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Levan production from sucrose using chicken feather peptone as a low cost supplemental nutrient source
Carbohydrate Polymers 227 (2020) 115361
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Levan production from sucrose using chicken feather peptone as a low cost supplemental nutrient source
T
Bhuvaneshwari Veerapandiana, Saravanan Ramiah Shanmugama, Srivathsan Varadhana, ⁎ Kartik Kumar Sarwareddyb, Krishna Priya Manib, V. Ponnusamia, a Biomass conversion and Bioproducts Laboratory, Center for Bioenergy, School of Chemical & Biotechnology, SASTRA Deemed University, Thirumalaisamudram, Tamil Nadu, India b Cardiomyocyte Toxicity and Oncology Research Laboratory, School of Chemical & Biotechnology, SASTRA Deemed University, Thirumalaisamudram, Tamil Nadu, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Chicken feathers Acid hydrolysis Chicken feather peptone Levan production
Chicken feather peptone (CFP) derived from poultry waste is a rich source of essential minerals and amino acids. This, along with suitable carbon source, can be used as a low cost complex supplemental nutrient source for microbial fermentation. In the present work, CFP blended with sucrose was evaluated for the production of levan using Bacillus subtilis MTCC 441. Amount of CFP added to the medium significantly influenced levan production and it was found that at a concentration 2 g/L, maximum levan yield of 0.26 ± 0.04 g/g sucrose was obtained. The levan yield obtained with CFP as a low cost supplemental nutrient source was comparable with that obtained from commercial medium (0.31 ± 0.02 g/g sucrose). Levan produced using CFP was tested on primary cell lines at various concentrations (100–1000 μM) and found to be non-toxic and bio-compatible in nature. This indicates that CFP could be used as low cost nutrient source for levan production.
1. Introduction Exopolysaccharides are important secondary metabolites produced by microorganisms. They are used in many food, medicine and cosmetic industries and are becoming industrially important owing to their biodegradable, renewable, biocompatible, economical and environmental friendly nature. Levan is one such exopolysaccharide which has gained increased research attention recently. Levan is produced by both microorganisms and plants (Matsuhira, Tamura, Tamagake, & Sato, 2014). While levan synthesised from plants exhibits low degree of polymerisation and low intrinsic viscosity, microbial levan shows higher degree of polymerisation and high intrinsic viscosity. Microbial levan have wide application due to their high molecular weight, mechanical and rheological properties. Levan is composed of D-fructofuranosyl residues linked by β-(2,6) and β-(2,1) linkages in the main and side chains respectively with D - glucosyl terminal residue (Nasir, Wahyuningrum, & Hertadi, 2015). Microorganisms such as Bacillus subtilis, Lactobacillus johnsonii, Lactobacillus gasserii, Aerobacter levaium, etc. are known to produce levan (Srikanth, Reddy, Siddartha, Ramaiah, & Uppuluri, 2015). Microbial biosynthesis of levan is catalysed by the enzyme levansucrase. Levansucrase catalyses both the sucrose hydrolysis and fructan polymerisation reactions (Kuruppasamy,
⁎
Gunasekaran, & Velusamy, 2007; Nasir et al., 2015). Levan production is influenced by many factors such as source (bacteria or plant), species and environmental conditions like pH, temperature and incubation time. Media composition is another important factor that affects levan production and accounts for overall economy of the process. The amount of carbon, nitrogen, micronutrients and macronutrients used in the media add upto the production cost. About 35–60% of the production costs is contributed by the chemical ingredients used in microbial fermentation process as medium (Stanbury, Whitaker, & Hall, 2003). So, there is a pressing need to search for high yielding, low cost and abundant raw materials for levan production (Yezza, Tyagi, Valéro, & Surampalli, 2006). Towards satisfying this need, researchers have put on significant research efforts in the recent past to identify and suitable low cost carbon sources for levan production. This includes sugarcane molasses, sugar beet molasses, sugarcane syrup (de Oliveira, da Silva, Buzato, & Celligoi, 2007), sugarcane juice, beet molasses (Han & Watson, 1992), date syrup (Moosavi-Nasab, Layegh, Aminlari, & Hashemi, 2010) and starch molasses (Küçükaşik et al., 2011). However, research on finding alternative low-cost micro- and macro-nutrients that needs to be supplemented along with the fermentation media is lacking in literature. Previous studies in literature have shown that CFP contains
Corresponding author. E-mail address: [email protected] (V. Ponnusami).
https://doi.org/10.1016/j.carbpol.2019.115361 Received 19 August 2019; Received in revised form 19 September 2019; Accepted 20 September 2019 Available online 23 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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Table 2 Amino acids composition of chicken feather peptone. Amino acids (mg/ 100 g)
Percent of amino acids
Proline Glutamic acid Valine Aspartic acid Glycine Leucine Alanine Serine Isoleucine Phenylalanine Lysine Threonine Histidine Tyrosine Cystine Methionine Tryptophan
(Taskin & Kurbanoglu, 2011)
(Orak et al., 2018)
(Baltaci et al., 2018)
7.76 13.02 14.28 6.78 11.56 11.12 8.35 7.94 6.25 5.70 4.67 0.00 2.57 0.00 0.00 ND ND
6.49 12.56 13.16 5.97 10.95 9.09 8.57 8.34 5.89 5.48 3.93 4.31 0.00 3.01 2.24 ND ND
6.87 11.74 12.97 6.16 11.04 9.94 7.57 7.19 5.68 5.12 4.26 4.45 0.00 2.51 4.49 ND ND
Table 3 Elemental composition of Chicken feather peptone. Elements
P K Ca Mg Na Cu Fe Zn Mn B S
Fig. 1. CFP production from chicken feathers. Table 1 Different Medium Used for Levan Production (All quantities are given in g).
Sucrose Ammonium sulphate Bacterial Peptone Yeast extract Potassium dihydrogen phosphate Magnesium sulphate Manganese sulphate CFPd Levan
FM1
FM2
FM3a
FM3b
FM3c
FM3d
100 3 – 2 1 0.6 0.2 – 30.43
100 – 3
100 – – – – – – 1 20.08
100 – – – – – – 2 25.83
100 – – – – – – 3 14.65
100 – – – – – – 4 13.45
– 21.99
CFP (g/ 100 g) (Orak et al., 2018)
(Taskin & Kurbanoglu, 2011)
0.34 16.34 0.20 0.17 0.18 0.00 0.15 0.00 0.00 0.00 3.82
2.06 12.52 6.08 4.21 6.96 0.22 0.83 0.67 0.51 0 3.07
CFP for levan production using Bacillus subtilis MTCC441; (iii) to compare the effectiveness of CFP as a sole complex nutrient source in comparison with optimized levan production media using defined nutrients and (iv) to characterize the levan produced using CFP as sole nutrient source.
