium represent auxotrophic strains.

 

 

 Replica plating technique

 

 

 

Materials required: Gradient Plate Technique

 

1.       24 hour old nutrient broth culture of Escherichia coli.

2.      Two nutrient agar deep tubes (10 ml per tube/culture).

3.      1% Streptomycin sulphate solution (100 µg/ml).

4.      A beaker with 90% ethanol.

5.      Sterile Petri plates.

6.      Sterile 1 ml pipette.

7.      Glass rod spreader.

8.      Water bath.

Procedure: I) Preparation of gradient plate:

 

1.      Melt two nutrient agar plates maintained at 96⁰C and cool to 55⁰ C

2.      Pour the contents of one agar tube into a sterile petriplate. Allow the medium to solidify in a slanting position by placing either a glass rod under one side.

3.      After the agar medium is solidified remove the glass rod and place the plate in the horizontal position.

4.      Pipette out 0.1mL of 1% Streptomycin solution into the second tube of the second nutrient agar medium.

5.      Rotate the tube between the palms and pour contents to cover the gradient layer agar and allow to the medium to solidify on a level table.

6.      Label the low and high antibiotic concentration area on the bottom of the plate.

 

II) Inoculation of culture:

 

1.      Pipette out 200µl (0.2ml) of the overnight Escherichia coli culture onto the gradient plate after 24 hours of its preparation.

2.      Spread the inoculums evenly over the agar surface With a sterile bent glass rod by rotating the plate.

3.      Incubate the inoculated plate in an inverted position at 37^o C for 48-72 hours. 

4.      Observe the plate for appearance of E.coli colonies in the area of low streptomycin concentration (LSC) and high streptomycin concentration (HSC) and record the results.
 

Results: Colonies which appear in the area of high concentration streptomycin region will be streptomycin resistant mutants.

Replica plating method:

 Materials required:

1.      24 hour old nutrient broth culture of Escherichia coli.

2.      Minimal salt agar with glucose.

3.      Three 10ml Nutrient agar deeps.

4.      1% Streptomycin sulphate solution (10mg /100ml of sterile water).

5.      Sterile petridishes.

6.      Sterile velveteen colony carrier.

7.      Glass rod or wooden dowel stick .

8.      Beaker with 95% ethanol.

9.      Bent glass rod.

10.  Quebec colony counter

 

 

Procedure:

DAY 1

1.      Melt the nutrient agar deeps tubes in a hot water bath maintained at 96⁰ C.

2.      Allow the molten medium to cool to 55⁰ C.

3.      Pour the molten agar medium to two sterile petriplates and allow to solidify in a horizontal position.

4.      Add 0.1% of Streptomycin, using sterile pipette into the third tube of molten nutrient agar (maintained at 55⁰C ), properly mix by rotating between the hands and pour the contents into a sterile petriplate. Allow to solidify.

5.      Add 200 µL of(0.2mL) of the the E.coli test culture to the surface of the nutrient agar plate.

6.      Using an alchohol dipped and flamed bent glass rod spread the inoclum evenly on the plate.

7.      Incubate the plate in an inverted position for 24-48 hours at 37 ⁰ C.

8.      After incubation observe the colonies of E.coli on the plate and this plate was considered as the master plate.

DAY 2

 

1.      A reference mark (at 12 O'clock position) was noted on the bottom of the master plate, plate with nutrient agar and plate supplemented with Streptomycin.

2.      The sterile velveteen colony carrier was carefully lowered and gently pressed onto the colonies of the E.coli on the master plate.

3.      Without altering the position of the carrier, the sterile velveteen was gently pressed on to the nutrient agar plate followed by the Nutrient agar plate supplemented with Streptomycin.

4.      Incubate both the inoculated plates, nutrient agar and streptomycin agar plates in an inverted position for 48-72 hours at 37⁰C. Master plate is refrigerated.

5.      Following incubation number of colonies in the replica plates with nutrient agar and streptomycin agar was counted using Quebec Colony Counter.

6.      The colonies appearing on the nutrient agar plates and Streptomycin plates were noted and compared.

Results:

E.coli colonies which appear on the Streptomycin supplemented agar was confirmed as Streptomycin resistant mutants and the number of colonies were counted using Quebec Colony Counter.