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Why sucrose is the most suitable substrate for pullulan fermentation by Aureobasidium pullulans CGMCC1234?
Enzyme and Microbial Technology 92 (2016) 49–55
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Why sucrose is the most suitable substrate for pullulan fermentation by Aureobasidium pullulans CGMCC1234? Long Sheng a , Qunyi Tong b , Meihu Ma a,∗ a b
National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
a r t i c l e
i n f o
Article history: Received 15 March 2016 Received in revised form 23 June 2016 Accepted 24 June 2016 Available online 25 June 2016 Keywords: Aureobasidium pullulans Pullulan Sucrose -Fructofuranosidase
a b s t r a c t This paper studies the metabolic pathway of sucrose in pullulan fermentation by Aureobasidium pullulans. Because of its high pullulan production, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234 (36.3 g/L). Compared to other carbon sources, A. pullulans cells reached the stationary phase more quickly in the presence of sucrose. The specific sugar types and concentrations occurring during pullulan fermentation were detected using High Performance Liquid Chromatography (HPLC). HPLC results revealed that sucrose did not simply break down into glucose and fructose in the medium employed. Kestose (22.69 g/L) also accumulated during early stages of fermentation (24 h), which reduced the osmotic pressure of the extracellular fluid and diminished the inhibition of cell growth and pullulan production. -Fructofuranosidase activity strongly depended on the carbon source. Sucrose was the best inducer of -fructofuranosidase production. However, -fructofuranosidase production did not directly and/or proportionally correlate with the growth of A. pullulans cells. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Aureobasidium pullulans is generally known for producing pullulan, an exocellular homopolysaccharide. Pullulan is widely utilized in the food and medical industries because of its advantageous chemical and physical properties, including low viscosity, nontoxicity, slow digestibility, high plasticity and excellent film-forming capabilities [1]. Pullulan is a linear homopolysaccharide composed of maltotriose reduplicative units connected by ␣-1,4-linkages. This particular linkage pattern confers pullulan with excellent solubility in water compared to other polysaccharides [2]. Because of its nontoxic and nonimmunogenic properties, pullulan has been used in biological materials for targeted drug and gene deliveries in recent years [3]. All of these factors indicate that pullulan could become the most widely used biopolymer in a large number of high-value technological platforms [4]. Pullulan biosynthesis is a complex metabolic process controlled by environmental conditions. Factors known to affect pullulan production in A. pullulans include the fungal strain used, the carbon source, the nitrogen source, the incubation pH, the culture temperature, the dissolved oxygen level and the fermenter configuration
∗ Corresponding author at: College of Food Science and Technology of Huazhong, Agricultural University, Wuhan, Hubei Province, China. E-mail address: [email protected] (M. Ma). http://dx.doi.org/10.1016/j.enzmictec.2016.06.016 0141-0229/© 2016 Elsevier Inc. All rights reserved.
[5]. The proposed pathway of pullulan biosynthesis has been summarized as Fig. 1 [1]. Pullulan can be synthesized from a diverse number of carbon sources, including glucose, sucrose, mannose, galactose, fructose, and agriculture waste. Although published reports regarding pullulan biosynthesis contradict one another because of differences among the numerous strains of A. pullulans studied, it has been clearly demonstrated that pullulan yields strongly depend on the rate of substrate conversion [6]. Generally for microorganisms, monosaccharide carbon sources are superior to disaccharide and polysaccharide carbon source. Nevertheless, sucrose is predominantly used as the carbon source for pullulan production medium. Numerous reports have demonstrated the superiority of sucrose as the substrate for pullulan production by A. pullulans [7–12]. Prasongsuk et al. [10] investigated pullulan production using five different isolates of A. pullulans using sucrose or glucose as the carbon source and reported that sucrose could support 2–4 times more pullulan production than glucose. Recently, Ma et al. [11] tested the effects of six different carbon sources on pullulan production by A. pullulans var. melanogenum P16 and found that sucrose was the most suitable substrate for pullulan production. The current understanding was that sucrose would be degraded to glucose and fructose, and then fructose would be isomerized to glucose. However, such a process would require metabolic pathways beyond those of glucose or fructose for pullulan synthesis.