2. Materials and methods 2.1. Media components
numerous amino acids, minerals and trace elements. Hence, it is hypothesized that addition of CFP to the fermentation media will eliminate the need for additional micro-and macro nutrients. Thus, chicken feather peptones can serve as low cost nutrient supplement to reduce medium cost. Owing to their rich nutritional content, CFP along with suitable carbon source had been used as low cost fermentation substrate for the production of microbial metabolites like xanthan gum and citric Acid (Ozdal & Başaran Kurbanoglu, 2019). Chicken feathers are considered as a waste material in poultry processing industries (Akpor, Odesola, Thomas, & Oluba, 2019; Orak, Caglar, Ortucu, Ozkan, & Taskin, 2018; Ozdal & Kurbanoglu, 2018). Feathers account for 10% of total chicken weight. Several million tons of these wastes are generated every year throughout the world. Chicken feathers constitute about 90% protein that is composed of keratin. To the best of our knowledge, CFP has not been investigated as the source of nitrogen and other essential nutrients for levan production. Hence, the objectives of the current study are as follows (i) to synthesize CFP from chicken feather waste; (ii) to optimize the composition of
Isopropanol was purchased from M/s Molychem., Mumbai, India. Sucrose and all other chemicals used in this study were purchased from M/s Himedia, India. All the chemical ingredients used in this study are of analytical grade with high purity (> 99%).
2.2. Microorganism Bacillus subtilis MTCC441 was purchased from Institute of Microbial Technology (IMT), Chandigarh, India. The pure culture of B. subtilis MTCC 441 was made to grow on Luria Bertani (LB) agar using petriplates at 37 °C and stored at 20 °C. A colony was picked up from the culture grown on petriplates, inoculated and incubated at 150 RPM, 37 °C, overnight culture on LB broth which was used as inoculum for production throughout the study. The glycerol stock was maintained at –80 °C. 2
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Fig. 2. Effect of different concentration of CFP, bacterial peptone, control medium on (a) levan production and (b) biomass concentration. Notes: (1). Fermentation conditions: sucrose-100 g/L, 37 °C, 150 RPM, 20 h, CFP (1–4) g/L. (2). FM1 – synthetic medium, FM2 – sucrose + bacterial peptone, FM3a – sucrose + 1 g/L CFB, FM3b – sucrose + 2 g/L CFB, FM3c – sucrose + 3 g/L CFB, FM3d – sucrose + 4 g/L CFB. (3) NS = Not Significant at α = 0.05.
detector temperatures were maintained at 35 °C and 30 °C, respectively. For molecular weight determination, Ultrahydrogel 1000 Gel Permeation Chromatography (GPC) column was used with 0.6 mL/min 0.5 N sodium nitrite solution as mobile phase. Column and detector were maintained at 35 °C and 30 °C, respectively.
2.3. Production of chicken feather peptone (CFP) Chicken feathers were collected from local market at Tiruchirapalli, Tamil Nadu, India. Feathers were washed with distilled water and dried in sunlight. Hydrolysis of chicken feather was carried out using the process as outlined in Fig. 1. Chicken feathers were grinded and powered using pulverizer. 50 g of chicken feather powder was taken and treated with 6 N hydrochloric acid in two steps at 70 °C and 130 °C, as shown in the Fig. 1. The resulting sample was neutralised using 5 N sodium hydroxide solution and filtered using sintered crucible under vacuum. The chicken feather hydrolysate (liquid fraction) was collected and concentrated in rotary evaporator. The residue collected after evaporation is oven dried at 60 °C for 24 h. The dried powder was labelled as CFP (Chicken feather peptone) and stored for further use.
2.7. Cell culture Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from HiMedia (India) and cultured in endothelial cell expansion medium (HiEndoXL, Himedia) supplemented with endothelial cell growth supplement (AL517B) and 1% penicillin/streptomycin antibiotics. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
2.4. Levan production
2.8. Cytotoxicity analysis
Levan production was carried out in an Erlenmeyer’s flask with the culture volume of 50 mL in a 250 mL flask. Levan production was carried out with different media composition as shown in Table 1. The initial pH was adjusted to 7.0 before autoclaving. The culture was incubated at optimised conditions 37 °C, 150 RPM, for 20 h. All the experiments were carried out in triplicates and the experimental means were statistically compared using Tukey’s test at α = 0.05.
HUVEC cells were seeded in a 96 well plate (coated with 0.5% gelatine) at an approximate density of 1 × 104 per well and incubated overnight. The next day, they were treated with different concentrations of isolated levan compound (100, 250,500,750 and 1000 μM) for 24 h. Cells treated with N-acetyl cysteine (NAC - 1000 μM) for 24 h was used as positive control. Subsequently, cells were washed with 1X PBS and incubated with 1 mg/mL of MTT. After 4 h of incubation at 37 °C, formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm by using microplate reader spectrophotometer (Synergy H1).
2.5. Recovery of levan At the end of the fermentation process the culture was collected and centrifuged at 13,230 × g for 10 min and pellet was dried at 60 °C for biomass estimation. Levan was recovered from the supernatant. The pH of the supernatant was adjusted to 9.0. Levan was precipitated by adding ice cold isopropanol in the ratio of 1:5 (supernatant to isopropanol) and the mixture was centrifuged at 16,538 × g for 10 min. The pellet was dried at room temperature overnight (35 °C) and weighed.
3. Results and discussion Comparison of defined media and complex media for levan production was performed as described in materials and methods section. 3.1. CFP production Chicken feather peptone was prepared using the process given in Fig. 1. Investigation showed that the CFP would contain organic and inorganic substances required for the microbial growth and metabolism (EPS production) (Ozdal & Kurbanoglu, 2018; Taskin & Kurbanoglu, 2011). CFP produced from chicken feather would give stimulatory effect for EPS production because it contain essential amino acids and minerals such as Na, Fe, Ca, Mn, Mg required for the microbial cultivation list in the Tables 2 and 3 respectively. Since CFP contain all essential amino acids it can be exploited as a complex nutrient source along with suitable carbon source to support microbial growth and production metabolites. CFP is rich in amino acids such as glutamic acid, alanine, glycine, cysteine which is essential for growth and sporulation of Bacillus subtilis. Bacillus subtilis MTCC 441 is a sporulating
2.6. Analytical methods 1
H- NMR and 13C- NMR analyses of the polymer were carried out in Bruker 300 MHz instrument at room temperature. Samples for 1H-NMR and 13C-NMR were prepared using D2O. Fourier Transform Infrared Spectroscopy (FTIR) spectrum was recorded using Perkin- Elmer spectrum one instrument, with a resolution of 5 cm−1. Residual sugar composition and molecular weight of the samples were determined using Waters High Performance Liquid Chromatography (HPLC) system equipped with a Refractive Index detector. Amino Spherisorb (5 μm, 4.6 × 250 mm) analytical column was used with a mobile phase 80% acetonitrile at 1 mL/min for residual sugar analysis. Column and 3
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Fig. 3.
