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Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023
New Biotechnology Volume 28, Number 6 October 2011
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Research Paper
Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023 Chuan-Lu Wang1, Tzu-Huang Huang3, Tzu-Wen Liang2,3, Chun-Yong Fang3 and San-Lang Wang2,3,4, 1
Department of Cosmetic Science and Application, Lan-Yang Institute of Technology, I-Lan 261, Taiwan Life Sciences Development Center, Tamkang University, Taipei 25137, Taiwan 3 Department of Chemistry, Tamkang University, Taipei 25137, Taiwan 2
Using squid pen powder (SPP) as the sole C/N source, Paenibacillus sp. TKU023 produced exopolysaccharides (EPS) and antioxidant. With medium containing 1.5% SPP, 0.1% K2HPO4, and 0.05% MgSO47H2O, pH 7.23, the culture was incubated at 378C in liquid (50 mL/250 mL) for five days. The resultant culture supernatant had higher EPS productivity (4.55 g/L). The crude EPS were isolated by centrifugation, methanol precipitation and deproteinization. The characterization of the EPS demonstrated that it was mainly composed of glucose and maltose. In addition, the culture supernatant incubated for four days by using baffled base flask showed the strongest antioxidant activities and the highest total phenolic content, but maximum EPS production was found at the fifth day by using flat base flask. The production of two invaluable environmental-friendly biomaterials (EPS and antioxidant) is unprecedented. Besides, the use of SPP (waste) is green that made the whole process more valuable and attractive.
Introduction Polysaccharides are composed of many monosaccharide units that are joined one another by a glycosidic linkage to give a long chain. Traditionally, polysaccharides are extracted from plant seeds, plant exudates, marine algae and animals [1]. Over the past few decades, the number of exopolysaccharides (EPS) produced by microbial fermentation has been gradually increasing. In recent years, microbial EPS have found multifarious applications in food, pharmaceutical, other industries and had physiological activity different from natural gums and synthetic polymers [2,3]. Moreover, they are highly susceptible to biodegradation in nature and less harmful than synthetic polymer. The most used carbon sources for EPS production have been sugars, namely glucose and sucrose [4,5]. However, the high cost Corresponding author: Wang, S.-L. ([email protected]) 4 Present address: 151 Ying-chuan Road Tamsui, New Taipei City 25137, Taiwan, ROC.
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of these carbon sources has a direct impact on production costs, which limits the market potential of these biopolymers. To decrease the production costs, it is important to look for less expensive carbon sources, like wastes or industrial by-products [2]. Squid pen waste is an important source of bioactive molecules. The major components (on dry weight basis) of squid pen waste are protein (61%), chitin (38%) and minerals (1%) [6]. Fermentation technique can be used for the utilization of squid pen waste [7]. During fermentation, owing to liquefaction of protein and chitin, bioactive material rich liquor is formed including peptide, amino acids and chitooligosaccharides [7]. Literature survey found that the shellfish waste is a rich source of phenolic compounds, such as shrimp shell waste [8]. Phenolic compounds play an important role in the antioxidative properties and phenolic substances were also reported to possess a wide range of biological effects, including antioxidant, antimicrobial, anti-inflammatory and anticancer activities [9,10]. It is expected that this bioactive material rich liquor will have beneficial biological functions due www.elsevier.com/locate/nbt
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to the inherent protein and chitin hydrolysis and other bioactive material production occurring during fermentation. In this study, the EPS and antioxidant were obtained during fermentation of squid pen waste. Till now, there are many reports on the culture conditions for the production of EPS from microorganism [11,12]. However, reports about the production of EPS and antioxidant by Paenibacillus species seem to be scarce. Therefore, the objectives of present study were to investigate the culture conditions of the EPS and antioxidant from Paenibacillus sp. TKU023, and the antioxidant properties were assessed using different in vitro systems. Besides, the crude EPS obtained from the culture were characterized by thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy. In addition, we also assessed the phenolic content of the fermented supernatant. Furthermore, the correlation between the antioxidant activity and phenolic content was also considered.
Measurement of total sugars
Materials and methods Materials
Deproteinization of EPS
The squid pen powder (SPP) and shrimp head powder (SHP) used in these experiments were prepared as described earlier [7]. Squid pens and shrimp shells were purchased from Shin-Ma Frozen Food Co. (I-Lan, Taiwan). The squid pen and shrimp head were washed thoroughly with tap water and then dried at 358C. The dried materials obtained were milled to powder for using as the carbon source for EPS production. Paenibacillus sp. TKU023 was a biosurfactant producing strain isolated from soils and maintained on nutrient agar. The a,a-diphenyl-b-picrylhydrazyl (DPPH) and Folin-Ciocalteu reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents and solvents used were of the highest grade available.
Identification of the strain TKU023 The bacterial strain TKU023 was identified on the basis of morphological, physiological and biochemical parameters as well as on the basis of 16S rDNA based sequence analysis after PCR amplification with primers and cloning. Nucleotide bases of the DNA sequence obtained were compiled and compared with sequences in the GenBank databases using BLAST program.
Biowaste fermentation for EPS production In the investigation of the culture condition, growth was carried out in a basal medium containing 0.1% K2HPO4 and 0.05% MgSO47H2O (pH 7), and supplemented with 0.5–1.5% (w/v) of various carbon sources for one to ten day fermentation. The carbon sources investigated included sucrose, SPP and SHP. Factors affecting cell growth and EPS production were investigated using one-factor-at-a-time method. To determine the optimum initial pH for EPS production, medium was adjusted to the required pH by the addition of 1 N HCl and 1 N NaOH before sterilization. Then, the media including not adjusted pH and adjusted to pH 2–12 were sterilized before inoculating with 1% (v/v) of the seed culture. To find out the optimum temperature and cultivation volume for EPS production, 25–200 mL of medium was incubated at 25, 30 and 378C, respectively. Time course experiment was carried out in a 250-mL flask containing the screened culture medium based on the results of single factor experiments. 560
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To evaluate total sugars, the phenol–sulfuric acid method was used [13]. Briefly, 25 mL of 5% phenol was added to 1 mL of sample. After shaking, 2.5 mL of concentrated H2SO4 was added. The mixture was left to stand for 10 min and absorbance was read at 490 nm. Pure D-glucose was employed as standard.
