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Dual Role of Acidic Diacetate Sophorolipid as Biostabilizer for ZnO Nanoparticle Synthesis and Biofunctionalizing Agent Against Salmonella enterica and Candida albicans
Dual Role of Acidic Diacetate Sophorolipid as Biostabilizer for ZnO Nanoparticle Synthesis and Biofunctionalizing Agent Against Salmonella enterica and Candida albicans
Journal of Microbiology and Biotechnology. 2014. Jan, 24(1): 87-96
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : July 30, 2013
  • Accepted : October 12, 2013
  • Published : January 28, 2014
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About the Authors
Geetanjali Basak
Devlina Das
Nilanjana Das
nilanjana00@lycos.com

Abstract
In the present study, a yeast species isolated from CETP, Vellore, Tamilnadu was identified as Cryptococcus sp. VITGBN2 based on molecular techniques and was found to be a potent producer of acidic diacetate sophorolipid in mineral salt media containing vegetable oil as additional carbon source. The chemical structure of the purified biosurfactant was identified as acidic diacetate sophorolipid through GC-MS analysis. This sophorolipid was used as a stabilizer for synthesis of zinc oxide nanoparticles (ZON). The formation of biofunctionalized ZON was characterized using UV-visible spectroscopy, XRD, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy. The antimicrobial activities of naked ZON and sophorolipid functionalized ZON were tested based on the diameter of inhibition zone in agar well diffusion assay, microbial growth rate determination, protein leakage analysis, and lactate dehydrogenase assay. Bacterial pathogen Salmonella enterica and fungal pathogen Candida albicans showed more sensitivity to sophorolipid biofunctionalized ZON compared with naked ZON. Among the two pathogens, S. enterica showed higher sensitivity towards sophorolipid biofunctionalized ZON. SEM analysis showed that cell damage occurred through cell elongation in the case of S. enterica , whereas cell rupture was found to occur predominantly in the case of C. albicans . This is the first report on the dual role of yeastmediated sophorolipid used as a biostabilizer for ZON synthesis as well as a novel functionalizing agent showing antimicrobial property.
Keywords
Introduction
Sophorolipids are biosurfactants produced by different yeast species, reported to be a mixture of mono- and diacetate and lactonic sophorolipids [3] . Applications of sophorolipids in various industries such as pharmaceutical, medical, cosmetics, environmental, and detergent industries have been reported [5] . The use of rhamnolipid biosurfactant derived from Pseudomonas aeruginosa has been reported as a biostabilizer during the synthesis of ZnS nanoparticles [11] . Synthesis of zinc oxide nanoparticles using sophorolipid as biostabilizer has not been reported so far.
Nanoparticles and other nanoscale materials are of great interest owing to their multiple potential applications in material science, medicine, and industry. Nanoparticles possess well-renowned antimicrobial activity against several microorganisms. However, it has some nonspecific toxicity. There is scope to increase the biological applicability of nanoparticles through functionalization. Nanostructures that are functionalized with biomolecules can display antimicrobial properties [8 , 12 , 18 , 19] . Although there are some reports related to the basic guidelines for functionalization and application of specialized nanomaterials, active research on functionalizing nanomaterials with biomolecules and their specific antimicrobial activity is poor. Reports are scanty on the application of functionlized nanoparticles for pathogenic microbes [14 , 20] .
Therefore, in the present study, an attempt has been made to employ the sophorolipid produced by yeast Cryptococcus sp. VITGBN2 as a biostabilizer for the synthesis of ZnO nanoparticles (ZON). In addition, the role of sophorolipids biofunctionlized ZON has also been tested as an antimicrobial agent.
Materials and Methods
- Chemicals
All the chemicals were of the highest purity grade. Zn(NO 3 ) 2 ·6H 2 O, NaOH, and zinc oxide nanoparticles were obtained from Sigma-Aldrich. Chemicals for minimal media preparation, including NH 4 NO 3 , KH 2 PO 4 , MgSO 4 ·7H 2 O, yeast extract, and glucose, were obtained from Hi Media, Mumbai, India.