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blade impeller (diameter = 4 cm) located 3 cm above the bottom of the vessel. The temperature, pH, dissolved oxygen, agitation speed and aeration rate in the vessel was monitored and controlled automatically. The fermentor with 3 L of production medium was sterilized at 121 ◦ C for 15 min. After cooling, the medium was inoculated with 150 mL of the seed culture. Pullulan production was carried out at a constant temperature of 28 ◦ C, an aeration rate of 3 L/min and an agitation speed of 300 rpm. The pH was maintained at 6.5 by adding either 2 M NaOH or 2 M HCl. 2.4. Determination of pullulan production and biomass
Fig. 1. Putative biosynthesis of pullulan. Referenced from Cheng et al. [1]
Two volumes of distilled water were used to dilute the fermentation liquor. The broth was heated and maintained at 80 ◦ C in a water bath for 30 min and then centrifuged at 10,000g for 20 min to separate the biomass and insoluble debris. Two volumes of precooled 95% (v/v) ethanol were added to the supernatant, which was then stirred and maintained at 4 ◦ C for 24 h to thoroughly precipitate the exopolysaccharide. The sediment was isolated by centrifugation at 15,000g for 20 min, washed with ethanol again, and dried at 80 ◦ C until the weight was constant. The biomass sediment from the initial centrifugation was washed three times with distilled water by centrifuging at 10,000g for 5 min and was then dried at 80 ◦ C until the dry cell weight (DCW) was constant [13]. Pullulan production and DCW were expressed as grams per liter. 2.5. Determination of residual sugar and specific sugar types
Although pullulan has been used in different fields in industry, the mechanisms by which pullulan is biosynthesized by A. pullulans are still poorly understood. To control the fermentation process, it is important to understand why sucrose is considered the most suitable substrate for pullulan fermentation by A. pullulans CGMCC1234. The objective of the present study was to compare differences in pullulan fermentation using various carbon sources and to describe how the sucrose is utilized by A. pullulans in culture. The influence of different carbon sources and sucrose concentrations on -fructofuranosidase activity was also investigated.
After removing the pullulan by ethanol precipitation from the cell-free broth, the supernatant was used for the estimation of residual sugar content according the method of Dubois et al. [14]. Specific sugar types and concentrations were determined using High Performance Liquid Chromatography (HPLC). The conditions for HPLC analysis were as follows: column, BIO-RAD Aminex HPX-87C (7.8 mm × 300 mm); mobile phase, water; flow rate, 0.4 mL/min; temperature, 78 ◦ C; detector, Evaporative Light Scattering Detector; detection temperature, 105 ◦ C; and carrier gas flow rate, 2.5 mL/min.
2. Materials and methods 2.6. Preparation of cell-free extract 2.1. Microorganism A. pullulans CGMCC1234 was maintained on potato dextrose agar (PDA) at 4 ◦ C and cultured every 2 weeks. 2.2. Seed culture The seed broth included 50.0 g of sucrose, 4.0 g of K2 HPO4 , 2.0 g of NaCl, 1.5 g of yeast extract, 0.8 g of (NH4 )2 SO4 , and 0.2 g of MgSO4 in 1 L of distilled water. The medium pH was 6.5, and the medium was sterilized at 121 ◦ C for 15 min.
The cells in 5.0 mL of the culture were collected by centrifugation at 10,000g for 20 min at 4 ◦ C. The resulting cell pellet was washed three times with ice-cold distilled water by centrifuging at 10,000g for 5 min. The pellet was suspended in 1.0 mL of ice-cold 1.0 M TrisHCl (pH 7.6) to make a thick paste. The paste was homogenized in a Homogenizer (DY89-I, Xinzhi, Zhejiang, China) for 1 h in an ice bath. Cell debris was removed by centrifugation at 14,000g for 30 min at 4 ◦ C [15]. The supernatant (the cell-free extract) was stored at −80 ◦ C and used as a source of enzymes. The protein concentration in the cell-free extract was determined using the method described by Bradford and bovine serum albumin as the standard [16].
2.3. Culture conditions Cultures were grown on PDA at 28 ◦ C for 4 days and then transferred to a 250 mL flask that contained 50 mL of the seed culture medium, in which they were subsequently incubated at 28 ◦ C for 2 days with shaking at 200 rpm. Pullulan was produced in a 5 L stirred tank fermentor (KF-5l, KoBioTech Bioengineering Equipment Co., Ltd.) containing a working volume of 3 L of the production medium, which contained (g/L) 80.0 g of different sugars (sucrose, maltose, glucose, fructose, mannose, galactose, xylose, soluble starch), 0.9 g of yeast extract, 6.0 g of K2 HPO4 , 0.6 g of (NH4 )2 SO4 , 0.5 g of MgSO4 and 4.0 g of NaCl. The fermentor consisted of a glass vessel with stainless-steel endplates and three equally spaced vertical baffles. Agitation was provided by a six-flat-
2.7. Assay for ˛-amylase and ˇ-fructofuranosidase activity Activities of ␣-amylase were determined using blue starch (Starch Azure, Sigma) as the substrate according to the method of Manitchotpisit et al. [17]. High-molecular-weight material was removed by centrifugation, and the absorbance of the reaction mixture was determined at 595 nm. The reaction mixture, which contained 200 L of 1% (w/v) substrate in 50 mM sodium acetate buffer (pH 5.0), 50 L of supernatant from each culture broth, and 2.5 L of 2% (w/v) sodium azide was incubated at 28 ◦ C for 20 h. Five-hundred microliters of precipitation buffer (4% (w/v) sodium acetate trihydrate, 0.4% (w/v) zinc acetate, 800 mL of 100% ethyl alcohol, and 200 mL of deionized water per liter) were added and
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mixed by briefly vortexing. The reaction mixture was incubated at room temperature for 10 min and then centrifuged at 10,000g for 10 min. Two-hundred microliters of the reaction mixture were applied to a microtiter plate, and the absorbance was measured as described above. The assays were calibrated using commercial enzymes with known activities. One unit of enzyme activity was defined as the amount necessary to release 1 mol of maltose equivalents per minute, as determined by the dinitrosalicylic acid method. A 0.1 mL aliquot of the enzyme solution was added to 0.9 mL sucrose solution [0.1 M in 0.03 M acetate buffer (pH 5.0)], after which the mixture was maintained at 50 ◦ C for 5 min. The reaction was terminated by adding 1 mL 3,5-dinitrosalicylic acid, and the mixture was maintained at 100 ◦ C for 5 min. The absorbance was measured at 540 nm using a spectrophotometer. The mixture containing the enzyme solution, which was heated at 100 ◦ C for 5 min, was used as the control [18]. Enzyme activity was defined as the amount of enzyme required to produce 1 mol glucose per min per mg protein. 2.8. High performance gel filtration chromatography (HPGFC) The molecular weight (MW) of the pullulan sample was determined via High Performance Gel Filtration Chromatography (HPGFC) using an Ultrahydrogel Size Exclusion Column (300 mm × 7.8 mm). Commercially available dextran standards (5.0 mg/mL, Sigma) were used to construct a calibration curve. For the mobile phase, NaNO3 (0.1 M) was used at a flow rate of 0.9 mL/min. The sample concentration and injection volume were 5.0 mg/mL and 20 L, respectively. 2.9. Determining the purity of pullulan samples The pullulan content was determined using the coupledenzyme assay technique described by Israilides et al. [19]. 2.10. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra of the pullulan sample were obtained via KBr pellets on an infrared spectrometer (Perkin-Elmer 16 PC spectrometer, Boston, USA) over a wavelength range of 650–4000 cm−1 . Commercial pullulan (PI20, MW 200000 Da), which was purchased from Hayashibara Biochemical Laboratories (Okayama, Japan) was used as a reference. 3. Results and discussion 3.1. Effect of different carbon sources on pullulan fermentation We investigated the effects of several kinds of representative carbon sources on pullulan fermentation. As shown in Fig. 2, several carbon sources are utilized by A. pullulans for cell growth. However, the biomass and exopolysaccharide accumulation obtained differed among different carbon sources. Although the fungus could grow in medium containing galactose, almost no pullulan was synthesized in its presence. It is possible that galactose cannot transform and participate in the structure of the pullulan chain. It was reported that Aureobasidium pullulans could utilize xylose, rhamnose, galactose, sucrose, maltose, cellobiose, lactose, inulin, soluble starch, glycerol and acetate for cell growth [20]. Among these, glucose, fructose, xylose, maltose, and sucrose were examined with regard to exopolysaccharide production [21]. Wang et al. also found that only minute quantities of pullulan (approximately 2 g/L) could be produced from galactose [22].
Fig. 2. Effects of various carbon sources on growth and pullulan production by A. pullulans CGMCC1234. Values are given as the means ± standard deviations (n = 3).
Duan et al. [23] reported that both biomass and pullulan production were low when A. pullulans was grown in a medium containing xylose. Interestingly, we found that the biomass obtained with xylose was higher than that obtained with other carbon sources. Duan et al. speculated that A. pullulans would require more energy to synthesize pullulan from xylose because of the complex metabolic pathway involved in converting pentose to hexose [23]. Similarly, Ravella et al. [12] also obtained 16.5 g/L of wet biomass but just 1.0 g/L pullulan from xylose. This phenomenon may be attributed to differences in the types of strains used. With regard to starch as a substrate, before it can be utilized, the soluble starch must first be hydrolyzed by amylase secreted from A. pullulans. Therefore, both biomass and pullulan yields were lower in medium containing soluble starch. The ␣-amylase activities at 24 h were 0.04 U/mL and 48 h were 0.05 U/mL. Manitchotpisit et al. [17] also found that low levels of ␣-amylase (<0.04 U/mL) in starch-grown cultures. Wu et al. [24] used raw potato starch hydrolysates as the substrates for pullulan production and extra ␣-amylase (60 U) was added to the reactor containing 500 mL of raw potato starch suspension. Among the carbon sources tested, the highest pullulan yield was obtained when cells were grown in a sucrose-containing medium. Because of the high conversion rate achieved with sucrose, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234. This result agrees with others, as most earlier reports have suggested that sucrose is a better substrate than glucose for EPS production [7–12]. A number of studies support sucrose as the best substrate for pullulan production by A. pullulans; nevertheless, some studies demonstrated that glucose was conducive to pullulan synthesis [25,26]. A possible reason for this contradiction may be the differences in A. pullulan strains and fermentation conditions. 3.2. Effects of mixed carbon sources on pullulan fermentation It has been reported that sucrose degrades into glucose and fructose in the pullulan fermentation broth and that fructose is isomerized into glucose [27,28]. In addition, pullulan is a single polysaccharide consisting of glucose units. It is presumed that sucrose, a disaccharide composed of glucose and fructose, must proceed through additional metabolic processes to generate pullulan [29]. Nonetheless, sucrose has proven more effective than
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Table 1 Effects of different carbon sources on pullulan fermentation by Aureobasidium pullulans. Carbon source
Sucrose
DCW (g/L) Pullulan (g/L) Residual sugar (g/L) YX/S (g/g) YP/S (g/g) YP/X (g/g)
5.59 39.02 12.54 0.083 0.578 6.964
Glucose ± ± ± ± ± ±
0.25 2.54 1.56 0.004 0.034 0.076
5.89 33.57 18.43 0.096 0.545 5.677
Fructose ± ± ± ± ± ±
0.15 3.11 2.35 0.003 0.021 0.046
6.39 31.01 20.43 0.107 0.521 4.869
± ± ± ± ± ±
Glucose + Fructose 0.31 1.74 3.35 0.006 0.031 0.049
6.05 34.98 17.42 0.097 0.559 5.763
± ± ± ± ± ±
0.21 2.97 2.98 0.003 0.052 0.065
Values are given as the means ± standard deviations (n = 3). Table 2 Comparisons of qualities of pullulan purified from fermentation broth containing different carbon sources. Carbon source Sucrose Glucose Fructose Glucose + Fructose
Pullulan purity(%, w/w) 95.39 94.83 95.24 94.89
± ± ± ±
0.86 0.74 0.92 0.83
Molecular weight( × 105 ) 9.32 10.17 11.63 10.73
± ± ± ±
0.44 0.57 0.73 0.75
Values are given as the means ± standard deviations (n = 3).
glucose or fructose in pullulan production. Therefore, a mixed carbon source (50% glucose and 50% fructose) was used to produce pullulan in the present study. As shown in Table 1, sucrose was the preferred substrate for pullulan biosynthesis, easily resulting in a higher pullulan yield. Compared to sucrose, the mixed carbon source provided no advantages in terms of pullulan synthesis. These results suggest that sucrose may not simply break down into glucose and fructose in the medium. Gibson and Coughlin [7] examined the effects of glucose and sucrose on EPS production by A. pullulans NRRLY-2311-1 in a medium containing 5% glucose and sucrose and found that sucrose was the better substrate in terms of the EPS yield and conversion efficiency of this isolate. Likewise, Ravella et al. [12] compared the effects of five different carbon sources (5% concentrations) on pullulan production and found that sucrose was the optimal carbon source, followed by fructose. Interestingly, glucose supported very little pullulan production with the isolate used in the latter study. Table 2 compares the quality of pullulan extracted from fermentation broth containing different carbon sources. Minimal differences were observed with regard to pullulan purity of the samples obtained from different media. However, using fructose as the carbon source resulted in the highest MW pullulan. Wiley et al. [30] also found that the MW of pullulan produced from fructose was higher than that from sucrose. The MW of pullulan ranges broadly, from hundreds to thousands of kilodaltons, depending on the strain, pH, cultivation techniques and substrates used [31,32]. 3.3. Metabolic pathway of sucrose in the fermentation broth via pullulans The specific types and concentrations of sugars in the medium were determined using HPLC. Interestingly, we found a number of low-molecular-weight sugars such as kestose in the fermentation liquor when sucrose was used as the carbon source (Fig. 3). Kestose is an excellent water-soluble dietary fiber contained in fruits, vegetables and honey. Given its superior physiological functions, kestose has been popular as a functional food in the international market [33]. Many microorganisms, including molds and yeasts (such as Aspergillus niger, Schwanniomyces occidentalis, Cryptococcus laurentii, etc.), can synthesize kestose [34–36]. Our results indicate that A. pullulans CGMCC1234 is also capable of producing kestose. Yoshikawa et al. [37] reported that A. pullulans DSM 2404 could convert sucrose to kestose via -fructofuranosidase. However, no details were provided with regard to how this strain might simultaneously produce pullulan.
As show in Fig. 4d, when sucrose was used as a single carbon source, the sucrose content rapidly decreased at 24 h, and all the sucrose was consumed before 48 h. Meanwhile, kestose accumulated during the early stages of fermentation, and the concentration of fructose was lower than that of glucose. At 24 h, kestose and glucose concentrations in the fermentation liquor reached 22.69 g/L and 30.02 g/L, respectively. After that, kestose and glucose were continuously consumed, and the concentration of fructose increased. These results implied the existence of -fructofuranosidase, which was confirmed by enzyme activity determinations. These observations revealed that sucrose did break down into glucose and fructose, after which fructose reacted (was connected) with the remaining sucrose to form kestose. Another noticeable phenomenon is that, when glucose and fructose simultaneously existed in the medium, the glucose concentration decreased faster than the fructose concentration (Fig. 4c). Therefore, it appears that, compared with fructose, glucose was preferentially utilized by A. pullulans. Compared to the other three kinds of substrates (Fig. 4a–c), the residual sugar content decreased fastest when sucrose was used as the carbon source (Fig. 4d). Due to the different molecular weights of sugars, the osmotic pressure caused by monosaccharides in the extracellular fluid was higher than that resulting from disaccharides and trisaccharides under the same weight. It has been suggested that a source providing excess carbon would exhibit an inhibitory effect on cell growth and pullulan production [38]. Shin et al. [39] found that the inhibitory effects of high sugar concentrations could be overcome and higher pullulan levels (58 g/L) might be achieved using a fed-batch culture system. Therefore, we speculated that, when grown in a medium containing a high concentration of sucrose, A. pullulans cells could hydrolyze sucrose and then synthesize kestose to reduce the extracellular fluid’s osmotic pressure; afterwards, kestose would be hydrolyzed as a carbon source when the residual sugar level decreased to an ineffective concentration. 3.4. Effects of different carbon sources on ˇ-fructofuranosidase activity -Fructofuranosidase is an enzyme responsible for the catalysis of sucrose to glucose and fructose in low-concentration sucrose solutions. This enzyme can also facilitate the generation of kestose by binding fructose to sucrose in high-concentration sucrose solutions [40]. The effects of various carbon sources on -fructofuranosidase activity are shown in Fig. 5. The fructofuranosidase activity was highest when sucrose was the carbon source and was lower when the strain was grown in media containing other carbon sources. Moreover, enzyme activity could not be detected when xylose or soluble starch were the substrates. This indicates that -fructofuranosidase activity is highly dependent on the carbon source. Sucrose was the best inducer of -fructofuranosidase synthesis. Chen et al. [41] and Kim et al. [42] found that -fructofuranosidase activities were highest when Aspergillus japonicas and Bacillus macerans EG6, respectively, were cultivated in media containing sucrose [41,42]. However, Kim
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Fig. 3. HPLC detection of residual sugars within the fermentation broth when sucrose was used as the carbon source.
Fig. 4. Types and concentrations of residual sugars within the fermentation broth by A. pullulans CGMCC1234 using the following different carbon sources: (a) glucose, (b) fructose, (c) glucose and fructose, and (d) sucrose. Values are given as the means ± standard deviations (n = 3).
et al. [42] reported that -fructofuranosidase production by Bacillus macerans EG6 could be improved with the use of soluble starch as well. This result is not consistent with our findings, which might be due to the differences in the organism. The results revealed that, in sucrose media, A. pullulans cells had the ability to secrete substantial amounts of -fructofuranosidase to decrease the extracellular fluid’s osmotic pressure. Therefore, A. pullulans cells grew faster during the initial stages of fermentation and produced more pullulan.
3.5. Kinetics of ˇ-fructofuranosidase activity and pullulan fermentation of Aureobasidium pullulans The time courses of -fructofuranosidase activity and pullulan fermentation by A. pullulans CGMCC1234 are shown in Fig. 6. There was a concomitant increase in the production of pullulan in the fermentation medium. The maximum pullulan yield was obtained at 96 h; thereafter, the pullulan yield slightly decreased. Similarly, the MW of pullulan showed the same trend, with a tendency to increase initially and then decrease later. These results agree with
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Fig. 5. Effects of various carbon sources on -fructofuranosidase activity by A. pullulans CGMCC1234. Values are given as the means ± standard deviations (n = 3).
Fig. 6. The time course of -fructofuranosidase activity and pullulan production by A. pullulans CGMCC1234 in fermentation broth containing 80 g/L sucrose. Values are given as the means ± standard deviations (n = 3).
our previous work, and Gibson and Coughlin also reported that the molecular weight of pullulan decreased during cultivation [7,43]. It has been reported that, when secreted into the fermentation broth, ␣-amylase attacked the minor maltotetraose subunits of pullulan [17]. This action would reduce the MW of pullulan. The fructofuranosidase activity rapidly increased before 36 h, and the highest enzyme activity (45.3 U/mg) was obtained at 48 h, after which enzyme activity was sustained at a high level for additional 24 h -fructofuranosidase maintained high enzyme activity during the last part of the logarithmic phase and the early stage of the stationary phase. The FT-IR spectra obtained for the sample obtained from the fermentation broth containing sucrose, using commercial pullulan as a reference, are compared in Fig. 7. In the specific range (1000–650 cm−1 ) characterizing the pullulan molecule as a whole, the spectra for commercial pullulan exhibited features that were similar to those observed for the evaluated sample. Absorptions at 930 cm−1 , 850 cm−1 and 755 cm−1 were characteristic of ␣-1,6-d-
Fig. 7. FT-IR spectra of pullulan produced by A. pullulans CGMCC1234 and commercial pullulan.
Fig. 8. Effects of different sucrose concentrations on -fructofuranosidase activity by A. pullulans CGMCC1234. Values are given as the means ± standard deviations (n = 3).
glucosidic bonds, ␣-configurations and ␣-1,4-d-glucosidic bonds, respectively. These results agree with those of previous reports and confirm the identity of the purified polysaccharide [44,45]. 3.6. Effects of sucrose concentrations on ˇ-fructofuranosidase activity Fig. 8 shows the influence of different sucrose concentrations on -fructofuranosidase activity. Increasing the sucrose content increased the biomass obtained; however, the biomass began to decrease when the sucrose concentration became greater than 120 g/L. A similar phenomenon was also observed using Sphingomonas paucimobilis [46]. Obviously, a high initial sucrose concentration was not beneficial to cell growth. Moreover, A. pullulans cell growth may be inhibited by high osmotic pressure. Whereas -fructofuranosidase activity could be detected regardless of how much sucrose was added to the culture, fructofuranosidase activity sustainably increased as the sucrose content increased. Consequently, -fructofuranosidase production
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did not directly and/or proportionally correlate with the growth of A. pullulans cells. 4. Conclusions Given the high conversion rate achieved using sucrose, sucrose constituted the best source of carbon for pullulan synthesis by A. pullulans CGMCC1234. Sucrose did not simply break down into glucose and fructose in the fermentation broth; instead, kestose was synthesized by the -fructofuranosidase secreted from A. pullulans cells during the early stages of fermentation. The resulting reduced osmotic pressure was beneficial to cell growth and pullulan production. -Fructofuranosidase activity strongly depended on the carbon source but did not correlate directly and/or proportionally with the growth of A. pullulans cells. These results improve our understanding of the physiological responses of A. pullulans and facilitate optimization of industrial pullulan fermentation. Acknowledgement This project was supported by the Fundamental Research Funds for the Central Universities (Program No.2015BQ042). References [1] K.C. Cheng, A. Emirci, J.M. Catchmark, Pullulan: biosynthesis, production, and applications, Appl. Microbiol. Biotechnol. 92 (2011) 29–44. [2] L. Jiang, S. Wu, J.M. Kim, Effect of different nitrogen sources on activities of UDPG-pyrophosphorylase involved in pullulan synthesis and pullulan production by Aureobasidium pullulans, Carbohydr. Polym. 86 (2011) 1085–1088. [3] R.S. Singh, N. Kaur, J.F. Kennedy, Pullulan and pullulan derivatives as promising biomolecules for drug and gene targeting, Carbohydr. Polym. 123 (2015) 190–207. [4] P. Dixit, A. Mehta, G. Gahlawat, G.S. Prasad, A.R. Choudhury, Understanding the effect of interaction among aeration, agitation and impeller positions on mass transfer during pullulan fermentation by Aureobasidium pullulans, RSC Adv. 5 (2015) 38984–38994. [5] K.I. Shingel, Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide pullulan, Carbohydr. Res. 339 (2004) 447–460. [6] R.S. Singh, G.K. Saini, J.F. Kennedy, Pullulan Microbial sources, production and applications, Carbohydr. Polym. 73 (2008) 515–531. [7] L.H. Gibson, R.W. Coughlin, Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations, Biotechnol. Progr. 18 (2002) 675–678. [8] R.S. Singh, H. Singh, G.K. Saini, Response surface optimization of the critical medium components for pullulan production by Aureobasidium pullulans FB-1, Appl. Biochem. Biotechnol. 152 (2009) 42–53. [9] H.P. Seo, K.I. Jo, C.W. Son, Continuous production of pullulan by Aureobasidium pullulans HP-2001 with feeding of high concentration of sucrose, J. Microbiol. Biotechnol. 16 (2006) 374–380. [10] S. Prasongsuk, M.A. Berhow, C.A. Dunlap, D. Weisleder, T.D. Leathers, D.E. Eveleigh, Pullulan production by tropical isolates of Aureobasidium pullulans, J. Ind. Microbiol. Biotechnol. 34 (2007) 55–61. [11] Z. Ma, W. Fu, G. Liu, Z. Wang, Z. Chi, High-level pullulan production by Aureobasidium pullulans var. melanogenium P16 isolated from mangrove system, Appl. Microbiol. Biotechnol. 98 (2014) 4865–4873. [12] S.R. Ravella, T.S. Quinones, A. Retter, M. Heiermann, T. Amon, P.J. Hobbs, Extracellular polysaccharide (EPS) production by a novel strain of yeast-like fungus Aureobasidium pullulans, Carbohydr. Polym. 82 (2010) 728–732. [13] L. Sheng, G. Zhu, Q. Tong, Comparative proteomic analysis of Aureobasidium pullulans in the presence of high and low levels of inorganic nitrogen compound, J. Agric. Food Chem. 43 (2014) 10529–10534. [14] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [15] L. Sheng, G. Zhu, Q. Tong, Mechanism study of Tween 80 enhancing the pullulan production by Aureobasidium pullulans, Carbohydr. Polym. 97 (2013) 121–123. [16] M.M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–253. [17] P. Manitchotpisit, C.D. Skory, T.D. Leathers, alpha-Amylase activity during pullulan production and alpha-amylase gene analyses of Aureobasidium pullulans, J. Ind. Microbiol. Biotechnol. 38 (2011) 1211–1218. [18] C. Aranda, A. Robledo, O. Loera, J.C. Contreras-Esquivel, R. Rodriguez, C.N. Aguilar, Fungal invertase expression in solid-state fermentation, Food Technol. Biotechnol. 44 (2006) 229–233.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Why sucrose is the most suitable substrate for pullulan fermentation by Aureobasidium pullulans CGMCC1234? Long Sheng a , Qunyi Tong b , Meihu Ma a,∗ a b
National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
a r t i c l e
i n f o
Article history: Received 15 March 2016 Received in revised form 23 June 2016 Accepted 24 June 2016 Available online 25 June 2016 Keywords: Aureobasidium pullulans Pullulan Sucrose -Fructofuranosidase
a b s t r a c t This paper studies the metabolic pathway of sucrose in pullulan fermentation by Aureobasidium pullulans. Because of its high pullulan production, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234 (36.3 g/L). Compared to other carbon sources, A. pullulans cells reached the stationary phase more quickly in the presence of sucrose. The specific sugar types and concentrations occurring during pullulan fermentation were detected using High Performance Liquid Chromatography (HPLC). HPLC results revealed that sucrose did not simply break down into glucose and fructose in the medium employed. Kestose (22.69 g/L) also accumulated during early stages of fermentation (24 h), which reduced the osmotic pressure of the extracellular fluid and diminished the inhibition of cell growth and pullulan production. -Fructofuranosidase activity strongly depended on the carbon source. Sucrose was the best inducer of -fructofuranosidase production. However, -fructofuranosidase production did not directly and/or proportionally correlate with the growth of A. pullulans cells. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Aureobasidium pullulans is generally known for producing pullulan, an exocellular homopolysaccharide. Pullulan is widely utilized in the food and medical industries because of its advantageous chemical and physical properties, including low viscosity, nontoxicity, slow digestibility, high plasticity and excellent film-forming capabilities [1]. Pullulan is a linear homopolysaccharide composed of maltotriose reduplicative units connected by ␣-1,4-linkages. This particular linkage pattern confers pullulan with excellent solubility in water compared to other polysaccharides [2]. Because of its nontoxic and nonimmunogenic properties, pullulan has been used in biological materials for targeted drug and gene deliveries in recent years [3]. All of these factors indicate that pullulan could become the most widely used biopolymer in a large number of high-value technological platforms [4]. Pullulan biosynthesis is a complex metabolic process controlled by environmental conditions. Factors known to affect pullulan production in A. pullulans include the fungal strain used, the carbon source, the nitrogen source, the incubation pH, the culture temperature, the dissolved oxygen level and the fermenter configuration
∗ Corresponding author at: College of Food Science and Technology of Huazhong, Agricultural University, Wuhan, Hubei Province, China. E-mail address: [email protected] (M. Ma). http://dx.doi.org/10.1016/j.enzmictec.2016.06.016 0141-0229/© 2016 Elsevier Inc. All rights reserved.
[5]. The proposed pathway of pullulan biosynthesis has been summarized as Fig. 1 [1]. Pullulan can be synthesized from a diverse number of carbon sources, including glucose, sucrose, mannose, galactose, fructose, and agriculture waste. Although published reports regarding pullulan biosynthesis contradict one another because of differences among the numerous strains of A. pullulans studied, it has been clearly demonstrated that pullulan yields strongly depend on the rate of substrate conversion [6]. Generally for microorganisms, monosaccharide carbon sources are superior to disaccharide and polysaccharide carbon source. Nevertheless, sucrose is predominantly used as the carbon source for pullulan production medium. Numerous reports have demonstrated the superiority of sucrose as the substrate for pullulan production by A. pullulans [7–12]. Prasongsuk et al. [10] investigated pullulan production using five different isolates of A. pullulans using sucrose or glucose as the carbon source and reported that sucrose could support 2–4 times more pullulan production than glucose. Recently, Ma et al. [11] tested the effects of six different carbon sources on pullulan production by A. pullulans var. melanogenum P16 and found that sucrose was the most suitable substrate for pullulan production. The current understanding was that sucrose would be degraded to glucose and fructose, and then fructose would be isomerized to glucose. However, such a process would require metabolic pathways beyond those of glucose or fructose for pullulan synthesis.
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blade impeller (diameter = 4 cm) located 3 cm above the bottom of the vessel. The temperature, pH, dissolved oxygen, agitation speed and aeration rate in the vessel was monitored and controlled automatically. The fermentor with 3 L of production medium was sterilized at 121 ◦ C for 15 min. After cooling, the medium was inoculated with 150 mL of the seed culture. Pullulan production was carried out at a constant temperature of 28 ◦ C, an aeration rate of 3 L/min and an agitation speed of 300 rpm. The pH was maintained at 6.5 by adding either 2 M NaOH or 2 M HCl. 2.4. Determination of pullulan production and biomass
Fig. 1. Putative biosynthesis of pullulan. Referenced from Cheng et al. [1]
Two volumes of distilled water were used to dilute the fermentation liquor. The broth was heated and maintained at 80 ◦ C in a water bath for 30 min and then centrifuged at 10,000g for 20 min to separate the biomass and insoluble debris. Two volumes of precooled 95% (v/v) ethanol were added to the supernatant, which was then stirred and maintained at 4 ◦ C for 24 h to thoroughly precipitate the exopolysaccharide. The sediment was isolated by centrifugation at 15,000g for 20 min, washed with ethanol again, and dried at 80 ◦ C until the weight was constant. The biomass sediment from the initial centrifugation was washed three times with distilled water by centrifuging at 10,000g for 5 min and was then dried at 80 ◦ C until the dry cell weight (DCW) was constant [13]. Pullulan production and DCW were expressed as grams per liter. 2.5. Determination of residual sugar and specific sugar types
Although pullulan has been used in different fields in industry, the mechanisms by which pullulan is biosynthesized by A. pullulans are still poorly understood. To control the fermentation process, it is important to understand why sucrose is considered the most suitable substrate for pullulan fermentation by A. pullulans CGMCC1234. The objective of the present study was to compare differences in pullulan fermentation using various carbon sources and to describe how the sucrose is utilized by A. pullulans in culture. The influence of different carbon sources and sucrose concentrations on -fructofuranosidase activity was also investigated.
After removing the pullulan by ethanol precipitation from the cell-free broth, the supernatant was used for the estimation of residual sugar content according the method of Dubois et al. [14]. Specific sugar types and concentrations were determined using High Performance Liquid Chromatography (HPLC). The conditions for HPLC analysis were as follows: column, BIO-RAD Aminex HPX-87C (7.8 mm × 300 mm); mobile phase, water; flow rate, 0.4 mL/min; temperature, 78 ◦ C; detector, Evaporative Light Scattering Detector; detection temperature, 105 ◦ C; and carrier gas flow rate, 2.5 mL/min.
2. Materials and methods 2.6. Preparation of cell-free extract 2.1. Microorganism A. pullulans CGMCC1234 was maintained on potato dextrose agar (PDA) at 4 ◦ C and cultured every 2 weeks. 2.2. Seed culture The seed broth included 50.0 g of sucrose, 4.0 g of K2 HPO4 , 2.0 g of NaCl, 1.5 g of yeast extract, 0.8 g of (NH4 )2 SO4 , and 0.2 g of MgSO4 in 1 L of distilled water. The medium pH was 6.5, and the medium was sterilized at 121 ◦ C for 15 min.
The cells in 5.0 mL of the culture were collected by centrifugation at 10,000g for 20 min at 4 ◦ C. The resulting cell pellet was washed three times with ice-cold distilled water by centrifuging at 10,000g for 5 min. The pellet was suspended in 1.0 mL of ice-cold 1.0 M TrisHCl (pH 7.6) to make a thick paste. The paste was homogenized in a Homogenizer (DY89-I, Xinzhi, Zhejiang, China) for 1 h in an ice bath. Cell debris was removed by centrifugation at 14,000g for 30 min at 4 ◦ C [15]. The supernatant (the cell-free extract) was stored at −80 ◦ C and used as a source of enzymes. The protein concentration in the cell-free extract was determined using the method described by Bradford and bovine serum albumin as the standard [16].
2.3. Culture conditions Cultures were grown on PDA at 28 ◦ C for 4 days and then transferred to a 250 mL flask that contained 50 mL of the seed culture medium, in which they were subsequently incubated at 28 ◦ C for 2 days with shaking at 200 rpm. Pullulan was produced in a 5 L stirred tank fermentor (KF-5l, KoBioTech Bioengineering Equipment Co., Ltd.) containing a working volume of 3 L of the production medium, which contained (g/L) 80.0 g of different sugars (sucrose, maltose, glucose, fructose, mannose, galactose, xylose, soluble starch), 0.9 g of yeast extract, 6.0 g of K2 HPO4 , 0.6 g of (NH4 )2 SO4 , 0.5 g of MgSO4 and 4.0 g of NaCl. The fermentor consisted of a glass vessel with stainless-steel endplates and three equally spaced vertical baffles. Agitation was provided by a six-flat-
2.7. Assay for ˛-amylase and ