chapter 4 result and dissicusion

RESULTS AND DISCUSSION

4.1. Separation of bran from endosperm of Finger millet.

Finger millet bran was separated from endosperm and the yield was found to be (~5-

7%) However, the bran contains associated starch (~10%). It was found necessary to

remove the starch portion before the use of bran for extraction of Xylo-

oligosaccharides. Hence bran was further subjected to destarching.

Figure 18: Finger millet bran after endosperm separation.

4.2. Removal of associated starch form the bran portion.Starch (~10%) was found to be associated with the finger millet bran and removed

by the successive treatments of thermostable bacterial alpha amylase and fungal

gluco amylase. Alpha amylase is an endoenzyme which hydrolyses alpha bonds of

large, alpha linked polysaccharide such as starch malto-oligosaccharides, it cleaves

via random fashion (endo attack) and liberates unattacked alpha 1-6 dextrin and

malto-oligosaccharides. Glucoamylase (1,4-alpha-D- glucan glucohydrolase) is an

exoenzyme catalyses the release of glucose from the non-reducing ends of starch. It

has the specificity for both alpha-1,4 and alpha 1-4 linkage thereby has the capacity

to degrade the alpha-1,6 linkage dextrin’s and alpha-1,4 linked malto-

oligosaccharides into glucose(). The destarched bran was dried by solvent exchange

and yield was found to be 69%.

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Figure 19: Destarching of Bran by using Alpha-amylase from thermostable

bacteria Lichenifromis (Image courtesy of Sigma-Aldrich.)

Figure 20: Finger millet bran after destarching process.

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4.3. Xylanse treatment of destarched finger millet bran.

Finger millet (25g) was treated with Xylanase endoenzyme which after treatment

yielded oligosaccharide (18%) in total. Xylanse enzyme degrades the linear

polysaccharide beta-1, 4-xylan into xylo-oligosaccharide. These oligosaccharides

were further concentrated and used for sugar composition analysis.

Figure 21: Xylanse action. (Lauren S. McKee, 6537–6542)

4.4. Composition of mixture of oligosaccharides

Sugar composition of xylo-oligosaccharide was determined by using gas liquid

chromatography and was found to be arabinose and xylose in 1:2.7 ratio. The uronic

acid content was found to be less than <2%. Reducing sugar was found to be

1.6mg/mL. Protein estimation was also performed and found to be absent in the

mixture.

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4.5. Composition of bound phenolic acids in the mixture of xylo-oligosaccharidesBound phenolic acids are the acids which are were attached to xylo-oligosaccharides

via ester linkage which were determined by HPLC as ferulic acid (83%) followed by

syringic acid (5%), gallic acid (4%), protocatechuic acid (1.5%), gentisic acid

(1.5%) and caffeic acid (0.5%).

4.6. Separation of mixture of xylo-oligosaccharides on Amberlite XAD-2 columnThe XOs mixture (30mg/5mL) was separated into three fractions; a) water eluent

(concentration 7mg/mL), b) methnol: water 1:1 ration eluent (1.75 mg/mL) and c)

methol eluent (<0.002mg/mL). Water eluent accounts for 70% were as methnol:

water accounts for 25%. The amount of sugar present in methanol elution is

negligible.

Mechanism by which separation takes place: “In the adsorption process, the

hydrophobic portion of the adsorbate molecule is favourably adsorbed on the

hydrophobic polystyrene surface of the resin, while the hydrophilic section of the

adsorbate remains oriented in the aqueous phase. Compound(s) being adsorbed

usually do not penetrate substantially into the microsphere phase, but remain

adsorbed at the surface. Therefore, with proper elution or regeneration techniques,

the adsorbed compound can be rapidly eluted, because of the high rate of diffusion

of the elution mobile phase through the porous structure of each bead. Since the

compound is bound to the outer and inner surfaces of the beads, penetration or

solvation of the microspheres by the eluting agent is neither involved nor obligatory.

The selectivity and extent of adsorption of soluble organic compounds by Amberlite

XAD-2 resin increases as the hydrophobicity of the adsorbate molecule increases.

The adsorption forces are primarily van der Waals type. Thus, you can change the

extent of adsorption of a compound by changing its hydrophobic/ hydrophilic

balance. For example, weak acids are more strongly adsorbed in the acid form than

in the salt form. A weak acid then can be eluted with caustic soda (sodium

hydroxide). In yet other cases, polar solvent(s)/water mixtures are very effective

elution mobile phases when such solvents have greater affinity for the adsorbed

compounds than does the resin.”( Amberlite, XAD – Rohm & Haas Co. Supelpak –

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Sigma-Aldrich Co.) The resultant elution has water eluent (neutral oligosaccharide)

and methanol : water eluent . Which was further subjected to compositional analysis

analysis by ESI-MS.