- Isolation and Identification of Yeast Strain
Effluent samples were collected aseptically from Common Effluent Treatment Plant (CETP) located in Ranipet, India. Yeast colonies were isolated by pure culture techniques and maintained on Yeast Extract Peptone Dextrose (YEPD) agar plates at 4℃. Gene sequencing and identification of yeast were done following the methodology as reported earlier [15] . The partial and complete 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 26S rRNA sequences of strain VITGBN2 after the assembly had been deposited in GenBank under Accession No. KC135884.
- Biosurfactant Production
The biosurfactant from Cryptococcus sp. VITGBN2 was produced using vegetable oil following the standard protocol [9] . The physiochemical properties of the biosurfactant, its purification, and characterization were done following the method of our earlier work [3] .
- Synthesis of Sophorolipid Biofunctionalized ZON
The synthesis of sophorolipid biofunctionalized ZON was carried out at a molar ratio of 1 × 10 -3 M (ZnNO 3 ) 2 ·6H 2 O:1 × 10 -3 M (sophorolipid). A white precipitate was obtained by dropwise addition of 2 × 10 -3 M NaOH solution. The precipitate was further separated by centrifugation at 6,708 × g for 20 min and calcined at 300℃ for 2 h to obtain sophorolipid biofunctionalized ZON in powder form. The sophorolipid biofunctionalized ZON were characterized by UV-VIS spectral analysis (Hitachi, U-2800), FTIR analysis (Shimadzu IR Affinity-1), and scanning electron microscopy (SEM, Stereo Scan LEO, Model -400).
- Antimicrobial studies
Determination of microbial growth rate. Microbial growth rate was examined in the presence of sophorolipid biofunctionalized ZON by growing the pathogens in liquid medium containing varying concentrations of sophorolipid biofunctionalized ZON. Blank controls were maintained containing the same concentration of sophorolipid biofunctionalized ZON without microorganisms. The absorbance of the cultures was noted at regular intervals of 4 to 24 h for bacteria and fungi using a UV-vis spectrophotometer at 595 nm [7] .
- Protein Leakage Analysis
The protein leakage assay was performed using the Bradford assay. The bacterial pathogen S. enterica and the fungal pathogen C. albicans were treated with 1, 3, and 5 mg/ml of sophorolipid biofunctionalized ZON for 12 and 24 h. After treatment, the tubes were centrifuged at 10,000 rpm for 5 min and the supernatant was collected. For each sample, 200 μl of the supernatant was mixed in 800 μl of Bradford reagent and kept for 10 min incubation in the dark. The optical density of the sample was measured at 595 nm using bovine serum albumin as a standard protein.
- Agar Well Diffusion Method
Antimicrobial assay was performed using the pathogenic bacterial strain Salmonella enterica and fungal strain Candida albicans collected from CMC, Department of Microbiology, Bagayam, Thorapadi, Vellore, Tamil Nadu, India, following the agar well diffusion method [13] . Approximately 20 ml of sterile molted and cooled Mueller Hinton Agar (MHA) medium was poured in sterilized petridishes. The plates were incubated at room temperature to check for any contamination to appear. The target bacterium and fungus ( viz ., S. enterica and C. albicans ) were inoculated and the wells were prepared using a sterilized steel cork borer of diameter 5 mm. About 0.1 ml of the target microbial pathogen along with 5 mg/ml of antimicrobial agent tetracycline, naked ZON, a nd b iofuntionaliz ed Z ON w ere added in t he w ells, respectively. The plates containing the pathogens, tetracycline, naked ZON, and biofuntionalized ZON were incubated at 37℃ and the antimicrobial activity was compared. The plates were examined for evidence of zones of inhibition, which appeared as a clear area around the wells. The diameter of such ZON was measured using a meter ruler and the mean values for each pathogen were recorded and expressed in mm.