13
C NMR and 1H-NMR of levan production using CFP- 2 g/L.
4
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concentration of 1.28 g/L. Further increase in CFP concentration gave less levan yield even though there is an increase in biomass concentration. This may be attributed to inhibitory effect of micronutrients in the medium as concentration of micronutrient increase with increase in concentrations of CFP. Similar trends were observed during xanthan production with increasing levels of CFP (Ozdal & Kurbanoglu, 2018). Among different organic nitrogen sources such as CFP, bacterial peptone and yeast extract, it was found that CFP showed comparable results with yeast extract and higher levels of levan production. These experiments demonstrated that the maximum levan yield of 0.31 g levan/g sucrose consumed while the biomass concentration of 1.32 g/L was obtained with control medium. In control medium levan production 0.26 g levan/g sucrose was obtained with CFP with a biomass concentration of 1.28 g/L. Bacterial peptone showed lower levan yield in comparison to cultures fed chicken feather peptone (Fig. 2a). This might be due to the difference in nutrient composition of bacterial peptone in comparison to the chicken feather peptone. Similar results were obtained during xanthan biopolymer production using bacterial peptone in comparison to CFP (Ozdal & Başaran Kurbanoglu, 2019). Researchers have reported that the addition of organic nitrogen sources (CFP) to production medium enhanced cell growth and product yield (Ozdal & Kurbanoglu, 2018, 2019; Taskin & Kurbanoglu, 2011).
Table 4 The chemical shifts of 13C- NMR spectra of levan produced by different bacteria. Carbon atom
C-1 C-2 C-3 C-4 C-5 C-6
Chemical shifts (ppm) From Bacillus subtilis MTCC 441 (present study)
From Bacillus licheniforms BK AG21 (Mamay et al., 2015)
From Chromohalobacter japonicus BK-AB18 (Nasir et al., 2015)
59.90 104.19 76.28 75.19 80.27 63.37
59.85 104.22 76.24 75.18 80.31 63.41
59.83 104.23 76.24 75.17 80.31 63.40
bacteria which is used to produce levan. Methionine and tryptophan are known for their inhibitory effect on sporulation. Thus, absence of methionine and tryptophan can inhibit sporulation and hence favourably influence the production of levan. Presence of micronutrients such as Fe, Ca, Mg enhance the activity of levansucrase (Chambert, Benyahia, & Glatron, 1990; Petit-Glatron, Monteil, Benyahia, & Chambert, 1990), the enzyme which catalyse sucrose hydrolysis and assist in transfructosylation for levan production. As CFP contains these nutrients it favours levan production.
3.3. Characterisation of levan The 13C NMR spectrum peak shown in Fig. 3(a) confirms the structure of levan produced by Bacillus subtilis MTCC 441 using CFP. The six carbon resonance at the chemical shifts of 104.17 ppm (C-2), 80.27 ppm (C-5), 76.28 ppm (C-3), 75.19 ppm (C-4), 63.37 ppm (C-6), 59.90 ppm (C-1) represents the carbon atoms in the levan polymer produced using CFP. The results obtained are consistent with the previous reports listed in the Table 4. The chemical shifts of 104.17 ppm (C-2) and 63.37 ppm (C-6) corresponded to the presence of β-(2–6) linkages of levan (Mamay, Wahyuningrum, & Hertadi, 2015; Nasir et al., 2015).The 1H-NMR spectrum of levan produced using CFP also represented the proton chemical shift signals related to the fructose as monomer of levan (Fig. 3(b)). The chemical shifts observed in the range of 3.46–3.86 ppm are in accordance with the previous reports (Mamay et al., 2015; Nasir et al., 2015). Fig. 4 shows the GPC results of produced levan. Molecular weight of the levan produced using CFP, bacterial peptone, control medium shows identical fraction listed in the Table 5. It was in the range of
3.2. Effect of CFP on levan production Comparisons of levan yield and biomass concentration obtained from different concentrations of CFP (1–4 g/L), bacterial peptone, and control medium are shown in Fig. 2a and b. Results show that CFP concentration strongly influenced bacterial growth and levan production. Cell growth and enzyme production patterns are strongly affected by nitrogen source, micronutrients and macronutrients. As expected, at 20 h of fermentation the levan yield in culture fed with 2 g/L CFP was almost similar (0.26 ± 0.04 g levan/g sucrose consumed) to that obtained from the control medium (FM1) (0.31 ± 0.02 g levan/g sucrose consumed). Fig. 2a shows that there is no significance difference (α = 0.05) in levan production between CFP 2 g/L and control medium. It is shown that CFP comprises amino acids, minerals and salts which could act as a complex nutrient source for the microorganism. Table 2 and 3 shows the composition of CFP reported in literature. The production medium containing 2 g/L CFP (FM3b) resulted in the biomass
Fig. 4. Gel Permeation chromatographs of levan. 5
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Biotechnology, Government of India for the financial support (Grant: BT/PR7511/BCE/8/973/2013) and Department of Science & Technology for support through FIST programme (Grant: SR/FST/ETI331/2013). Dr. Shanmugam S. R., and Dr. Krishna Priya Mani sincerely thank DST for the financial support through INSPIRE grants DST/INSPIRE/ 04/2017/002528 and DST/INSPIRE/04-I/2016/000003 respectively.
