The antimicrobial ability of Xylo-oligosaccharide

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CHAPTER 4: RESULTS AND DISCUSSION

CHAPTER 4

RESULT AND DISCUSSION

Antimicrobial Activity

Prebiotic affect the gut microflora (probiotic bacteria), by bringing the much desired effects such as decreasing the intestinal pH, production of SCFA and vitamins and immune activation (Schley and Field, 2002). It is also been considered that prebiotic/probiotics can modulate growth/activity of pathogenic bacteria by virtue of their antimicrobial activity.

In the present study, the inhibitory effect of different extracts (viz. Water and Methanol: Water (1:1)) of Xylo-oligosaccharides and its purified components (neutral oligosaccharide and Acidic oligosaccharides) for bacterial and fungal strains. The antimicrobial activity was evaluated by using agar well diffusion method and micro dilution method summarized in Table 1-2. The activity was quantitatively estimated on the basis of inhibition zone and their activity index was also calculated along with minimum inhibitory concentration (MIC).

Measurement of antimicrobial activity using Agar well diffusion Method

The antimicrobial ability of Xylo-oligosaccharide and its purified components was determined according to their zone of inhibition against various pathogens and the results (zone of inhibition) were compared with the activity of the standards, viz., Ampicillin (1.0 mg/disc), and Cyclohexamide (). The results proclaimed that all the extracts are potent antimicrobials against all the microorganisms studied. Among the different solvents extracts studied mixture of xylo oligosaccharide, water extractable and methanol: water extractable along with standard sugars (arabinose, galactronic acid, glucuronic acid and xylose) showed high to intermediate degree of inhibition. For all the tested microorganisms’ mixture of xylo-oligosaccharide, methanol: water and standard sugar galactronic and glucuronis acid showed maximum antibacterial activity in Eleucina coracana. Table no. represents the antimicrobial activity of the samples and standard sugar .In samples tested with the highest concentration used maximum inhibition zone diameter was obtained in M.luteus with diameter 25±5 mm 13±16mm, and 17±3 for samples respectively and for standards used 2±0.5mm, 1±0.5mm, 16±2mm and17±1.5 mm respectively, is represented in figure no.

Sample

Concentrat-ion

Organism

M.luteus

B.subtlius

S.aureus

P.aeruginosa

E.coli

AMP

100µg/mL

50µg/mL

10µ/mL

+++++

++++

++++

+++++

++++

++++

+++++

++++

++++

+++++

++++

++++

+++++

++++

++++

XOS

12.6mg/mL

13.5mg/mL

15mg/mL

++

++

+++

±

±

+

++

++

+++

±

±

±

+

+

++

NOS

3.2mg/mL

7.4.mg/mL

8.5mg/mL

-

-

+

-

-

±

-

±

±

-

-

±

±

±

±

AOS

0.7mg/mL

1.5mg/mL

5.5mg/mL

+

++

+++

-

±

+

±

+

+

±

±

+

±

±

+

ARA

1g/mL

±

±

±

±

±

GAL

1g/mL

++++

++

++

++

++

GUL

1g/mL

++++

++

++

++

++

XYL

1g/mL

±

±

±

±

±

Table: Antimicrobial activity of Xylo-oligosaccharides and its purified components agar diffusion method results.

Amp – Ampicillin (positive control)

XOS – mixture of xylo-oligosaccharide (Sample control)

NOS – Neutral Oligosaccharide

AOS – Acidic Oligosaccharide

ARA – Arabinose (Standard sugar)

GAL – Galactronic acid (Standard sugar)

GUL – Glucuronic acid (Standard sugar)

XYL – Xylose (Standard sugar)

Determination of MIC values

Minimum Inhibitory Concentration (MIC) is defined as the highest dilution or least concentration of the sample used that inhibit growth of organisms. Determination of the MIC is important in diagnostic laboratories because it assists in confirming resistance of micro-organism to an antimicrobial agent and it guides the activity of new antimicrobial agents. MIC was determined of the sample using ELISA plate in microtiter wells. Ampicillin was used as the positive control to determine the MIC against five different organisms used. Sample was tested with four different dilutions and the reading was read at 600nm.

Organism

Blank

Control

Concentration g/mL

0.5

1

1.5

2

2.5

M.luteus

0.057

0.215

0.168

0.060

0.059

0.058

0.057

B.subtilis

0.056

0.267

0.105

0.099

0.082

0.068

0.058

S.aureus

0.056

0.462

0.064

0.061

0.060

0.059

0.058

P.aeruginosa

0.056

0.448

0.392

0.247

0.079

0.076

0.068

E.coli

0.056

0.316

0.304

0.299

0.249

0.214

0.146

Table: MIC reading of positive control Ampicillin

Blank: only culture media.

Control: Test organism along with culture media but without antibiotic.

Reading: All the O.D. readings were read at 600nm and subtracted from the blank reading.