- Lactate Dehydrogenase Assay
Lactate dehydrogenase (LDH) activity in the cell medium was determined using an LDH Kit (Sigma-Aldrich, St. Louis, MO, USA). One hundred microliter of cell medium was used for LDH analysis. Absorption was measured using a Hitachi U-2800 UVvisible spectrophotometer at 340 nm following the standard procedure [21] . Released LDH catalyzed the oxidation of lactate to pyruvate with simultaneous reduction of NAD + to NADH. The rate of NAD + reduction was measured as an increase in absorbance at 340 nm. The rate of NAD + reduction was directly proportional to LDH activity in the cell medium.
Results and Discussion
- Identification of the Yeast and Phylogenetic Analysis
Colonies of strain VITGBN2 ( Fig. 1 A) on YEPD agar after 48 h of incubation were creamy colored and about 1-1.5 mm in diameter, circular, mucoid, glossy colonies. Staining revealed ovoid cells with the presence of a thin layer of glycoprotein capsular material having a gelatinlike consistency surrounding each cell ( Fig. 1 B). Using designed primers for PCR, the18S rRNA, ITS1, 5.8S rRNA, ITS2, and 26S rRNA regions were amplified and sequenced. The size of the sequence was 615 nucleotides long. The sequence analysis of the 18S rRNA gene showed 99% sequence coverage and 96% homology with Cryptococcus laurentii strain RY1 (Accession No. EF063363.2) in similarity search using the BLAST program. The ITS1 and ITS2, 5.8S rRNA region showed 98% sequence coverage and 96% homology with Cryptococcus aff. laurentii IMUFRJ 51980 (Accession No. FN428898.1). The sequence of the 26S rRNA gene (D1/D2/D3 region) showed 98% sequence coverage and 96% homology with Cryptococcus laurentii (Accession Nos. FN428934.1, FN428921.1, FN428909.1, and FN428903.1). Therefore, the isolate VITGBN2 was identified as a Cryptococcus species and designated as Cryptococcus sp. strain VITGBN2. Phylogenetic analysis ( Fig. 1 C) revealed that strain VITGBN2 was closely related to Cryptococcus laurentii strain RY1 (Accession No. EF063363.2).
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Characterization of Cryptococcus sp. VITGBN2. (A) Colony morphology on YEPD agar plates after 48 h of incubation. (B) Under 100× magnification of microscope. (C) Phylogenetic relationships of the yeast strain VITGBN2 isolated in this study and related species.
- Biosurfactant Characterization
The biosurfactant produced by the yeast contained carbohydrate (37.97%) and lipid (51.66%) as major constituents. MS spectra of the biosurfactant isolated from Cryptococcus sp. VITGBN2 are shown in Fig. 2 . The significant ions occurred at m/z 706 on mass spectra of the biosurfactant and corresponding chemical structures were determined as diacetate acidic sophorolipid with fatty acid moiety C18:1 ( Fig. 2 ). A similar structure of sophorolipid was reported in the case of Rhodotorula muciliginosa isolated from a hydrocarbon-contaminated site [3] . Sophorolipid biosurfactants are nontoxic and biodegradable, and can be isolated in huge quantity [2] . The yeast species Cryptococcus sp. VITGBN2 was found to be a potent producer of diacetate acidic sophorolipid.
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Structure of the purified biosurfactant through GC-MS.
- Characterization of ZON
The synthesis of ZON using acidic diacetate sophorolipids produced from Cryptococcus sp. VITGBN2 as a biostabilizer was confirmed by UV-Vis spectrum. An intensive absorption in the ultraviolet band of about 200-400 nm was obtained. An excitonic absorption peak was found at about 365 nm of ZnO nanoparticles (data not shown). Similar results were reported in the case of ZON synthesized by leaf extracts of Calotropis procera [16] .
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X-ray diffraction pattern of (A) naked ZON and (B) sophorolipid biofunctionalized ZON.