Table 5 Comparison of molecular weight distribution of levan samples. Molecular weight (kDa)
CFP - 2 g/L Bacterial peptone Control medium
1
2
3
4
2646.68 2765.57 2658.34
8.66 9.41 8.98
ND ND 5.39
3.36 3.75 3.78
References Note: ND- Not Determined. Akpor, O. B., Odesola, D. E., Thomas, R. E., & Oluba, O. M. (2019). Chicken feather hydrolysate as alternative peptone source for microbial cultivation. F1000Research, 7, 1918. https://doi.org/10.12688/f1000research.17134.2. Chambert, R., Benyahia, F., & Glatron, M. F. P. (1990). Secretion of Bacillus subtilis levansucrase - Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes. The Biochemical Journal, 265, 375–382. de Oliveira, M. R., da Silva, R. S. S. F., Buzato, J. B., & Celligoi, M. A. P. C. (2007). Study of levan production by Zymomonas mobilis using regional low-cost carbohydrate sources. Biochemical Engineering Journal, 37, 177–183. https://doi.org/10.1016/j.bej. 2007.04.009. Han, Y. W., & Watson, M. A. (1992). Production of microbial levan from sucrose, sugarcane juice and beet molasses. Journal of Industrial Microbiology, 9, 257–260. https://doi.org/10.1007/BF01569633. Küçükaşik, F., Kazak, H., Güney, D., Finore, I., Poli, A., Yenigün, O., et al. (2011). Molasses as fermentation substrate for levan production by Halomonas sp. Applied Microbiology and Biotechnology, 89, 1729–1740. https://doi.org/10.1007/s00253010-3055-8. Kuruppasamy, G., Gunasekaran, P., & Velusamy, S. K. (2007). Strategies for the improvement of levan and fructooligosaccharides production by zymomonas mobilis. Chapter 12Advances in bioprocessing in food industry. Haworth Press. Mamay, Wahyuningrum, D., & Hertadi, R. (2015). Isolation and characterization of Levan from moderate halophilic Bacteria Bacillus licheniformis BK AG21. Procedia Chemistry, 16, 292–298. https://doi.org/10.1016/j.proche.2015.12.055. Matsuhira, H., Tamura, K., Tamagake, H., & Sato, Y. (2014). High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes. Journal of Biotechnology, 192, 215–222. https://doi.org/10.1016/j.jbiotec.2014.09. 025. Moosavi-Nasab, M., Layegh, B., Aminlari, L., & Hashemi, M. B. (2010). Microbial production of levan using date syrup and investigation of its properties. World Academy of Science, Engineering and Technology, 44, 1248–1254. Nasir, D. Q., Wahyuningrum, D., & Hertadi, R. (2015). Screening and characterization of levan secreted by halophilic bacterium of Halomonas and Chromohalobacter genuses originated from Bledug Kuwu mud crater. Procedia Chemistry, 16, 272–278. https:// doi.org/10.1016/J.PROCHE.2015.12.050. Orak, T., Caglar, O., Ortucu, S., Ozkan, H., & Taskin, M. (2018). Chicken feather peptone: A new alternative nitrogen source for pigment production by Monascus purpureus. Journal of Biotechnology, 271, 56–62. https://doi.org/10.1016/j.jbiotec.2018.02.010. Ozdal, M., & Başaran Kurbanoglu, E. (2019). Use of chicken feather peptone and sugar beet molasses as low cost substrates for xanthan production by Xanthomonas campestris MO-03. Fermentation, 5, 9. https://doi.org/10.3390/fermentation5010009. Ozdal, M., & Kurbanoglu, E. B. (2019). Citric acid production by Aspergillus niger from agro-industrial by-products: Molasses and chicken feather peptone. Waste and Biomass Valorization, 10, 631–640. https://doi.org/10.1007/s12649-018-0240-y. Ozdal, M., & Kurbanoglu, E. B. (2018). Valorisation of chicken feathers for xanthan gum production using Xanthomonas campestris MO-03. Journal, Genetic Engineering & Biotechnology, 16, 259–263. https://doi.org/10.1016/j.jgeb.2018.07.005. Petit-Glatron, M. F., Monteil, I., Benyahia, F., & Chambert, R. (1990). Bacillus subtilis levansucrase: Amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals. Molecular Microbiology, 4, 2063–2070. https:// doi.org/10.1111/j.1365-2958.1990.tb00566.x. Shih, I. L., Chen, L. D., & Wu, J. Y. (2010). Levan production using Bacillus subtilis natto cells immobilized on alginate. Carbohydrate Polymers, 82, 111–117. https://doi.org/ 10.1016/j.carbpol.2010.04.030. Srikanth, R., Reddy, C. H. S. S. S., Siddartha, G., Ramaiah, M. J., & Uppuluri, K. B. (2015). Review on production, characterization and applications of microbial levan. Carbohydrate Polymers, 120, 102–114. https://doi.org/10.1016/j.carbpol.2014.12. 003. Stanbury, P. F., Whitaker, A., & Hall, S. J. (2003). Priniciple of fermentation technology. Vasa 367. Taskin, M., & Kurbanoglu, E. B. (2011). Evaluation of waste chicken feathers as peptone source for bacterial growth. Journal of Applied Microbiology, 111, 826–834. https:// doi.org/10.1111/j.1365-2672.2011.05103.x. Yezza, A., Tyagi, R. D., Valéro, J. R., & Surampalli, R. Y. (2006). Bioconversion of industrial wastewater and wastewater sludge into Bacillus thuringiensis based biopesticides in pilot fermentor. Bioresource Technology, 97, 1850–1857. https://doi.org/10. 1016/j.biortech.2005.08.#.
Fig. 5. Levan compound effect on HUVEC viability. Notes: Cells were treated with different concentrations of levan (C1 – 100 μM, C2 – 250 μM, C3 – 500 μM, C4 – 750 μM and C5 – 1000 μM) for 24 h, and cell viability was measured by MTT assay. The experiment was repeated three times and the values are represented as mean ± SEM. N-Acetyl Cysteine (NAC) was used as a positive control (PC).
2600–2800 kDa for the high fraction and 3–9.5 kDa for the low fraction. These results are consistent with the previous reports (Shih, Chen, & Wu, 2010). 3.4. Effect of levan on HUVECs viability The cytotoxicity of isolated levan compound was examined by using MTT assay. As shown in Fig. 5 levan compound (100, 250,500,750 and 1000 μM) did not altered the cell viability of HUVECs in 24 h, whereas 1000 μM NAC (Positive control) significantly increased the cell viability (P < 0.05). These results indicate that the produced levan using CFP doesn’t pose any risk to the cell lines. Hence the produced levan can be utilised for biomedical application such as carrier vehicle for drugs, etc. 4. Conclusions The current study evaluates the potential of CFP as a low cost nutrient source for levan production using Bacillus subtilis MTCC 441. Since chicken feathers are proteinaceous in nature and are available in large quantities they could be utilized as an effective nutrient for the growth of microorganisms and production process. The results from this study support this hypothesis. Studies conducted on primary cell lines to test the cytotoxicity indicate that levan produced using CFP does not show any negative effect on cell lines indicating that the produced levan is compatible for biomedical applications. Chicken feathers which are discarded as waste from poultry industry can be thus put into the production of levan and this would simultaneously solve the solid disposal problem and improve the economic potential of poultry industry. Acknowledgement Dr.