Isolation of EPS After fermenting, the sample was immediately autoclaved for 20 min to reduce ropy condition, followed by centrifugation (12,000 g for 20 min) to remove the remaining SPP and biomass. The supernatant was filtered through a 0.45 mm membrane filter, mixed with two volumes of methanol, stirred vigorously and kept overnight at 48C. The precipitate from the methanol dispersion was collected by centrifugation at 12,000 g for 15 min, redissolved in distilled water and lyophilized to afford the crude EPS.
The crude EPS were redissolved in distilled water and stirred vigorously at 808C for 30 min, mixed with four volumes of anhydrous ethanol, stirred vigorously and kept overnight at 48C. The precipitate from the ethanol dispersion was collected by centrifugation at 12,000 g for 15 min, redissolved in distilled water and followed by deproteinization with 1/5 volume of Sevag reagent (CHCl3–BuOH, v/v = 5/1) for seven times [14]. The deproteinized solution was then dialyzed against distilled water, concentrated and lyophilized to afford the deproteinized EPS.
Analysis of monosaccharide compositions of deproteinized EPS For monosaccharide composition analysis, the deproteinized EPS (25 mg) were dissolved in 50 mL distilled water and hydrolyzed with 25 U/mL of a-amylase at 208C, pH 6.9 for 24 hours. The hydrolyzate was then dialyzed against distilled water, concentrated and lyophilized. The hydrolyzate was analyzed by silica gel thin layer chromatography (TLC) using 5:4:3 (v/v/v) n-butanol/ methanol/16% aqueous ammonia as the mobile phase [15]. Silica gel TLC plates (0.25 mm) were obtained from E. Merck. After developing the TLC plates, the compounds were visualized by spraying with an aqueous solution of 2.4% (w/v) phosphomolybdic acid, 5% (v/v) H2SO4, and 1.5% (v/v) H3PO4 (phosphomolybdic acid reagent) or ethanol containing 0.5% (w/v) ninhydrin (ninhydrin reagent) or DPPH solution described as above method, followed by heating. Structural characterization of monosaccharide of EPS was analyzed further by NMR spectrum.
Antioxidant assays of EPS Measurement of DPPH radical scavenging activity The sample (150 mL) was mixed with 37.5 mL of methanolic solution containing 0.75 mM DPPH radical. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank [16]. The scavenging ability was calculated as follows: scavenging activity (%) = [(A517 of control A517 of sample)/A517 of control] 100.
Measurement of Fe2+ chelating ability The method of Decker and Welch [17] was adopted. Five milliliters of the diluted sample was spiked with 0.1 mL of 2 mM FeCl2 and 0.2 mL of 5 mM ferrozine solutions. After reaction for 10 min, the
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The total phenolics of the culture supernatants were determined [19] and expressed as micrograms of gallic acid equivalents per milliliter of culture supernatant. Two milliliters of deionized water and 1 mL of Folin-Ciocalteu’s phenol reagent were added to 0.3 mL of each sample. Five milliliters of 20% aqueous sodium carbonate solution (w/v) was added and mixed well, and then the mixture was allowed to stand at ambient temperature for 20 min. Absorbance of the developed dark bluepurple color was measured by spectrophotometer at 735 nm. The content of total phenolics in each sample was determined using a standard curve prepared with gallic acid at varied concentrations (0, 50, 100, 200, 400, 600 and 800 mg/mL).
Statistical analysis In this study, each experiment was conducted in triplicate. The statistical analysis was calculated and expressed. All statistical analyses were carried out using SPSS 11.01 for Windows. To determine whether there were any differences between activities of samples, variance analysis was applied to the result. Values of P < 0.05 were considered as significantly different (a = 0.05).
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Effects of carbon/nitrogen sources on total sugar (solid line) and EPS (dashed line) production by Paenibacillus sp. TKU023. (*) SPP; (~) SHP; (&) sucrose.
suitable carbon source for total sugar and EPS production, various carbon sources were separately provided in the basal medium. Besides, to further utilize the chitin/protein-containing biowastes, we incubated Paenibacillus sp. TKU023 for one to five days at 378C by using 1% sucrose, 1% SPP, or 1% SHP as carbon sources and analyzed the amount of total sugar and EPS. As shown in Fig. 1, SPP was the most suitable carbon source for EPS production. This is different from the finding of many other investigators [5,11,12,20]. To select the optimal SPP concentration for total sugar and EPS production, 0.5–1.5% SPP was added into the basal medium. As shown in Fig. 2a, the increase of the SPP concentration in the
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Results and discussion Identification of the strain TKU023 To characterize strain TKU023, 16S rDNA and phylogenetic analyses were utilized. According to the analysis of 16S rDNA gene sequence, TKU024 was most closely aligned to Paenibacillus sp. and close to Paenibacillus elgii, Paenibacillus ehimensis with 99% similarity. To characterize strain TKU023 further, standard morphological, physiological and biochemical plates showed that strain TKU023 was a Gram-positive and non-spore-forming bacillus, which grows in both aerobic and anaerobic environments. The phylogenetic identification indicated that the strain TKU023 belongs to Paenibacillus sp.