4.7. Compositional Analysis

4.7.1 Water eluent Neutral oligosaccharides.

Sugar composition of neutral oligosaccharide indicated arabinose and xylose in

different ratio. ESI-MS mass spectra indicated molecular weight of xylose (at 151)

and xylotriose (at 413) respectively.

4.7.2 Methano:Water eluent Acidic oligosaccharides.

Sugar composition of neutral oligosaccharide indicates arabinose and xylose in the ratio 1:2.69 and acidic oligosaccharide to be 1:3.68 respectively.

Table 6: Structural Analysis by ESI-MS

Eluents % Yield Nature of XOS

Sugar

Composition by

GLC(Ara: Xyl)

Neutral oligosaccharide

(Amberlite XAD- 2)70 Neutral XOS 1:2.69

Acidic oligosaccharide

(Amberlite XAD- 2)20- 25

Feruloylated and

acetylated XOS1:3.68

4.8. HPLC analysis for determination of bound phenolic acids.

High performance liquid chromatography indicated presence of Ferulic acid (~80%) and other bound pheolic acids (~20%).

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Figure 22: BPA HPLC profile isolated from methanol- water eluent (Amberlite XAD- 2).

4.3. Prebiotic Activity

In vitro experiments was investigated out using mixture of XOs and purified

components (NOs and AOs) along with four sugar standards, galactronic acid,

glucuronic acid, arabinose and xylose demonstrated their prebiotic nature with

respect to Bifidobacteria and Lactobacilli sp. in terms of the growth characteristics

pattern.In vitro experiments allow the comparison of the rates at which

oligosaccharides are broken down and consumed in fermentation experiments.

SCFAs produced as a result of the fermentation of NDOs result in the decline of pH

of culture broth. Such decrease in pH can be used as an indication of the prebiotic

effect of the oligosaccharides incorporated in the culture broth. A decrease in the pH

of the culture broth and increase in OD were observed for all the strains grown on

mixture of XOs, purified components and sugar standards after 24 h incubation.

Lactobacillus sp. showed maximum O.D. in the present investigation. A

simultaneous increase in the dry cell mass was also observed compared to the control

after 24 h of incubation proving that XOs and other samples were utilized by the

beneficial microbes and has enhanced their growth. Purified neutral oligosaccharides

is more effective than acidic oligosaccharides wherein the OD of the culture broth

and bacterial cell mass were found to be marginally high. But as compared to the

mixture of XOs it was significantly low. The growth of the microorganism on

particular oligosaccharide may be strain specific. With respect to four different

strains used for the present study lactobacillus spp. as compared to bifidobacteria

spp. had slightly more activity however the difference is very marginal.

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4.3.1 Enzyme Activities in the 24 h Microbial Culture Broth

The 24 h cultures showed xylanase, xylosidase, and feruloly esterase activities in

present study. High activity of xylanase was identified in the culture broth of all the

tested microorganisms grown on standard xylose as compared to the test sample,

amongst which XOs showed more xylanase activity. Other standard sugars didn’t

have any xylanase activity. Beta- D-Xylopyranosidase enzyme assay was also

examined and found that there was not much difference in mixture of XOs and the

purified components, however when standards are compared only xylose showed the

activity. Feruloyl esterase assay was performed to see its presence and it was

reported only in the culture filtrate of mixture of xylo-oligosaccharides and acidic

oligosaccharide as it has bound phenolic acids (ferulic acid).

4.3.2. Short chain fatty acid

The hydrolytic enzymes formed by the microorganisms help in the digestion of

NDOs, which escape digestion in the upper gastrointestinal tract. SCFA are

produced as a consequence of the fermentation of NDOs. SCFA in the 24 h

microbial culture broth was analysed by GLC Acetic, propionic and butyric acids are

the major SCFA produced during fermentation of XOs. Acetate was the foremost

SCFA released by the microorganisms due to fermentation of culture filtrate. These

three major SCFA are absorbed at comparable rate in different region of colon. Once

absorbed, SCFA are metabolized at three major sites in the body, 1) cells of the

ceco-colonic epithelium that use butyrate as the major substance for the maintenance

of energy producing pathway, 2) liver cells that metabolize residual butyrate with

propionate used for gluconeogenesis, 50% to 70% of acetat is also taken up by the

liver, 3) muscles cells that generates energy from thr oxidation of residual acetate, 4)

acetate being the major SCFA in the colon is readily absorbed and transported to

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liver and therefore less metabolized in the colon and helps in the production of

Acetyl-coA which is further responsible for liponeogenesis.

Figure 23: At 5.24 retention time

Acetic acid is been estimated,

standard of Acetic acid


Propionic acid is been estimated,

standard of Propionic acid


Butyric acid is been estimated,

standard of Butyric acid.

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4.3.3. Table 7: 24 hour’s incubation growth characteristic of microorganism on mixture of xylo-oligosaccharide and its purified components extracted from Finger Millet.

Sample O.D. at 600 nm pH Cell MassControl Sample Control Sample Control Sample

XOS B1 0.617 ± 0.2 1.155 5.73 5.27 1.2mg 1.7mgXOS B2 0.620 ± 0.2 1.128 5..8 5.43 1.6mg 1.8mgXOS L1 0.615±0.1 1.3 5.81 5.59 1.3mg 1.6mgXOS L2 0.617±0.2 1.280 5.85 5.50 1.5mg 1.7mg

NOS B1 0.451±0.2 0.533 6.19 5.81 1.6mg 1.9mgNOS B2 0.452±0.2 0.580 6.15 5.75 1.5mg 1.7mgNOS L1 0.201±0.1 0.637 6.29 5.82 1.2mg 1.5mgNOS L2 0.203±0.2 0.634 6.32 5.08 1.3mg 1.5mg

AOS B1 0.017±0.1 0.085 7.3 6.49 1.7mg 1.9.mAOS B2 0.018±0.1 0.105 7.5 6.84 1.1mg 1.5mgAOS L1 0.019±0.1 0.364 7.38 6.97 1.3mg 1.4mgAOS L2 0.017±0.1 0.165 7.4 6.99 1.2mg 1.6mg

ARA B1 0.025±0.01 0.107 7.12 6.92 1.4mg 1.8mgARA B2 0.026±0.01 0.099 7.14 6.82 1.5mg 1.7mgARA L1 0.034±0.01 0.252 7.29 7.18 1.2mg 1.3mgARA L2 0.036±0.01 0.183 7.30 7.12 1.1mg 1.4mg

GAL B1 0.061±0.01 0.215 5.58 5.01 1.1mg 1.5mgGAL B2 0.063±0.01 0.156 5.60 4.77 1.3mg 1.6mgGAL L1 0.083±0.02 0.464 5.79 5.31 1.1mg 1.6mgGAL L2 0.085±0.01 0.188 5.70 5.11 1.5mg 1.8mg

GLcA B1

0.035±0.01 0.174 6.05 5.07 1.3mg 1.7mg

GLcA B2

0.034±0.01 0.274 6.10 5.20 1.3mg 1.9mg

GLcA L1

0.019±0.02 0.318 6.31 5.21 1.4mg 1.5mg

GLcA L2

0.017±0.01 0.187 6.33 5.37 1.5mg 1.6mg

XYL B1 0.041±0.01 0.180 7.35 5.91 1mg 1.4mgXYL B2 0.043±0.01 0.233 7.40 5.26 1.3mg 1.9mgXYL L1 0.016±0.01 0.291 7.31 5.23 1.4mg 1.7mg

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XYL L2 0.018±0.01 0.101 7.40 5.18 1.3mg 1.3mgAll the results are the average of two experiments and the values are been subtracted from blank values before its consideration.Blank only consist of fermentation media and organism without carbon source. (XOS-xylo-oligosaccharide, NOS- neutral oligosaccharide, AOS-acidic oligosaccharide, ARA-arabinose, GAL- galactronic acid, GLcA- glucuronic acid and XYL- xylose; B1-B. adolescentist 236 and B2-B. bifdum 235 strain and L1-L. brevis 01 and L2-L. acidophilus 011)4.3.4. Table 8: Enzyme activity (mU/mL) in 24 hours culture broth of microorganism grown on mixture of xylo-oligosaccharide and its purified components.

SAMPLE ENZYME ACTIVITY

XYLANASEmU/min/mL

XYLOPYARANOSIDASEmU/min/mL

FERULOLY ESTERASEµU/min/mL

XOS B1 0.436 0.020 6.7XOS B2 0.31 0.021 2.7XOS L1 1.35 0.008 6.9XOS L2 0.596 0.008 2.6

NOS B1 0.67 0.013 NDNOS B2 0.59 0.016 NDNOS L1 0.32 0.012 NDNOS L2 0.52 0.014 ND

AOS L1 0.60 0.015 6.68AOS L2 0.31 0.025 6.9 AOS B1 0.27 0.017 6.9 AOS B2 0.56 0.015 7.2

ARA B1 ND ND NDARA B2 ND ND NDARA L1 ND ND NDARA L2 ND ND ND

GAL B1 ND ND NDGAL B2 ND ND NDGAL L1 ND ND NDGAL L2 ND ND ND

GLcA B1 ND ND NDGLcA B2 ND ND NDGLcA L1 ND ND NDGLcA L2 ND ND ND

XYL B1 5.6 0.023 NDXYL B2 4.9 0.025 ND

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XYL L1 5.80 0.021 NDXYL L2 5.86 0.019 ND

Xylanse and Xylopyaranisidase activity were estimated spectrophotomectrically were as feruloly esterase activity was estimated by HPLCND: Not detected. (XOS-xylo-oligosaccharide, NOS- neutral oligosaccharide, AOS-acidic oligosaccharide, ARA-arabinose, GAL- galactronic acid, GLcA- glucuronic acid and XYL- xylose; B1-B. adolescentist 236 and B2-B. bifdum 235 strain and L1-L. brevis 01 and L2-L. acidophilus 011)Table 9: Proportions of acetic, propionic and butyric acid as a percentage of total SCFA formed after 24 hours incubation in the culture broth of microbes incorporated with mixture of xylo-oligosaccharides, its purified components and also standard sugars. The values are presented in µmol.

SAMPLE SHORT CHAIN FATTY ACIDAcetic acid Butyric acid Propionic acid

XOS B1 5.8 ND NDXOS B2 4.8 ND NDXOS L1 8.8 0.24 0.2XOS L2 1.1

NOS B1 15.3 0.024 NDNOS B2 4.36 0.138 NDNOS L1 2.4 ND NDNOS L2 4.66 0.054 ND

AOS B1 15.5 0.4 NDAOS B2 13.24 0.63 NDAOS L1 1.14 0.13 NDAOS L2 1.12

ARA B1 3.81 0.25 0.040ARA B2 ND ND NDARA L1 4.82 0.4 0.058ARA L2 19.86 0.52 0.098

GAL B1 18.89 0.83 0.16GAL B2 1.32 0.97 NDGAL L1 11.94 0.22 0.64GAL L2 15.49 0.81 0.14

GLcA B1 23.32 0.129 1.65GLcA B2 53.17 2.15 0.05GLcA L1 2.47 0.016 NDGLcA L2 6 1.7 ND

XYL B1 34.66 0.24 0.05XYL B2 50.134 ND NDXYL L1 49.63 0.240 NDXYL L2 3.19 0.048 0.029

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(XOS-xylo-oligosaccharide, NOS- neutral oligosaccharide, AOS-acidic oligosaccharide, ARA-arabinose, GAL- galactronic acid, GLcA- glucuronic acid and XYL- xylose; B1-B. adolescentist 236 and B2-B. bifdum 235 strain and L1-L. brevis 01 and L2-L. acidophilus 011

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-oligosaccharides and the 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 (10mg/mL). 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. 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.

81


Figure 26: Agar well diffusion assay performed by using sample to study antimicrobial activity of the xylo-oligosaccharides and its purified components on M.luteus.

Figure 27: Agar well diffusion assay performed by using standard sugars to study antimicrobial activity of the Galactronic acid, Glucuronic acid, Arabinose and Xylose on M.luteus

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Table 10: Antimicrobial activity of Xylo-oligosaccharides and its purified components agar diffusion method results.

Sample Concentrat-ion

Organism

M.luteus B.subtlius S.aureus P.aeruginosa E.coli

AMP 100µg/mL50µg/mL10µ/mL

+++++++++++++

+++++++++++++

+++++++++++++

+++++++++++++

+++++++++++++

XOS 12.6mg/mL13.5mg/mL15mg/mL

+++++++

±±+

+++++++

±±±

++++

NOS 3.2mg/mL7.4.mg/mL8.5mg/mL

--+

--±

-±±

--±

±±±

AOS 0.7mg/mL1.5mg/mL5.5mg/mL

++++++

-±+

±++

±±+

±±+

ARA 1g/mL ± ± ± ± ±

GAL 1g/mL ++++ ++ ++ ++ ++

GUL 1g/mL ++++ ++ ++ ++ ++

XYL 1g/mL ± ± ± ± ±

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)

++++ represents high activity+++ represents medium activity++ represents intermediate activity+ represents low activity± represents slight activity but not considered as inhibitory concentration.

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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.

Table 11: MIC reading of positive control Ampicillin

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

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.

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Standard graphs of MIC of Ampicillin

0 0.5 1 1.5 2 2.5 30

0.020.040.060.08

0.10.12 0.111

0.003 0.001 0.001 0.002

M.luteus

CONCENTRATION µg/mL

O.D

AT

600n

m

Figure 28: M.luteus MIC Ampicillin graph

0 0.5 1 1.5 2 2.5 30

0.020.040.060.08

0.10.12 0.105 0.099

0.0820.068 0.058

B.subtilius

CONCENTRATIO µg/mL

O.D

. AT

600n

m

Figure 29: B.subtilius MIC Ampicillin graph

0 0.5 1 1.5 2 2.5 30

0.0020.0040.0060.008 0.007

0.0040.003

0.002 0.002

S.aureus

CONCENTRATION µg/ml

O.D

. AT

600

nm

Figure 30: S.aureus MIC Ampicillin graph

0 0.5 1 1.5 2 2.5 30

0.10.20.30.4 0.335

0.19

0.022 0.019 0.011

Psuedomonas aerug-inosa


O.D

. AT

600

nm

Figure 31: Pseudomonas aeruginosa MIC Ampicillin graph

0 0.5 1 1.5 2 2.5 30

0.050.1

0.150.2

0.250.3 0.247 0.242

0.1920.157

0.089

E.coli


O.D

. AT

600n

m

Figure 32: E.coli MIC Ampicillin graph

Ampicillin is an antibiotic which is used against a number of bacterial infections. It is a beta-lactam antibiotic. It is active against many Gram-positive and Gram-

85


negative bacteria. Belonging to the penicillin group of beta-lactam antibiotics, ampicillin is able to penetrate Gram-positive and few Gram-negative bacteria Ampicillin acts as an irreversible inhibitor of the enzyme transpeptidase, which is required by bacteria to make their cell walls. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, which eventually leads to cell lysis; hence ampicillin was used as bacteriocidal , positive control in the present study purpose. After conducting minimal inhibitory concentration studies it was found that less concentration of ampicillin is required for inhibiting gram positive bacteria then gram negative bacteria due to cell wall structure. M.luteus required least concentration whereas E.coli required the highest concentration for it inhibition amongst all the pathogens investigated in this studies.

Sample MIC reading

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Table 12: MIC reading of the sample used.

Oraganism/sample

Blank Control Dilutions

25 50 75 100M.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.luteusAOS

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

XOS - mixture of xylo-oligosaccharide (Sample control), NOS – Neutral Oligosaccharide and AOS – Acidic Oligosaccharide.Blank: Culture mediaControl: Organism along with culture media but without sample.All the readings were subtracted from blank reading.

Concentration of the sample used for the experiment.

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Table 13: Concentration of sample used for MIC.

SAMPLE / VOLUME

25µL 50µL 75µL 100µL

XOS→CONC. 370µg 740µg 1120µg 1500µgNOS→CONC. 175µg 350µg 525µg 700µgAOS→CONC. 125µg 250µg 375µg 500µg

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 shows more antimicrobial activity. Butyl esters of the phenolic acids effectively inhibits the gram positive microorganism.

Figure 33: Minimal inhibitory concentration studied carried out in flat bottom ELISA plate and O.D. was read at 600nm.

Graph of MIC results of the experimental sample.

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M.luteus

Figure 34: Xylo-oligosaccharide MIC graph for M.luteus

Figure 35: Neutral oligosaccharide MIC graph for M.luteus

Figure 36: Acidic oligosaccharide MIC graph for M.luteus

Figure 37: Comparison of MIC reading of sample for M.luteus

M.luteus being gram positive pathogen xylo-oligosaccharide mixture showed maximum activity against it giving least concentration for MIC studies. Whereas neutral oligosaccharides showed more increase in concentration to show the desired effect and acidic oligosaccharide could inhibit the test pathogen efficiently with used concentration for the study.

B.subtilius

89

20 30 40 50 60 70 80 90 100 1100

0.020.040.060.08

0.10.12 0.098 0.093

0.0790.056

XOS

VOLUME µL

O.D

. AT

600

nm


Figure 38: Xylo-oligosaccharide MIC graph for B.subtilius

Figure 39: Neutral oligosaccharide MIC graph for B.subtilius

Figure 40: Acidic oligosaccharide MIC graph for B.subtilius

Figure 41: Comparison of MIC reading of sample for B.subtilius

Bacillus subtilius even though being gram positive bacteria differs from the rest test pathogens as it can form endospores to protect it from extreme environmental conditions of temperature and other elements. As endospore is a dormant, tough, and non-reproductive structure it’s difficult for antibacteriocidals to inhibit it. In the present it was found that more concentration would require for the all the sample to shown effective minimal inhibitory concentration.

S.auerus

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Figure 42: Xylo-oligosaccharide MIC graph for S.aureus

Figure 43: Neutral oligosaccharide MIC graph for S.aureus

Figure 44: Acidic oligosaccharide MIC graph for S.aureu

Figure 45: Comparison of MIC reading of sample for S.aureus

S.aureus is gram positive bacteria which was effectively inhibited by mixture of xylo-oligosaccharides in present study but required increase in concentration for neutral and acidic oligosaccharides for effective inhibition with least concentration.

P.aeruginosa

91


Figure 46: Xylo-oligosaccharide MIC graph for P.aeruginosa

Figure 47: Neutral oligosaccharide MIC graph for P.aeruginosa

Figure 48: Acidic oligosaccharide MIC graph for P.aeruginosa

Figure 49: Comparison of MIC reading of sample for P.aeruginosa

P.aeruginosa being gram negative bacteria required more concentration of all the test sample used for the study purpose for efficient inhibition to be considered. The reason for increase in concentration is the cell wall structure of gram negative bacteria.

E.coli

92

20 30 40 50 60 70 80 90 100 1100

0.10.20.30.40.50.6

XOS

VOLUME µL

O.D

. AT

600

nm

20 30 40 50 60 70 80 90 100 1100

0.10.20.30.40.50.6 0.548

0.4870.42

0.34

NOS

VOLUME µL

O.D

AT

600

nm


Figure 50: Xylo-oligosaccharide MIC graph for E.coli

Figure 51: Neutral oligosaccharide MIC graph for E.coli

Figure 52: Acidic oligosaccharide MIC graph for E.coli

Figure 53: Comparison of MIC reading of sample for E.coli

E.coli was effectively inhibited by mixture of xylo-oligosaccharide and neutral oligosaccharide but required increase in concentration of acidic oligosaccharide to show productive results.

Table 14: Minimal inhibitory concentration of the sample used against the test organism

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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

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 finger millet Elucina coracana had antimicrobial activity against test microbial pathogen. In accordance with studies carried out on xylo-oligosaccharide and 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.

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 negative 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 pass 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 probable reason for the antimicrobial activity of xylo-oligosaccharides is presence of bound phenolic compound present in them. It has been already reported that phenolic compounds are related to inactivation of cellular enzymes, which depends on the rate of penetration of the substance into cell or caused by membrane permeability changes. Increased membrane permeability is the main cause in the mechanism of antimicrobial action where compounds may disrupt membrane and cause loss of cellular integrity and eventual cell death. This cellular enzymes are essential for chemical reactions inside the cell, they are proteins. They are made up of special chains of amino acid that come together in different shapes to do distinctive jobs, like breaking down sugar and fat molecules or to make more enzyme. Enzyme activity can be inhibited by binding to specific small molecules and ions. Enzyme inhibition serves as a major control mechanism in biological system.

Hence results revealed significant results against antimicrobial activity and in variability in the inhibitory concentration of each extract against test pathogen.

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Antifungal activity

The fungal infections still remain one of the most common as well as the imperative problem associated with the daily life. The proficient, lesser toxic and more effective drug against them is yet to be taken in concern as most of the fungi are developing resistance against the antifungal drugs which are being used comprehensively and some of them are showing toxic effect on human as well. In such consequence, the natural phenolic compounds have come into noticed as they are proving to be very potent antifungal agents with lesser or no toxic effect. Not only can they themselves be used as antifungal agent but also in synergism with the currently used antifungal agents. In the current studies mixture of xylo-oligosaccharides and purified components was used against Penicillum and Monascus using agar well diffusion assay. It was found that were was slight activity against the fungal pathogen but not that significant to report its MIC. Further investigation would be necessary to report any significant activity by increasing its concentration of the test sample.

Figure 54: Agar well diffusion of Monascus

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Figure 55: Agar well diffusion of Penicillum

Figure56: Front view of the assay plate.

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chapter 4 result and dissicusion

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