V.
Ponnusami
gratefully
thank
the
Department
of
6
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Levan production from sucrose using chicken feather peptone as a low cost supplemental nutrient source
T
Bhuvaneshwari Veerapandiana, Saravanan Ramiah Shanmugama, Srivathsan Varadhana, ⁎ Kartik Kumar Sarwareddyb, Krishna Priya Manib, V. Ponnusamia, a Biomass conversion and Bioproducts Laboratory, Center for Bioenergy, School of Chemical & Biotechnology, SASTRA Deemed University, Thirumalaisamudram, Tamil Nadu, India b Cardiomyocyte Toxicity and Oncology Research Laboratory, School of Chemical & Biotechnology, SASTRA Deemed University, Thirumalaisamudram, Tamil Nadu, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Chicken feathers Acid hydrolysis Chicken feather peptone Levan production
Chicken feather peptone (CFP) derived from poultry waste is a rich source of essential minerals and amino acids. This, along with suitable carbon source, can be used as a low cost complex supplemental nutrient source for microbial fermentation. In the present work, CFP blended with sucrose was evaluated for the production of levan using Bacillus subtilis MTCC 441. Amount of CFP added to the medium significantly influenced levan production and it was found that at a concentration 2 g/L, maximum levan yield of 0.26 ± 0.04 g/g sucrose was obtained. The levan yield obtained with CFP as a low cost supplemental nutrient source was comparable with that obtained from commercial medium (0.31 ± 0.02 g/g sucrose). Levan produced using CFP was tested on primary cell lines at various concentrations (100–1000 μM) and found to be non-toxic and bio-compatible in nature. This indicates that CFP could be used as low cost nutrient source for levan production.
1. Introduction Exopolysaccharides are important secondary metabolites produced by microorganisms. They are used in many food, medicine and cosmetic industries and are becoming industrially important owing to their biodegradable, renewable, biocompatible, economical and environmental friendly nature. Levan is one such exopolysaccharide which has gained increased research attention recently. Levan is produced by both microorganisms and plants (Matsuhira, Tamura, Tamagake, & Sato, 2014). While levan synthesised from plants exhibits low degree of polymerisation and low intrinsic viscosity, microbial levan shows higher degree of polymerisation and high intrinsic viscosity. Microbial levan have wide application due to their high molecular weight, mechanical and rheological properties. Levan is composed of D-fructofuranosyl residues linked by β-(2,6) and β-(2,1) linkages in the main and side chains respectively with D - glucosyl terminal residue (Nasir, Wahyuningrum, & Hertadi, 2015). Microorganisms such as Bacillus subtilis, Lactobacillus johnsonii, Lactobacillus gasserii, Aerobacter levaium, etc. are known to produce levan (Srikanth, Reddy, Siddartha, Ramaiah, & Uppuluri, 2015). Microbial biosynthesis of levan is catalysed by the enzyme levansucrase. Levansucrase catalyses both the sucrose hydrolysis and fructan polymerisation reactions (Kuruppasamy,
⁎
Gunasekaran, & Velusamy, 2007; Nasir et al., 2015). Levan production is influenced by many factors such as source (bacteria or plant), species and environmental conditions like pH, temperature and incubation time. Media composition is another important factor that affects levan production and accounts for overall economy of the process. The amount of carbon, nitrogen, micronutrients and macronutrients used in the media add upto the production cost. About 35–60% of the production costs is contributed by the chemical ingredients used in microbial fermentation process as medium (Stanbury, Whitaker, & Hall, 2003). So, there is a pressing need to search for high yielding, low cost and abundant raw materials for levan production (Yezza, Tyagi, Valéro, & Surampalli, 2006). Towards satisfying this need, researchers have put on significant research efforts in the recent past to identify and suitable low cost carbon sources for levan production. This includes sugarcane molasses, sugar beet molasses, sugarcane syrup (de Oliveira, da Silva, Buzato, & Celligoi, 2007), sugarcane juice, beet molasses (Han & Watson, 1992), date syrup (Moosavi-Nasab, Layegh, Aminlari, & Hashemi, 2010) and starch molasses (Küçükaşik et al., 2011). However, research on finding alternative low-cost micro- and macro-nutrients that needs to be supplemented along with the fermentation media is lacking in literature. Previous studies in literature have shown that CFP contains
Corresponding author. E-mail address: [email protected] (V. Ponnusami).
https://doi.org/10.1016/j.carbpol.2019.115361 Received 19 August 2019; Received in revised form 19 September 2019; Accepted 20 September 2019 Available online 23 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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Table 2 Amino acids composition of chicken feather peptone. Amino acids (mg/ 100 g)
Percent of amino acids
Proline Glutamic acid Valine Aspartic acid Glycine Leucine Alanine Serine Isoleucine Phenylalanine Lysine Threonine Histidine Tyrosine Cystine Methionine Tryptophan
(Taskin & Kurbanoglu, 2011)
(Orak et al., 2018)
(Baltaci et al., 2018)
7.76 13.02 14.28 6.78 11.56 11.12 8.35 7.94 6.25 5.70 4.67 0.00 2.57 0.00 0.00 ND ND
6.49 12.56 13.16 5.97 10.95 9.09 8.57 8.34 5.89 5.48 3.93 4.31 0.00 3.01 2.24 ND ND
6.87 11.74 12.97 6.16 11.04 9.94 7.57 7.19 5.68 5.12 4.26 4.45 0.00 2.51 4.49 ND ND
Table 3 Elemental composition of Chicken feather peptone. Elements
P K Ca Mg Na Cu Fe Zn Mn B S
Fig. 1. CFP production from chicken feathers. Table 1 Different Medium Used for Levan Production (All quantities are given in g).
Sucrose Ammonium sulphate Bacterial Peptone Yeast extract Potassium dihydrogen phosphate Magnesium sulphate Manganese sulphate CFPd Levan
FM1
FM2
FM3a
FM3b
FM3c
FM3d
100 3 – 2 1 0.6 0.2 – 30.43
100 – 3
100 – – – – – – 1 20.08
100 – – – – – – 2 25.83
100 – – – – – – 3 14.65
100 – – – – – – 4 13.45
– 21.99
CFP (g/ 100 g) (Orak et al., 2018)
(Taskin & Kurbanoglu, 2011)
0.34 16.34 0.20 0.17 0.18 0.00 0.15 0.00 0.00 0.00 3.82
2.06 12.52 6.08 4.21 6.96 0.22 0.83 0.67 0.51 0 3.07
CFP for levan production using Bacillus subtilis MTCC441; (iii) to compare the effectiveness of CFP as a sole complex nutrient source in comparison with optimized levan production media using defined nutrients and (iv) to characterize the levan produced using CFP as sole nutrient source.
2. Materials and methods 2.1. Media components
numerous amino acids, minerals and trace elements. Hence, it is hypothesized that addition of CFP to the fermentation media will eliminate the need for additional micro-and macro nutrients. Thus, chicken feather peptones can serve as low cost nutrient supplement to reduce medium cost. Owing to their rich nutritional content, CFP along with suitable carbon source had been used as low cost fermentation substrate for the production of microbial metabolites like xanthan gum and citric Acid (Ozdal & Başaran Kurbanoglu, 2019). Chicken feathers are considered as a waste material in poultry processing industries (Akpor, Odesola, Thomas, & Oluba, 2019; Orak, Caglar, Ortucu, Ozkan, & Taskin, 2018; Ozdal & Kurbanoglu, 2018). Feathers account for 10% of total chicken weight. Several million tons of these wastes are generated every year throughout the world. Chicken feathers constitute about 90% protein that is composed of keratin. To the best of our knowledge, CFP has not been investigated as the source of nitrogen and other essential nutrients for levan production. Hence, the objectives of the current study are as follows (i) to synthesize CFP from chicken feather waste; (ii) to optimize the composition of
Isopropanol was purchased from M/s Molychem., Mumbai, India. Sucrose and all other chemicals used in this study were purchased from M/s Himedia, India. All the chemical ingredients used in this study are of analytical grade with high purity (> 99%).
2.2. Microorganism Bacillus subtilis MTCC441 was purchased from Institute of Microbial Technology (IMT), Chandigarh, India. The pure culture of B. subtilis MTCC 441 was made to grow on Luria Bertani (LB) agar using petriplates at 37 °C and stored at 20 °C. A colony was picked up from the culture grown on petriplates, inoculated and incubated at 150 RPM, 37 °C, overnight culture on LB broth which was used as inoculum for production throughout the study. The glycerol stock was maintained at –80 °C. 2
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Fig. 2. Effect of different concentration of CFP, bacterial peptone, control medium on (a) levan production and (b) biomass concentration. Notes: (1). Fermentation conditions: sucrose-100 g/L, 37 °C, 150 RPM, 20 h, CFP (1–4) g/L. (2). FM1 – synthetic medium, FM2 – sucrose + bacterial peptone, FM3a – sucrose + 1 g/L CFB, FM3b – sucrose + 2 g/L CFB, FM3c – sucrose + 3 g/L CFB, FM3d – sucrose + 4 g/L CFB. (3) NS = Not Significant at α = 0.05.
detector temperatures were maintained at 35 °C and 30 °C, respectively. For molecular weight determination, Ultrahydrogel 1000 Gel Permeation Chromatography (GPC) column was used with 0.6 mL/min 0.5 N sodium nitrite solution as mobile phase. Column and detector were maintained at 35 °C and 30 °C, respectively.
2.3. Production of chicken feather peptone (CFP) Chicken feathers were collected from local market at Tiruchirapalli, Tamil Nadu, India. Feathers were washed with distilled water and dried in sunlight. Hydrolysis of chicken feather was carried out using the process as outlined in Fig. 1. Chicken feathers were grinded and powered using pulverizer. 50 g of chicken feather powder was taken and treated with 6 N hydrochloric acid in two steps at 70 °C and 130 °C, as shown in the Fig. 1. The resulting sample was neutralised using 5 N sodium hydroxide solution and filtered using sintered crucible under vacuum. The chicken feather hydrolysate (liquid fraction) was collected and concentrated in rotary evaporator. The residue collected after evaporation is oven dried at 60 °C for 24 h. The dried powder was labelled as CFP (Chicken feather peptone) and stored for further use.
2.7. Cell culture Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from HiMedia (India) and cultured in endothelial cell expansion medium (HiEndoXL, Himedia) supplemented with endothelial cell growth supplement (AL517B) and 1% penicillin/streptomycin antibiotics. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
2.4. Levan production
2.8. Cytotoxicity analysis
Levan production was carried out in an Erlenmeyer’s flask with the culture volume of 50 mL in a 250 mL flask. Levan production was carried out with different media composition as shown in Table 1. The initial pH was adjusted to 7.0 before autoclaving. The culture was incubated at optimised conditions 37 °C, 150 RPM, for 20 h. All the experiments were carried out in triplicates and the experimental means were statistically compared using Tukey’s test at α = 0.05.
HUVEC cells were seeded in a 96 well plate (coated with 0.5% gelatine) at an approximate density of 1 × 104 per well and incubated overnight. The next day, they were treated with different concentrations of isolated levan compound (100, 250,500,750 and 1000 μM) for 24 h. Cells treated with N-acetyl cysteine (NAC - 1000 μM) for 24 h was used as positive control. Subsequently, cells were washed with 1X PBS and incubated with 1 mg/mL of MTT. After 4 h of incubation at 37 °C, formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm by using microplate reader spectrophotometer (Synergy H1).
2.5. Recovery of levan At the end of the fermentation process the culture was collected and centrifuged at 13,230 × g for 10 min and pellet was dried at 60 °C for biomass estimation. Levan was recovered from the supernatant. The pH of the supernatant was adjusted to 9.0. Levan was precipitated by adding ice cold isopropanol in the ratio of 1:5 (supernatant to isopropanol) and the mixture was centrifuged at 16,538 × g for 10 min. The pellet was dried at room temperature overnight (35 °C) and weighed.
3. Results and discussion Comparison of defined media and complex media for levan production was performed as described in materials and methods section. 3.1. CFP production Chicken feather peptone was prepared using the process given in Fig. 1. Investigation showed that the CFP would contain organic and inorganic substances required for the microbial growth and metabolism (EPS production) (Ozdal & Kurbanoglu, 2018; Taskin & Kurbanoglu, 2011). CFP produced from chicken feather would give stimulatory effect for EPS production because it contain essential amino acids and minerals such as Na, Fe, Ca, Mn, Mg required for the microbial cultivation list in the Tables 2 and 3 respectively. Since CFP contain all essential amino acids it can be exploited as a complex nutrient source along with suitable carbon source to support microbial growth and production metabolites. CFP is rich in amino acids such as glutamic acid, alanine, glycine, cysteine which is essential for growth and sporulation of Bacillus subtilis. Bacillus subtilis MTCC 441 is a sporulating
2.6. Analytical methods 1
H- NMR and 13C- NMR analyses of the polymer were carried out in Bruker 300 MHz instrument at room temperature. Samples for 1H-NMR and 13C-NMR were prepared using D2O. Fourier Transform Infrared Spectroscopy (FTIR) spectrum was recorded using Perkin- Elmer spectrum one instrument, with a resolution of 5 cm−1. Residual sugar composition and molecular weight of the samples were determined using Waters High Performance Liquid Chromatography (HPLC) system equipped with a Refractive Index detector. Amino Spherisorb (5 μm, 4.6 × 250 mm) analytical column was used with a mobile phase 80% acetonitrile at 1 mL/min for residual sugar analysis. Column and 3
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Fig. 3.
13
C NMR and 1H-NMR of levan production using CFP- 2 g/L.
4
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concentration of 1.28 g/L. Further increase in CFP concentration gave less levan yield even though there is an increase in biomass concentration. This may be attributed to inhibitory effect of micronutrients in the medium as concentration of micronutrient increase with increase in concentrations of CFP. Similar trends were observed during xanthan production with increasing levels of CFP (Ozdal & Kurbanoglu, 2018). Among different organic nitrogen sources such as CFP, bacterial peptone and yeast extract, it was found that CFP showed comparable results with yeast extract and higher levels of levan production. These experiments demonstrated that the maximum levan yield of 0.31 g levan/g sucrose consumed while the biomass concentration of 1.32 g/L was obtained with control medium. In control medium levan production 0.26 g levan/g sucrose was obtained with CFP with a biomass concentration of 1.28 g/L. Bacterial peptone showed lower levan yield in comparison to cultures fed chicken feather peptone (Fig. 2a). This might be due to the difference in nutrient composition of bacterial peptone in comparison to the chicken feather peptone. Similar results were obtained during xanthan biopolymer production using bacterial peptone in comparison to CFP (Ozdal & Başaran Kurbanoglu, 2019). Researchers have reported that the addition of organic nitrogen sources (CFP) to production medium enhanced cell growth and product yield (Ozdal & Kurbanoglu, 2018, 2019; Taskin & Kurbanoglu, 2011).
Table 4 The chemical shifts of 13C- NMR spectra of levan produced by different bacteria. Carbon atom
C-1 C-2 C-3 C-4 C-5 C-6
Chemical shifts (ppm) From Bacillus subtilis MTCC 441 (present study)
From Bacillus licheniforms BK AG21 (Mamay et al., 2015)
From Chromohalobacter japonicus BK-AB18 (Nasir et al., 2015)
59.90 104.19 76.28 75.19 80.27 63.37
59.85 104.22 76.24 75.18 80.31 63.41
59.83 104.23 76.24 75.17 80.31 63.40
bacteria which is used to produce levan. Methionine and tryptophan are known for their inhibitory effect on sporulation. Thus, absence of methionine and tryptophan can inhibit sporulation and hence favourably influence the production of levan. Presence of micronutrients such as Fe, Ca, Mg enhance the activity of levansucrase (Chambert, Benyahia, & Glatron, 1990; Petit-Glatron, Monteil, Benyahia, & Chambert, 1990), the enzyme which catalyse sucrose hydrolysis and assist in transfructosylation for levan production. As CFP contains these nutrients it favours levan production.
3.3. Characterisation of levan The 13C NMR spectrum peak shown in Fig. 3(a) confirms the structure of levan produced by Bacillus subtilis MTCC 441 using CFP. The six carbon resonance at the chemical shifts of 104.17 ppm (C-2), 80.27 ppm (C-5), 76.28 ppm (C-3), 75.19 ppm (C-4), 63.37 ppm (C-6), 59.90 ppm (C-1) represents the carbon atoms in the levan polymer produced using CFP. The results obtained are consistent with the previous reports listed in the Table 4. The chemical shifts of 104.17 ppm (C-2) and 63.37 ppm (C-6) corresponded to the presence of β-(2–6) linkages of levan (Mamay, Wahyuningrum, & Hertadi, 2015; Nasir et al., 2015).The 1H-NMR spectrum of levan produced using CFP also represented the proton chemical shift signals related to the fructose as monomer of levan (Fig. 3(b)). The chemical shifts observed in the range of 3.46–3.86 ppm are in accordance with the previous reports (Mamay et al., 2015; Nasir et al., 2015). Fig. 4 shows the GPC results of produced levan. Molecular weight of the levan produced using CFP, bacterial peptone, control medium shows identical fraction listed in the Table 5. It was in the range of
3.2. Effect of CFP on levan production Comparisons of levan yield and biomass concentration obtained from different concentrations of CFP (1–4 g/L), bacterial peptone, and control medium are shown in Fig. 2a and b. Results show that CFP concentration strongly influenced bacterial growth and levan production. Cell growth and enzyme production patterns are strongly affected by nitrogen source, micronutrients and macronutrients. As expected, at 20 h of fermentation the levan yield in culture fed with 2 g/L CFP was almost similar (0.26 ± 0.04 g levan/g sucrose consumed) to that obtained from the control medium (FM1) (0.31 ± 0.02 g levan/g sucrose consumed). Fig. 2a shows that there is no significance difference (α = 0.05) in levan production between CFP 2 g/L and control medium. It is shown that CFP comprises amino acids, minerals and salts which could act as a complex nutrient source for the microorganism. Table 2 and 3 shows the composition of CFP reported in literature. The production medium containing 2 g/L CFP (FM3b) resulted in the biomass
Fig. 4. Gel Permeation chromatographs of levan. 5
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Biotechnology, Government of India for the financial support (Grant: BT/PR7511/BCE/8/973/2013) and Department of Science & Technology for support through FIST programme (Grant: SR/FST/ETI331/2013). Dr. Shanmugam S. R., and Dr. Krishna Priya Mani sincerely thank DST for the financial support through INSPIRE grants DST/INSPIRE/ 04/2017/002528 and DST/INSPIRE/04-I/2016/000003 respectively.
Table 5 Comparison of molecular weight distribution of levan samples. Molecular weight (kDa)
CFP - 2 g/L Bacterial peptone Control medium
1
2
3
4
2646.68 2765.57 2658.34
8.66 9.41 8.98
ND ND 5.39
3.36 3.75 3.78
References Note: ND- Not Determined. Akpor, O. B., Odesola, D. E., Thomas, R. E., & Oluba, O. M. (2019). Chicken feather hydrolysate as alternative peptone source for microbial cultivation. F1000Research, 7, 1918. https://doi.org/10.12688/f1000research.17134.2. Chambert, R., Benyahia, F., & Glatron, M. F. P. (1990). Secretion of Bacillus subtilis levansucrase - Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes. The Biochemical Journal, 265, 375–382. de Oliveira, M. R., da Silva, R. S. S. F., Buzato, J. B., & Celligoi, M. A. P. C. (2007). Study of levan production by Zymomonas mobilis using regional low-cost carbohydrate sources. Biochemical Engineering Journal, 37, 177–183. https://doi.org/10.1016/j.bej. 2007.04.009. Han, Y. W., & Watson, M. A. (1992). Production of microbial levan from sucrose, sugarcane juice and beet molasses. Journal of Industrial Microbiology, 9, 257–260. https://doi.org/10.1007/BF01569633. Küçükaşik, F., Kazak, H., Güney, D., Finore, I., Poli, A., Yenigün, O., et al. (2011). Molasses as fermentation substrate for levan production by Halomonas sp. Applied Microbiology and Biotechnology, 89, 1729–1740. https://doi.org/10.1007/s00253010-3055-8. Kuruppasamy, G., Gunasekaran, P., & Velusamy, S. K. (2007). Strategies for the improvement of levan and fructooligosaccharides production by zymomonas mobilis. Chapter 12Advances in bioprocessing in food industry. Haworth Press. Mamay, Wahyuningrum, D., & Hertadi, R. (2015). Isolation and characterization of Levan from moderate halophilic Bacteria Bacillus licheniformis BK AG21. Procedia Chemistry, 16, 292–298. https://doi.org/10.1016/j.proche.2015.12.055. Matsuhira, H., Tamura, K., Tamagake, H., & Sato, Y. (2014). High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes. Journal of Biotechnology, 192, 215–222. https://doi.org/10.1016/j.jbiotec.2014.09. 025. Moosavi-Nasab, M., Layegh, B., Aminlari, L., & Hashemi, M. B. (2010). Microbial production of levan using date syrup and investigation of its properties. World Academy of Science, Engineering and Technology, 44, 1248–1254. Nasir, D. Q., Wahyuningrum, D., & Hertadi, R. (2015). Screening and characterization of levan secreted by halophilic bacterium of Halomonas and Chromohalobacter genuses originated from Bledug Kuwu mud crater. Procedia Chemistry, 16, 272–278. https:// doi.org/10.1016/J.PROCHE.2015.12.050. Orak, T., Caglar, O., Ortucu, S., Ozkan, H., & Taskin, M. (2018). Chicken feather peptone: A new alternative nitrogen source for pigment production by Monascus purpureus. Journal of Biotechnology, 271, 56–62. https://doi.org/10.1016/j.jbiotec.2018.02.010. Ozdal, M., & Başaran Kurbanoglu, E. (2019). Use of chicken feather peptone and sugar beet molasses as low cost substrates for xanthan production by Xanthomonas campestris MO-03. Fermentation, 5, 9. https://doi.org/10.3390/fermentation5010009. Ozdal, M., & Kurbanoglu, E. B. (2019). Citric acid production by Aspergillus niger from agro-industrial by-products: Molasses and chicken feather peptone. Waste and Biomass Valorization, 10, 631–640. https://doi.org/10.1007/s12649-018-0240-y. Ozdal, M., & Kurbanoglu, E. B. (2018). Valorisation of chicken feathers for xanthan gum production using Xanthomonas campestris MO-03. Journal, Genetic Engineering & Biotechnology, 16, 259–263. https://doi.org/10.1016/j.jgeb.2018.07.005. Petit-Glatron, M. F., Monteil, I., Benyahia, F., & Chambert, R. (1990). Bacillus subtilis levansucrase: Amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals. Molecular Microbiology, 4, 2063–2070. https:// doi.org/10.1111/j.1365-2958.1990.tb00566.x. Shih, I. L., Chen, L. D., & Wu, J. Y. (2010). Levan production using Bacillus subtilis natto cells immobilized on alginate. Carbohydrate Polymers, 82, 111–117. https://doi.org/ 10.1016/j.carbpol.2010.04.030. Srikanth, R., Reddy, C. H. S. S. S., Siddartha, G., Ramaiah, M. J., & Uppuluri, K. B. (2015). Review on production, characterization and applications of microbial levan. Carbohydrate Polymers, 120, 102–114. https://doi.org/10.1016/j.carbpol.2014.12. 003. Stanbury, P. F., Whitaker, A., & Hall, S. J. (2003). Priniciple of fermentation technology. Vasa 367. Taskin, M., & Kurbanoglu, E. B. (2011). Evaluation of waste chicken feathers as peptone source for bacterial growth. Journal of Applied Microbiology, 111, 826–834. https:// doi.org/10.1111/j.1365-2672.2011.05103.x. Yezza, A., Tyagi, R. D., Valéro, J. R., & Surampalli, R. Y. (2006). Bioconversion of industrial wastewater and wastewater sludge into Bacillus thuringiensis based biopesticides in pilot fermentor. Bioresource Technology, 97, 1850–1857. https://doi.org/10. 1016/j.biortech.2005.08.#.
Fig. 5. Levan compound effect on HUVEC viability. Notes: Cells were treated with different concentrations of levan (C1 – 100 μM, C2 – 250 μM, C3 – 500 μM, C4 – 750 μM and C5 – 1000 μM) for 24 h, and cell viability was measured by MTT assay. The experiment was repeated three times and the values are represented as mean ± SEM. N-Acetyl Cysteine (NAC) was used as a positive control (PC).
2600–2800 kDa for the high fraction and 3–9.5 kDa for the low fraction. These results are consistent with the previous reports (Shih, Chen, & Wu, 2010). 3.4. Effect of levan on HUVECs viability The cytotoxicity of isolated levan compound was examined by using MTT assay. As shown in Fig. 5 levan compound (100, 250,500,750 and 1000 μM) did not altered the cell viability of HUVECs in 24 h, whereas 1000 μM NAC (Positive control) significantly increased the cell viability (P < 0.05). These results indicate that the produced levan using CFP doesn’t pose any risk to the cell lines. Hence the produced levan can be utilised for biomedical application such as carrier vehicle for drugs, etc. 4. Conclusions The current study evaluates the potential of CFP as a low cost nutrient source for levan production using Bacillus subtilis MTCC 441. Since chicken feathers are proteinaceous in nature and are available in large quantities they could be utilized as an effective nutrient for the growth of microorganisms and production process. The results from this study support this hypothesis. Studies conducted on primary cell lines to test the cytotoxicity indicate that levan produced using CFP does not show any negative effect on cell lines indicating that the produced levan is compatible for biomedical applications. Chicken feathers which are discarded as waste from poultry industry can be thus put into the production of levan and this would simultaneously solve the solid disposal problem and improve the economic potential of poultry industry. Acknowledgement Dr.
V.
Ponnusami
gratefully
thank
the
Department
of
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