Standard graphs of MIC of Ampicillin

Fig: M.luteus MIC Ampicillin graph

Fig: B.subtilius MIC Ampicillin graph

Fig: S.aureus MIC Ampicillin graph

Fig: Pseudomonas aeruginosa MIC Ampicillin graph

Fig: E.coli MIC Ampicillin graph

  • Sample MIC reading

Oraganism

/sample

Blank

Control

Dilutions

25

50

75

100

M.luteus XOS

0.047

0.214

0.098

0.093

0.079

0.056

M.luteus NOS

0.047

0.174

0.151

0.151

0.133

0.116

M.luteus

AOS

0.047

0.179

0.176

0.136

0.094

0.099

B.subtilius XOS

0.047

0.344

0.324

0.188

0.121

0.121

B.subtilius NOS

0.047

0.267

0.457

0.358

0.359

0.104

B.subtilius AOS

0.047

0.307

0.227

0.169

0.151

0.071

S.aureus

XOS

0.090

0.757

0.405

0.207

0.173

0.168

S.aureus

NOS

0.089

0.723

0.469

0.456

0.332

0.293

S.aureus

NOS

0.089

0.782

0.496

0.476

0.465

0.438

P.aeruginos XOS

0.086

0.818

0.494

0.365

0.291

0.284

P.aeruginos NOS

0.086

0.820

0.548

0.487

0.420

0.340

P.aeruginos AOS

0.088

0.817

0.543

0.525

0.520

0.361

E.coli

XOS

0.047

0.407

0.299

0.103

0.092

0.064

E.coli

NOS

0.048

0.386

0.337

0.137

0.124

0.085

E.coli

AOS

0.047

0.398

0.271

0.166

0.137

0.091

Table: MIC reading of the sample used.

XOS – mixture of xylo-oligosaccharide (Sample control), NOS – Neutral Oligosaccharide and AOS – Acidic Oligosaccharide.

Blank: Culture media

Control: Organism along with culture media but without sample.

All the readings were subtracted from blank reading.

  • Concentration of the sample used for the experiment.

SAMPLE / VOLUME

25µL

50µL

75µL

100µL

XOS→CONC.

370µg

740µg

1120µg

1500µg

NOS→CONC.

175µg

350µg

525µg

700µg

AOS→CONC.

125µg

250µg

375µg

500µg

Table: concentration of sample used for MIC.

For experiment carried out with three different samples different concentrations of the sample was used to check the MIC of it against five different test organisms. Due to presence of different level of concentration of the sample there was difference amongst each concentration use. Mixture of xylo-oligosaccharide being the high in sugar concentration, high concentration was easy to use, were as neutral and acidic oligosaccharides being the purified components of xylo-oligosaccharide its concentration varies accordingly. The ability of each of the sample used depends upon the phenolic compound present in the sample. Phenolic are been reported to have more antimicrobial activity than any other compound present in the oligosaccharide composition. Phenolic acid derivatives having long alkyl chain shows more antimicrobial activity. Butyl esters of the phenolic acids effectively inhibits the gram positive microorganism.

  • Graph of MIC results of the experimental sample.
  • M.luteus

Fig: Xylo-oligosaccharide MIC graph

Fig: Neutral oligosaccharide MIC graph

Fig: Acidic oligosaccharide MIC graph

Fig: comparison of MIC reading of sample.

  • B.subtilius

Fig: Xylo-oligosaccharide MIC graph

Fig: Neutral oligosaccharide MIC graph

Fig: Acidic oligosaccharide MIC graph

Fig: comparison of MIC reading of sample

  • S.auerus

Fig: Xylo-oligosaccharide MIC graph

Fig: Neutral oligosaccharide MIC graph

Fig: Acidic oligosaccharide MIC graph

Fig: comparison of MIC reading of sample

  • P.aeruginosa

Fig: Xylo-oligosaccharide MIC graph

Fig: Neutral oligosaccharide MIC graph

Fig: Acidic oligosaccharide MIC graph

Fig: comparison of MIC reading of sample

  • E.coli

Fig: Xylo-oligosaccharide MIC graph

Fig: Neutral oligosaccharide MIC graph

Fig: Acidic oligosaccharide MIC graph

Fig: comparison of MIC reading of sample

Sample

Organism

M.luteus

B.subtilius

S.aureus

P.aeruginosa

E.coli

XOS

1500µg

+1500µg

1500µg

+1500µg

1500µg

NOS

+700µg

+700µg

+700µg

+700µg

700µg

AOS

500µg

+500µg

+500µg

+500µg

+500µg

Table: Minimal inhibitory concentration of the sample used against the test organism

The effect of the three sample on the test pathogen were summarized in the table. Average of the three experiment were carried out and it was antimicrobial activity of these samples was reported in the present study. The result showed that sample from Elucina coracana had antimicrobial activity against test microbial pathogen. In accordance with studies carried out on xylo-oligosaccharide ad its purified components show high activity against gram positive organisms. M.luteus was found to be more sensitive amongst all were as other were comparatively resistant against the sample, sensitivity of these pathogenic bacteria would increase with increase in the concentration of the sample as noticeable activity was reported.

It was observed that the antimicrobial activity of different sample varies from one sample to other in different research carried all around. This may be due to various factors like, effect of climate, soli composition, age and vegetation cycle stage, sample source, quality, quantity and composition of the extracted product and also on the different test pathogens used. (Masotti, V. et.al. 2003). Furthermore, different studies found that the type of solvent has an important role in the process of extracting.

In the present study it was reported that gram positive bacteria are more resistant special B.subtilius as spores from it more resistant to environmental condition than any other tested bacteria. E.coli and S.aureus which are already known to be multi-resistant pathogens showed low sensitivity. The activity difference between gram positive and gram negative bacteria is due to lipopolysaccharide layer of gram naegative bacteria in the outer membrane have a high hydrophobicity which acts as a strong permeability barrier against hydrophobic molecules (Smith Palmer et. al., 1998). Hydrophobic molecules can apss through cell of the gram positive bacteria easier than the gram negative bacteria because cell wall of the gram positive bacteria contains only peptidoglycan ( Nikaido H. et.al., 1985).

The results revealed variability in the inhibitory concentration of each extract against test pathogen.

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