The structural facts of both naked and biofunctionalized ZON were elucidated by the XRD spectrum ( Fig. 3 ). The peaks at 2θ had values of 31.81°, 34.46°, 36.25°, 47.59°, 56.63°, 62.91°, 67.99°, and 69.12° in the case of naked ZON ( Fig. 3 A), whereas the peaks at 2θ had values of 31.76°, 34.43°, 36.26°, 47.55°, 58.59°, 62.87°, 67.96°, and 69.08° for biofunctionalized ZON ( Fig. 3 B), corresponding to the crystal planes of (100), (002), (101), (102), (110), and (103), (112), and (201) hexagonal wurtzite phase of ZnO nanoparticle [10 , 24] . The synthesized ZnO nanoparticle diameter was calculated using the Debye-Scherrer formula [4] .
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where 0.89 is Scherrer’s constant, λ is the wavelength of X-rays, θ is the Bragg diffraction angle, and β is the full width at half-maximum (FWHM) of the diffraction peak corresponding to plane (101). The average particle sizes of naked ZON and biofunctionalized ZON were determined from the FWHM of the more intense peak corresponding to the 101 plane located at 36.24° and 36.27°, and respectively were found to be 6.55 and 6.02 nm.
- Scanning Electron Microscopy
The surface morphology of naked ZON and sophorolipid stabilized ZON was studied using SEM ( Figs. 4 A and 4 B). A difference in surface smoothness was noted. Biofunctionalization by sophorolipids resulted in the enhancement of surface smoothness.
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Scanning electron micrograph of (A) naked ZON and (B) biofunctionalized ZON synthesized at 300℃.
- Microbial Growth Rate Determination and Protein Leakage Analysis
Figs. 5 A and 5 B represent the effect of sophorolipid biofunctionalized ZON on the growth of bacterial and fungal pathogens with respect to time, studied by measuring OD at 595 nm. The antimicrobial activity was achieved through electrostatic attraction obtained between the positively charged sophorolipid biofunctionalized ZON and the negatively charged cell membrane [6] , interaction of zinc ions with microbes [1] , and location of ZON [22] . The sophorolipid biofunctionalized ZON exhibited significant inhibitory effect on the growth of pathogens during the 24 h incubation period. The optical density was noted as the number of microbes after contact with the sophorolipid biofunctionalized ZON. It was clear from the figure that sophorolipid biofunctionalized ZON at a concentration of 5 mg/ml inhibited the growth of the pathogens, whereas the effect was much less at low concentrations.
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Antimicrobial effect of biofunctionalized ZON. Growth curves of (A) S. enterica and (B) C. albicans treated with biofunctionalized ZON. (C) Protein leakage analysis on interaction of pathogens with different concentrations of naked ZON and biofunctionalized ZON. (D) Protein leakage analysis on interaction of pathogens with 5 mg/ml of naked and biofunctionalized ZON at 12 and 24 h.
Fig. 5 C shows the amount of protein released in the suspension by the treated cells at different concentrations ranging from 1-5 mg/ml of ZON and sophorolipid biofunctionalized ZON. The amount of protein released from the microbial cells increased along with increase in concentration of ZON and biofunctionalized ZON for a contact period of 24 h. Fig. 5 D depicts the amount of protein leakage by the treated cells at a concentration of 5 mg/ml of ZON and sophorolipid biofunctionalized ZON at 12 and 24 h. The quantity of protein released for both pathogens was more in the case of 5 mg/ml of sophorolipid biofunctionalized ZON compared with naked ZON. This might be due to the coating of sophorolipid on ZON that helped the sophorolipid biofunctionalized ZON to penetrate into the pathogen cells more easily. Similar results were reported by other workers. Oleic acid was used in the synthesis of silver nanoparticles. That oleic-acid-stabilized silver nanoparticles exhibited high antibacterial activity against both gram-negative E. coli and gram-positive S. aureus bacteria [8] . It was demonstrated that the oleicacid-derived sophorolipid-capped silver nanoparticles were highly potent antibacterial agents, against both grampositive and gram-negative bacteria [17] . Other authors also found that many silver nanoparticles were located around the cell membrane of the bacterial strains as well as inside the cells. The increased permeability of the silver nanoparticles into bacterial cells led to a loss of cellular transport through the plasma membrane, which ultimately resulted in cell death [25] .
- Mechanism of Cell Damage
The mechanism of cell damage was well elucidated by SEM analysis of the biomass of S. enterica and C. albicans after a treatment with 5 mg/ml of naked ZON and biofunctionalized ZON ( Figs. 6 A-B and 7 A-B). In the case of S. enterica , cell elongation occurred, followed by cell wall disruption, causing protein leakage ( Fig. 6 A). Protein leakage was found to be more in the case of sophorolipid biofunctionalized ZON compared with the cells treated with naked ZON ( Fig. 6 B). No cell elongation was noted in the case of C. albicans ( Fig. 7 A) and complete cell rupture occurred followed by protein leakage ( Fig. 7 B). Among both species, S. enterica was found to be more susceptible to the treatment with naked ZON and biofunctionalized ZON compared with C. albicans . This could be probably due to the difference in nature of the cell machinery. The antibacterial activity of the sophorolipid biofunctionalized ZON mostly appeared owing to the exposure of thiol (-SH) groups of protein existing in the cell wall. This interaction decreased the cell permeability, which led to cell lyses and finally resulted in cell damage [26] .
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Scanning electron micrographs of S. enterica treated with 5 mg/ml of (A) naked ZON and (B) biofunctionalized ZON.
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Scanning electron micrographs of C. albicans treated with 5 mg/ml of (A) naked ZON and (B) biofunctionalized ZON.
- Evaluation of Antimicrobial Activity Through Agar Well Diffusion Method
Figs. 8 A and 8 B show the antimicrobial effects of ZON and sophorolipid biofunctionalized ZON as indicated by zone of inhibition for S. enterica and C. albicans . The sophorolipid biofunctionalized ZON showed a greater effect on S. enterica compared with C. albicans . The order of zone of inhibition was as follows: biofunctionalized ZON (23.5 ± 0.05/21.7 ± 0.1 mm) > Tetracycline (22.1 ± 0.1/18.4 ± 0.12 mm) > naked ZON (commercially purchased) (20.1 ± 0.1/11.9 ± 0.3 mm) > control (0.0 ± 0.0/0.0 ± 0.0 mm) for ( S. enterica / C. albicans ), respectively. This could be due to the enhanced smoothness of sophorolipid biofunctionalized ZON resulting in a higher penetration potential [20] .
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Zone of inhibition against (A) S. enterica and (B) C. albicans with respect to different antimicrobial agents (tetracycline, naked ZON, and biofunctionalized ZON) and control (distilled water).
- Lactate Dehydrogenase Assay
The extent of cell membrane breakage of S. enterica and C. albicans cells was revealed by LDH levels in the cell medium. The LDH levels in the cell culture were increased in all treatment groups after exposure to sophorolipid biofunctionalized ZON for a period of 24 h in the case of both the pathogens C. albicans ( Fig. 9 A) and S. enterica ( Fig. 9 B). From Fig. 9 , it was observed that the production of LDH from S. enterica was higher than from C. albicans , thereby causing higher cell membrane damage in the case of S. enterica compared with C. albicans . LDH increased from 47.0% to 81.3% with increase in concentration of sophorolipid biofunctionalized ZON upto 5 mg/ml in the case of C. albicans , whereas for S. enterica , LDH increased from 67.0% to 89.4% with an increase in concentration of sophorolipid biofunctionalized ZON upto 5 mg/ml. The LDH levels were found to be maximum at a concentration of 5.0 mg/ml of sophorolipid biofunctionalized ZON for both the pathogens. The damage to the cell membrane directly led to the loss of minerals, proteins, and genetic material, causing stress in the cell wall. As a consequence, the cell walls produced more lactate dehydrogenase enzymes, leading to cell membrane damage with respect to time [23] .
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LDH activities of pathogens (A) C. albicans and (B) S. enterica in the cell culture medium after 24 h exposure to 1, 3.0, and 5.0 mg/ml of biofunctionalized ZON.
- Fourier Transform Infrared Spectroscopy
Table 1 shows the FTIR spectra of sophorolipid biofunctionalized ZON and microbial pathogens before and after their interaction with sophorolipid biofunctionalized ZON. In the case of sophorolipid biofunctionalized ZON, the peaks obtained at 3,420.23, 2,926.45, 1,657.78, 1,247.21, and 1,054.95 cm -1 indicated the O-H stretching vibration, strong CH antisym and sym stretching of the (methyl) CH 3 group, C=O stretching, C-O-C antisym stretch in esters and lactones, and C-N stretching vibrations of aliphatic and aromatic amines, respectively.
FTIR spectra displayed several vibrational bands indicating the complex nature of the native pathogens S. enterica and C.albicans . The broad and strong bonds at 3,429.08 and 3,396.46 cm -1 , respectively, indicated bound amino (-NH) groups. The peaks at 2,927.94 cm -1 ( S. enterica ) and 2,871.87 cm -1 ( C. albicans ) were attributed to the stretching vibrations of –CH 2 groups. The absorption peaks at 2,562.34, 6,26.87, and 5,78.64 cm -1 ( S. enterica ), and 2,558.78 and 658.78 cm -1 ( C. albicans ) were due to the strong S-H stretch of the thiol group. T he a bsorption b ands o bserved at 1 ,647.51 cm -1 ( S. enterica ) and 1,671.09 cm -1 ( C. albicans ) were attributed mainly to a C=O stretch broad band. The moderately strong bonds at 1,354.65 and 1,082.07 cm -1 ( S. enterica ) and 1,048.78 cm -1 ( C. albicans ) could be assigned to the C-N stretching vibration of aliphatic and aromatic amines.
FTIR spectral analysis of pathogens before and after interaction with biofunctionalized ZON.
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FTIR spectral analysis of pathogens before and after interaction with biofunctionalized ZON.
FTIR spectra of S. enterica interacted with sophorolipid biofunctionalized ZON showed that the peaks expected at 3,429.08, 2,562.34, 626.87, 578.64, 1,647.51, 1,354.65, and 1,082.07 cm -1 had shifted, respectively, to 3,447.21, 2,563.97, 663.87, 563.97, 1,661.78, 1,364.60, and 1,063.08 cm -1 . As for C. albicans , the peaks expected at 3,396.46, 2,558.78, 658.78, 1,671.09, and 1,048.78 cm -1 had shifted to 3,464.71, 2,564.09, 671.87, 1,654.09, and 1,058.78 cm -1 , respectively. The spectral analysis before and after microbial pathogen interaction with sophorolipid biofunctionalized ZON clearly indicated that amino (-NH), thiol (S-H), and carbonyl (C=O) groups are the predominant contributors for binding of sophorolipid biofunctionalized ZON for both the pathogens. Moreover, the presence of increased number of thiol groups in the cell wall surface of S. enterica compared with C. albicans shows the enhanced antimicrobial activity of sophorolipid biofunctionalized ZON against S. enterica than against C. albicans .
In conclusion, the present study summarized the production of acidic diacetate sophorolipid by Cryptococcus sp. VITGBN2 and its dual role as a biostabilizer for ZnO nanoparticle synthesis and a biofunctionalizing agent against S. enterica and C. albicans . The biostabilized ZON were characterized by UV-vis, XRD, SEM, and FTIR analysis. Zinc oxide nanoparticles stabilized by sophorolipids were quite stable and no visible changes were observed even after a month. The growth inhibition was higher in the case of sophorolipid biofunctionalized ZON compared with naked ZON and other antimicrobial agents. The enhanced bioactivity of these sophorolipid biofunctionalized ZON was attributed to the increased permeability through the microbial cells that led to a loss of cellular transport through the plasma membrane, resulting in cell death.
Acknowledgements
The authors would like to thank VIT University for providing laboratory facility and financial support for the smooth conduct of the work. We also extend our gratitude towards Pondicherry University for the assistance in SEM analysis.
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