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A method developed by Oyaizu [18] for testing reducing power was used. The diluted sample or distilled water (control) (0.5 mL) was spiked with 0.5 mL of sodium phosphate buffer (0.02 M, pH 7) and 0.5 mL of 1% potassium ferricyanide. The mixture was then kept in a 508C water bath for 20 min. The resulting solution was cooled rapidly, spiked with 0.5 mL of 10% trichloroacetic acid, and centrifuged at 800 g for 10 min. The supernatant (1.5 mL) was then mixed with 0.2 mL of 0.1% ferrichloride. After reaction for 10 min, the absorbance at 700 nm was measured. The reducing power of each sample was determined using a standard curve prepared with cysteine at varied concentrations.
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absorbance (at 562 nm) of the resulting solution was recorded. A complex of Fe2+/ferrozine has a strong absorbance at 562 nm. A high ferrous ion chelating ability in the test sample results in a low absorbance. The ferrous ion chelating ability was calculated as follows: chelating ability (%) = [1 (test sample absorbance/blank sample absorbance)] 100.
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Effects of SPP concentration (A) and initial temperature (B) on total sugar (solid line) and EPS (dashed line) production by Paenibacillus sp. TKU023. www.elsevier.com/locate/nbt
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Effects of initial pH on total sugar (&) and EPS (&) production by Paenibacillus sp. TKU023.
medium led to the increases of both total sugar and EPS production. During six-day fermentation, the SPP decreased gradually in the medium, indicating that most of the SPP in the medium had been utilized for EPS production. The highest EPS production (4.18 g/L) was obtained at the SPP concentration of 1.5%.
Effect of cultivation volume, temperature and initial pH To study the effect of cultivation volume on EPS production, we found that 50 mL of medium was more suitable for EPS production than 100 mL, 150 mL and 200 mL. This might be related to the oxygen dissolved in the medium. The dissolved oxygen increased with the decrease of cultivation volume at a constant agitation speed. The dissolved oxygen can influence the physiology and the metabolism of the aerobic microorganisms. In a fermentation system, the dissolved oxygen is typically controlled by the variation of the agitation speed [21,22] or by changing the air flow rate
Time course of EPS production Using SPP as the sole C/N source, the relationship between the EPS production and cell growth was investigated. As shown in Fig. 4, maximum total sugar (5555 mg/mL) and EPS production (4.55 g/L) were both found at the fifth day by using 50 mL of the media and then decreased gradually. These results were similar to those found in Fig. 2b. Besides, the bacterium grew rapidly in the first four days, and we also found that total sugar and EPS production were closely related to the cell growth. The EPS yield reached the maximum level (the fifth day) after the cell growth reached the stationary phase. These results indicate that the production of EPS is cell growth dependent and Paenibacillus sp. TKU023 is a promising EPS producer. In Paenibacillus spp., P. polymyxa EJS-3 was also reported
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[22]. To affirm the result, we further investigated the effect of dissolved oxygen on EPS production by fermentor. There was no EPS production when cultured at 50 rpm, but EPS production was found at 150 rpm. The dissolved oxygen increases with agitation speed in the same medium. Therefore, it was inferred that the dissolved oxygen might affect EPS production of Paenibacillus sp. TKU023. Incubation temperature is a crucial factor in EPS biosynthesis [5]. The production of EPS was investigated, using 1.5% SPP as the sole C/N source at various culture temperatures. As shown in Fig. 2b, the optimal temperature for EPS production was 378C with EPS production reached 4.55 g/L. The initial culture pH is also an important factor that may affect cell membrane, cell morphology and structure, the uptake of various nutrients and EPS biosynthesis [5,23]. The production of EPS was also investigated, using SPP as the sole C/N source, in media of various pH values controlling namely, not adjusted, adjusted to 2–12. As shown in Fig. 3, the optimal pH value before sterilization for EPS production was 7.23 (not adjusted pH), with the corresponding EPS production of 4.55 g/L. This result was similar to P. polymyxa strains from soil [11,12], but unlike P. polymyxa EJS-3 (pH 8, slightly alkaline) for EPS production [5].
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Time courses of total sugar and EPS production in a culture of Paenibacillus sp. TKU023 on squid pen containing media: (*) total sugar (mg/mL); (*) EPS (g/L); (&) cell growth. 562
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TABLE 1
Comparison of EPS produced from other kinds of microorganisms Sources
Carbon/nitrogen sources
Yield of EPS (g/L)
Cultivation time (hour)
Productivity of EPS (g/(L hour))
Reference
1.5% squid pen powder
4.55
120
0.038
This study
16% sucrose/1% yeast extract
22.82
60
0.380
[5]
Pseudomonas oleovorans NRRL B-14682a
4% glycerol-rich product
12.18
120
0.102
[24]
a
Rhodotorula acheniorum MC
5% sucrose
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96
0.065
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Agrocybe cylindracea ASI-9002
6% maltose
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240
0.013
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Azotobacter sp. SSB81
2% glucose, 0.0001% riboflavin and 0.2% casamino acid
2.52
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0.084
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Paecilomyces tenuipes C240a
3% glucose
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0.010
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Halomonas eurihalina H212
MY medium
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192
0.008
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Bacillus licheniformis
MRS-0.2% glucose
0.88
12
0.073
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Halomonas anticariensis Al16
1% glucose
0.2895
120
0.002
[29]
Pseudomonas aeruginosa B1
Nutrient broth
0.075
72
0.001
[30]
The EPS was produced by a jar fermentor.
The composition of EPS In the present study, EPS was isolated from the fermentation broth of Paenibacillus sp. TKU023 by centrifugation, methanol precipitation and deproteinization. To determine the monosaccharide composition, the isolated EPS was hydrolyzed with 0.5 U/mL aamylase at 208C, pH 6.9 for 24 hours. The hydrolysates of EPS were developed on the TLC plates, and two compounds were visualized by spraying with phosphomolybdic acid reagent (data not shown). The results of TLC showed that the compositions of the EPS might contain glucose and maltose. Furthermore, the hydrolysates were characterized by 1H NMR and 13C NMR. The 1H NMR and 13C NMR spectra show peaks identical to maltose. The results indicated that the monosaccharide composition of the EPS was mainly glucose, and maltose molecules also appeared in the composition of the EPS.
production has been well documented [22]. Therefore, we also chose baffled base flask to increase levels of oxygenation and mixing. As shown in Fig. 5, it was found that TKU023 culture supernatant (1.5% SPP) incubated for four days by using baffled base flask has the highest antioxidant activity, the DPPH scavenging ability of TKU023 culture supernatant was about 92%. However, maximum EPS production (4.55 g/L) was found at the fifth day by using flat base flask (Fig. 5). The EPS produced by P. polymyxa EJS-3 had strong antioxidant activity [5]. However, our results indicated that the antioxidant was not mainly the EPS and it may include other materials. This may be also due to the presence of other antioxidant components in the culture supernatant, such as proteins, amino acids, peptides, organic acids and microelements. Among these antioxidant components, there may be some interactions and synergistic effects for antioxidant properties.
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DPPH scavenging ability To utilize the chitin/protein-containing biowastes, we incubated Paenibacillus sp. TKU023 for one to six days at 378C by using 1.5% SPP as carbon/nitrogen source and analyzed the antioxidant activity of the culture supernatant using DPPH scavenging ability. Besides, the importance of oxygenation for the enhancement of biopolymer
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as an EPS producing strain [5]. P. polymyxa EJS-3 produced EPS by using 16% sucrose and 1% yeast extract as the carbon and nitrogen sources. Compared with above, the EPS was produced merely by using cheaper medium from Paenibacillus sp. TKU023 than P. polymyxa EJS-3 (Table 1). The production of EPS reported in various documents is shown in Table 1. In most of the previous reports, the effect of sugars was investigated on the production of EPS by microorganisms and found EPS was quickly produced with the increase of sucrose or glucose concentration in the medium (Table 1). However, in this study, Paenibacillus sp. TKU023 adjusted with culture conditions and could use fish waste SPP as C/N source to produce a respectable amount of EPS (4.55 g/L).
DPPH free radical scavenging activity (%)
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Paenibacillus sp. TKU023 Paenibacillus polymyxa EJS-3
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Antioxidant activity (&) and EPS production (*) from Paenibacillus sp. TKU023 by using flat base flask (solid line) and baffled base flask (dashed line), respectively.
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Ferrous ion chelating ability (%)
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FIGURE 6
The ferrous ion chelating ability (^) and reducing power (~) of the fermented supernatants from Paenibacillus sp. TKU023.
Chelating effects on ferrous ion Ferrous ion chelating abilities of the supernatants fermented from 1.5% SPP for one to six days at 378C by using baffled base flask were analyzed. As shown in Fig. 6, it was found that the culture supernatants incubated for four to six days have the highest chelating ability (71–77%) on ferrous ion. The chelating ability did not decrease with the culture time. Therefore, the antioxidant in the culture supernatant is stable for the culture time. Because ferrous ions are the most effective pro-oxidants, the higher chelating abilities of the culture supernatant would be beneficial. For the culture supernatant, the difference between DPPH scavenging ability and ferrous ion chelating ability might be related to the antioxidant compounds. Previous studies showed that the chelating to ferrous was dependent on the numbers of hydroxyl, and the hydroxyl substitution in the ortho position was desirable [31].
shown in Fig. 6. It was found that the culture supernatants incubated for three to five days have the highest reducing power with 354–383 mg of cysteine equivalents per milliliter of the fermented supernatant. The reducing power could be attributed mainly to the bioactive compounds associated with antioxidant activity [8,32– 34]. These bioactive compounds might be present in the culture supernatant, including phenolics, oligopeptides or chitooligosaccharides, are good electron donors and can terminate the radical chain reactions by converting free radicals to more stable products.
Total phenolic content It has been reported that shrimp shell waste contains natural antioxidants, mainly phenolic compounds [8]. The contents of total phenolics in the fermented supernatant from SPP by Paenibacillus sp. TKU023 were investigated. It was found that the fermented supernatants (1.5% SPP) incubated at the fourth day have the highest total phenolics contents, with 880 mg of gallic acid equivalents per milliliter of the fermented supernatant. The fermented supernatant at the fourth day with high phenolic content also showed high DPPH radical-scavenging activity (Fig. 5). This result suggested that phenolic compounds might be responsible for the activity.
Conclusion The success in the production of two environmental-friendly and invaluable biomaterials (EPS and antioxidant) by microbial fermentation using fishery processing wastes of SPP is unprecedented. The culture supernatant of Paenibacillus sp. TKU023 incubated for four days by using baffled base flask showed the highest antioxidant activity, but maximum EPS production (4.55 g/L) was found at the fifth day by using flat base flask. It may be a new source of natural antioxidants with potential value for health food and therapeutics. The findings appear useful for further research aiming to identify the structure of the EPS and antioxidant.
Reducing power
Acknowledgements
The reducing powers of the culture supernatants fermented from 1.5% SPP for one to six days at 378C by using baffled base flask are
This work was supported in part by a grant of the National Science Council, Taiwan (NSC99-2313-B-032-001-MY3).
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25 Pavlova, K. and Grigorova, D. (1999) Production and properties of exopolysaccharide by Rhodotorula acheniorum MC. Food Res. Int. 32, 473–477 26 Xu, C.P. and Yun, J.W. (2004) Influence of aeration on the production and the quality of the exopolysaccharides from Paecilomyces tenuipes C240 in a stirred-tank fermenter. Enzyme Microb. Technol. 35, 33–39 27 Be´jar, V. et al. (1998) Characterization of exopolysaccharides produced by 19 halophilic strains of the species Halomonas eurihalina. J. Biotechnol. 61, 135–141 28 Larpin, S. et al. (2002) Biosynthesis of exopolysaccharide by a Bacillus licheniformis strain isolated from ropy cider. Int. J. Food Microbiol. 77, 1–9 29 Mata, J.A. et al. (2006) Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis. Res. Microbiol. 157, 827–835 30 Onbasli, D. and Aslim, B. (2009) Effects of some organic pollutants on the exopolysaccharides (EPSs) produced by some Pseudomonas spp. strains. J. Hazard. Mater. 168, 64–67 31 Wang, H. et al. (2008) In vitro and in vivo antioxidant activity of aqueous extract from Choerospondias axillaries fruit. Food Chem. 106, 888–895 32 Lin, H.Y. and Chou, C.C. (2004) Antioxidant activities of water-soluble disaccharide chitosan derivatives. Food Res. Int. 37, 883–889 33 Xing, R. et al. (2005) Antioxidative activity of differently regioselective chitosan sulfates in vitro. Bioorg. Med. Chem. 13, 1387–1392 34 He, H. et al. (2006) Preparation and functional evaluation of oligopeptide-enriched hydrolysate from shrimp (Acetes chinensis) treated with crude protease from Bacillus sp. SM98011. Bioresour. Technol. 97, 385–390
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Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023 Chuan-Lu Wang1, Tzu-Huang Huang3, Tzu-Wen Liang2,3, Chun-Yong Fang3 and San-Lang Wang2,3,4, 1
Department of Cosmetic Science and Application, Lan-Yang Institute of Technology, I-Lan 261, Taiwan Life Sciences Development Center, Tamkang University, Taipei 25137, Taiwan 3 Department of Chemistry, Tamkang University, Taipei 25137, Taiwan 2
Using squid pen powder (SPP) as the sole C/N source, Paenibacillus sp. TKU023 produced exopolysaccharides (EPS) and antioxidant. With medium containing 1.5% SPP, 0.1% K2HPO4, and 0.05% MgSO47H2O, pH 7.23, the culture was incubated at 378C in liquid (50 mL/250 mL) for five days. The resultant culture supernatant had higher EPS productivity (4.55 g/L). The crude EPS were isolated by centrifugation, methanol precipitation and deproteinization. The characterization of the EPS demonstrated that it was mainly composed of glucose and maltose. In addition, the culture supernatant incubated for four days by using baffled base flask showed the strongest antioxidant activities and the highest total phenolic content, but maximum EPS production was found at the fifth day by using flat base flask. The production of two invaluable environmental-friendly biomaterials (EPS and antioxidant) is unprecedented. Besides, the use of SPP (waste) is green that made the whole process more valuable and attractive.
Introduction Polysaccharides are composed of many monosaccharide units that are joined one another by a glycosidic linkage to give a long chain. Traditionally, polysaccharides are extracted from plant seeds, plant exudates, marine algae and animals [1]. Over the past few decades, the number of exopolysaccharides (EPS) produced by microbial fermentation has been gradually increasing. In recent years, microbial EPS have found multifarious applications in food, pharmaceutical, other industries and had physiological activity different from natural gums and synthetic polymers [2,3]. Moreover, they are highly susceptible to biodegradation in nature and less harmful than synthetic polymer. The most used carbon sources for EPS production have been sugars, namely glucose and sucrose [4,5]. However, the high cost Corresponding author: Wang, S.-L. ([email protected]) 4 Present address: 151 Ying-chuan Road Tamsui, New Taipei City 25137, Taiwan, ROC.
1871-6784/$ - see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.03.003
of these carbon sources has a direct impact on production costs, which limits the market potential of these biopolymers. To decrease the production costs, it is important to look for less expensive carbon sources, like wastes or industrial by-products [2]. Squid pen waste is an important source of bioactive molecules. The major components (on dry weight basis) of squid pen waste are protein (61%), chitin (38%) and minerals (1%) [6]. Fermentation technique can be used for the utilization of squid pen waste [7]. During fermentation, owing to liquefaction of protein and chitin, bioactive material rich liquor is formed including peptide, amino acids and chitooligosaccharides [7]. Literature survey found that the shellfish waste is a rich source of phenolic compounds, such as shrimp shell waste [8]. Phenolic compounds play an important role in the antioxidative properties and phenolic substances were also reported to possess a wide range of biological effects, including antioxidant, antimicrobial, anti-inflammatory and anticancer activities [9,10]. It is expected that this bioactive material rich liquor will have beneficial biological functions due www.elsevier.com/locate/nbt
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to the inherent protein and chitin hydrolysis and other bioactive material production occurring during fermentation. In this study, the EPS and antioxidant were obtained during fermentation of squid pen waste. Till now, there are many reports on the culture conditions for the production of EPS from microorganism [11,12]. However, reports about the production of EPS and antioxidant by Paenibacillus species seem to be scarce. Therefore, the objectives of present study were to investigate the culture conditions of the EPS and antioxidant from Paenibacillus sp. TKU023, and the antioxidant properties were assessed using different in vitro systems. Besides, the crude EPS obtained from the culture were characterized by thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy. In addition, we also assessed the phenolic content of the fermented supernatant. Furthermore, the correlation between the antioxidant activity and phenolic content was also considered.
Measurement of total sugars
Materials and methods Materials
Deproteinization of EPS
The squid pen powder (SPP) and shrimp head powder (SHP) used in these experiments were prepared as described earlier [7]. Squid pens and shrimp shells were purchased from Shin-Ma Frozen Food Co. (I-Lan, Taiwan). The squid pen and shrimp head were washed thoroughly with tap water and then dried at 358C. The dried materials obtained were milled to powder for using as the carbon source for EPS production. Paenibacillus sp. TKU023 was a biosurfactant producing strain isolated from soils and maintained on nutrient agar. The a,a-diphenyl-b-picrylhydrazyl (DPPH) and Folin-Ciocalteu reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents and solvents used were of the highest grade available.
Identification of the strain TKU023 The bacterial strain TKU023 was identified on the basis of morphological, physiological and biochemical parameters as well as on the basis of 16S rDNA based sequence analysis after PCR amplification with primers and cloning. Nucleotide bases of the DNA sequence obtained were compiled and compared with sequences in the GenBank databases using BLAST program.
Biowaste fermentation for EPS production In the investigation of the culture condition, growth was carried out in a basal medium containing 0.1% K2HPO4 and 0.05% MgSO47H2O (pH 7), and supplemented with 0.5–1.5% (w/v) of various carbon sources for one to ten day fermentation. The carbon sources investigated included sucrose, SPP and SHP. Factors affecting cell growth and EPS production were investigated using one-factor-at-a-time method. To determine the optimum initial pH for EPS production, medium was adjusted to the required pH by the addition of 1 N HCl and 1 N NaOH before sterilization. Then, the media including not adjusted pH and adjusted to pH 2–12 were sterilized before inoculating with 1% (v/v) of the seed culture. To find out the optimum temperature and cultivation volume for EPS production, 25–200 mL of medium was incubated at 25, 30 and 378C, respectively. Time course experiment was carried out in a 250-mL flask containing the screened culture medium based on the results of single factor experiments. 560
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To evaluate total sugars, the phenol–sulfuric acid method was used [13]. Briefly, 25 mL of 5% phenol was added to 1 mL of sample. After shaking, 2.5 mL of concentrated H2SO4 was added. The mixture was left to stand for 10 min and absorbance was read at 490 nm. Pure D-glucose was employed as standard.
Isolation of EPS After fermenting, the sample was immediately autoclaved for 20 min to reduce ropy condition, followed by centrifugation (12,000 g for 20 min) to remove the remaining SPP and biomass. The supernatant was filtered through a 0.45 mm membrane filter, mixed with two volumes of methanol, stirred vigorously and kept overnight at 48C. The precipitate from the methanol dispersion was collected by centrifugation at 12,000 g for 15 min, redissolved in distilled water and lyophilized to afford the crude EPS.
The crude EPS were redissolved in distilled water and stirred vigorously at 808C for 30 min, mixed with four volumes of anhydrous ethanol, stirred vigorously and kept overnight at 48C. The precipitate from the ethanol dispersion was collected by centrifugation at 12,000 g for 15 min, redissolved in distilled water and followed by deproteinization with 1/5 volume of Sevag reagent (CHCl3–BuOH, v/v = 5/1) for seven times [14]. The deproteinized solution was then dialyzed against distilled water, concentrated and lyophilized to afford the deproteinized EPS.
Analysis of monosaccharide compositions of deproteinized EPS For monosaccharide composition analysis, the deproteinized EPS (25 mg) were dissolved in 50 mL distilled water and hydrolyzed with 25 U/mL of a-amylase at 208C, pH 6.9 for 24 hours. The hydrolyzate was then dialyzed against distilled water, concentrated and lyophilized. The hydrolyzate was analyzed by silica gel thin layer chromatography (TLC) using 5:4:3 (v/v/v) n-butanol/ methanol/16% aqueous ammonia as the mobile phase [15]. Silica gel TLC plates (0.25 mm) were obtained from E. Merck. After developing the TLC plates, the compounds were visualized by spraying with an aqueous solution of 2.4% (w/v) phosphomolybdic acid, 5% (v/v) H2SO4, and 1.5% (v/v) H3PO4 (phosphomolybdic acid reagent) or ethanol containing 0.5% (w/v) ninhydrin (ninhydrin reagent) or DPPH solution described as above method, followed by heating. Structural characterization of monosaccharide of EPS was analyzed further by NMR spectrum.
Antioxidant assays of EPS Measurement of DPPH radical scavenging activity The sample (150 mL) was mixed with 37.5 mL of methanolic solution containing 0.75 mM DPPH radical. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank [16]. The scavenging ability was calculated as follows: scavenging activity (%) = [(A517 of control A517 of sample)/A517 of control] 100.
Measurement of Fe2+ chelating ability The method of Decker and Welch [17] was adopted. Five milliliters of the diluted sample was spiked with 0.1 mL of 2 mM FeCl2 and 0.2 mL of 5 mM ferrozine solutions. After reaction for 10 min, the
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The total phenolics of the culture supernatants were determined [19] and expressed as micrograms of gallic acid equivalents per milliliter of culture supernatant. Two milliliters of deionized water and 1 mL of Folin-Ciocalteu’s phenol reagent were added to 0.3 mL of each sample. Five milliliters of 20% aqueous sodium carbonate solution (w/v) was added and mixed well, and then the mixture was allowed to stand at ambient temperature for 20 min. Absorbance of the developed dark bluepurple color was measured by spectrophotometer at 735 nm. The content of total phenolics in each sample was determined using a standard curve prepared with gallic acid at varied concentrations (0, 50, 100, 200, 400, 600 and 800 mg/mL).
Statistical analysis In this study, each experiment was conducted in triplicate. The statistical analysis was calculated and expressed. All statistical analyses were carried out using SPSS 11.01 for Windows. To determine whether there were any differences between activities of samples, variance analysis was applied to the result. Values of P < 0.05 were considered as significantly different (a = 0.05).
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Effects of carbon/nitrogen sources on total sugar (solid line) and EPS (dashed line) production by Paenibacillus sp. TKU023. (*) SPP; (~) SHP; (&) sucrose.
suitable carbon source for total sugar and EPS production, various carbon sources were separately provided in the basal medium. Besides, to further utilize the chitin/protein-containing biowastes, we incubated Paenibacillus sp. TKU023 for one to five days at 378C by using 1% sucrose, 1% SPP, or 1% SHP as carbon sources and analyzed the amount of total sugar and EPS. As shown in Fig. 1, SPP was the most suitable carbon source for EPS production. This is different from the finding of many other investigators [5,11,12,20]. To select the optimal SPP concentration for total sugar and EPS production, 0.5–1.5% SPP was added into the basal medium. As shown in Fig. 2a, the increase of the SPP concentration in the
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Results and discussion Identification of the strain TKU023 To characterize strain TKU023, 16S rDNA and phylogenetic analyses were utilized. According to the analysis of 16S rDNA gene sequence, TKU024 was most closely aligned to Paenibacillus sp. and close to Paenibacillus elgii, Paenibacillus ehimensis with 99% similarity. To characterize strain TKU023 further, standard morphological, physiological and biochemical plates showed that strain TKU023 was a Gram-positive and non-spore-forming bacillus, which grows in both aerobic and anaerobic environments. The phylogenetic identification indicated that the strain TKU023 belongs to Paenibacillus sp.
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A method developed by Oyaizu [18] for testing reducing power was used. The diluted sample or distilled water (control) (0.5 mL) was spiked with 0.5 mL of sodium phosphate buffer (0.02 M, pH 7) and 0.5 mL of 1% potassium ferricyanide. The mixture was then kept in a 508C water bath for 20 min. The resulting solution was cooled rapidly, spiked with 0.5 mL of 10% trichloroacetic acid, and centrifuged at 800 g for 10 min. The supernatant (1.5 mL) was then mixed with 0.2 mL of 0.1% ferrichloride. After reaction for 10 min, the absorbance at 700 nm was measured. The reducing power of each sample was determined using a standard curve prepared with cysteine at varied concentrations.
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absorbance (at 562 nm) of the resulting solution was recorded. A complex of Fe2+/ferrozine has a strong absorbance at 562 nm. A high ferrous ion chelating ability in the test sample results in a low absorbance. The ferrous ion chelating ability was calculated as follows: chelating ability (%) = [1 (test sample absorbance/blank sample absorbance)] 100.
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Effects of SPP concentration (A) and initial temperature (B) on total sugar (solid line) and EPS (dashed line) production by Paenibacillus sp. TKU023. www.elsevier.com/locate/nbt
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medium led to the increases of both total sugar and EPS production. During six-day fermentation, the SPP decreased gradually in the medium, indicating that most of the SPP in the medium had been utilized for EPS production. The highest EPS production (4.18 g/L) was obtained at the SPP concentration of 1.5%.
Effect of cultivation volume, temperature and initial pH To study the effect of cultivation volume on EPS production, we found that 50 mL of medium was more suitable for EPS production than 100 mL, 150 mL and 200 mL. This might be related to the oxygen dissolved in the medium. The dissolved oxygen increased with the decrease of cultivation volume at a constant agitation speed. The dissolved oxygen can influence the physiology and the metabolism of the aerobic microorganisms. In a fermentation system, the dissolved oxygen is typically controlled by the variation of the agitation speed [21,22] or by changing the air flow rate
Time course of EPS production Using SPP as the sole C/N source, the relationship between the EPS production and cell growth was investigated. As shown in Fig. 4, maximum total sugar (5555 mg/mL) and EPS production (4.55 g/L) were both found at the fifth day by using 50 mL of the media and then decreased gradually. These results were similar to those found in Fig. 2b. Besides, the bacterium grew rapidly in the first four days, and we also found that total sugar and EPS production were closely related to the cell growth. The EPS yield reached the maximum level (the fifth day) after the cell growth reached the stationary phase. These results indicate that the production of EPS is cell growth dependent and Paenibacillus sp. TKU023 is a promising EPS producer. In Paenibacillus spp., P. polymyxa EJS-3 was also reported
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[22]. To affirm the result, we further investigated the effect of dissolved oxygen on EPS production by fermentor. There was no EPS production when cultured at 50 rpm, but EPS production was found at 150 rpm. The dissolved oxygen increases with agitation speed in the same medium. Therefore, it was inferred that the dissolved oxygen might affect EPS production of Paenibacillus sp. TKU023. Incubation temperature is a crucial factor in EPS biosynthesis [5]. The production of EPS was investigated, using 1.5% SPP as the sole C/N source at various culture temperatures. As shown in Fig. 2b, the optimal temperature for EPS production was 378C with EPS production reached 4.55 g/L. The initial culture pH is also an important factor that may affect cell membrane, cell morphology and structure, the uptake of various nutrients and EPS biosynthesis [5,23]. The production of EPS was also investigated, using SPP as the sole C/N source, in media of various pH values controlling namely, not adjusted, adjusted to 2–12. As shown in Fig. 3, the optimal pH value before sterilization for EPS production was 7.23 (not adjusted pH), with the corresponding EPS production of 4.55 g/L. This result was similar to P. polymyxa strains from soil [11,12], but unlike P. polymyxa EJS-3 (pH 8, slightly alkaline) for EPS production [5].
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Time courses of total sugar and EPS production in a culture of Paenibacillus sp. TKU023 on squid pen containing media: (*) total sugar (mg/mL); (*) EPS (g/L); (&) cell growth. 562
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TABLE 1
Comparison of EPS produced from other kinds of microorganisms Sources
Carbon/nitrogen sources
Yield of EPS (g/L)
Cultivation time (hour)
Productivity of EPS (g/(L hour))
Reference
1.5% squid pen powder
4.55
120
0.038
This study
16% sucrose/1% yeast extract
22.82
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0.380
[5]
Pseudomonas oleovorans NRRL B-14682a
4% glycerol-rich product
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0.102
[24]
a
Rhodotorula acheniorum MC
5% sucrose
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0.065
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Agrocybe cylindracea ASI-9002
6% maltose
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0.013
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Azotobacter sp. SSB81
2% glucose, 0.0001% riboflavin and 0.2% casamino acid
2.52
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Paecilomyces tenuipes C240a
3% glucose
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0.010
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Halomonas eurihalina H212
MY medium
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192
0.008
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Bacillus licheniformis
MRS-0.2% glucose
0.88
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0.073
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Halomonas anticariensis Al16
1% glucose
0.2895
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0.002
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Pseudomonas aeruginosa B1
Nutrient broth
0.075
72
0.001
[30]
The EPS was produced by a jar fermentor.
The composition of EPS In the present study, EPS was isolated from the fermentation broth of Paenibacillus sp. TKU023 by centrifugation, methanol precipitation and deproteinization. To determine the monosaccharide composition, the isolated EPS was hydrolyzed with 0.5 U/mL aamylase at 208C, pH 6.9 for 24 hours. The hydrolysates of EPS were developed on the TLC plates, and two compounds were visualized by spraying with phosphomolybdic acid reagent (data not shown). The results of TLC showed that the compositions of the EPS might contain glucose and maltose. Furthermore, the hydrolysates were characterized by 1H NMR and 13C NMR. The 1H NMR and 13C NMR spectra show peaks identical to maltose. The results indicated that the monosaccharide composition of the EPS was mainly glucose, and maltose molecules also appeared in the composition of the EPS.
production has been well documented [22]. Therefore, we also chose baffled base flask to increase levels of oxygenation and mixing. As shown in Fig. 5, it was found that TKU023 culture supernatant (1.5% SPP) incubated for four days by using baffled base flask has the highest antioxidant activity, the DPPH scavenging ability of TKU023 culture supernatant was about 92%. However, maximum EPS production (4.55 g/L) was found at the fifth day by using flat base flask (Fig. 5). The EPS produced by P. polymyxa EJS-3 had strong antioxidant activity [5]. However, our results indicated that the antioxidant was not mainly the EPS and it may include other materials. This may be also due to the presence of other antioxidant components in the culture supernatant, such as proteins, amino acids, peptides, organic acids and microelements. Among these antioxidant components, there may be some interactions and synergistic effects for antioxidant properties.
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as an EPS producing strain [5]. P. polymyxa EJS-3 produced EPS by using 16% sucrose and 1% yeast extract as the carbon and nitrogen sources. Compared with above, the EPS was produced merely by using cheaper medium from Paenibacillus sp. TKU023 than P. polymyxa EJS-3 (Table 1). The production of EPS reported in various documents is shown in Table 1. In most of the previous reports, the effect of sugars was investigated on the production of EPS by microorganisms and found EPS was quickly produced with the increase of sucrose or glucose concentration in the medium (Table 1). However, in this study, Paenibacillus sp. TKU023 adjusted with culture conditions and could use fish waste SPP as C/N source to produce a respectable amount of EPS (4.55 g/L).
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Antioxidant activity (&) and EPS production (*) from Paenibacillus sp. TKU023 by using flat base flask (solid line) and baffled base flask (dashed line), respectively.
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Ferrous ion chelating ability (%)
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The ferrous ion chelating ability (^) and reducing power (~) of the fermented supernatants from Paenibacillus sp. TKU023.
Chelating effects on ferrous ion Ferrous ion chelating abilities of the supernatants fermented from 1.5% SPP for one to six days at 378C by using baffled base flask were analyzed. As shown in Fig. 6, it was found that the culture supernatants incubated for four to six days have the highest chelating ability (71–77%) on ferrous ion. The chelating ability did not decrease with the culture time. Therefore, the antioxidant in the culture supernatant is stable for the culture time. Because ferrous ions are the most effective pro-oxidants, the higher chelating abilities of the culture supernatant would be beneficial. For the culture supernatant, the difference between DPPH scavenging ability and ferrous ion chelating ability might be related to the antioxidant compounds. Previous studies showed that the chelating to ferrous was dependent on the numbers of hydroxyl, and the hydroxyl substitution in the ortho position was desirable [31].
shown in Fig. 6. It was found that the culture supernatants incubated for three to five days have the highest reducing power with 354–383 mg of cysteine equivalents per milliliter of the fermented supernatant. The reducing power could be attributed mainly to the bioactive compounds associated with antioxidant activity [8,32– 34]. These bioactive compounds might be present in the culture supernatant, including phenolics, oligopeptides or chitooligosaccharides, are good electron donors and can terminate the radical chain reactions by converting free radicals to more stable products.
Total phenolic content It has been reported that shrimp shell waste contains natural antioxidants, mainly phenolic compounds [8]. The contents of total phenolics in the fermented supernatant from SPP by Paenibacillus sp. TKU023 were investigated. It was found that the fermented supernatants (1.5% SPP) incubated at the fourth day have the highest total phenolics contents, with 880 mg of gallic acid equivalents per milliliter of the fermented supernatant. The fermented supernatant at the fourth day with high phenolic content also showed high DPPH radical-scavenging activity (Fig. 5). This result suggested that phenolic compounds might be responsible for the activity.
Conclusion The success in the production of two environmental-friendly and invaluable biomaterials (EPS and antioxidant) by microbial fermentation using fishery processing wastes of SPP is unprecedented. The culture supernatant of Paenibacillus sp. TKU023 incubated for four days by using baffled base flask showed the highest antioxidant activity, but maximum EPS production (4.55 g/L) was found at the fifth day by using flat base flask. It may be a new source of natural antioxidants with potential value for health food and therapeutics. The findings appear useful for further research aiming to identify the structure of the EPS and antioxidant.
Reducing power
Acknowledgements
The reducing powers of the culture supernatants fermented from 1.5% SPP for one to six days at 378C by using baffled base flask are
This work was supported in part by a grant of the National Science Council, Taiwan (NSC99-2313-B-032-001-MY3).
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New Biotechnology Volume 28, Number 6 October 2011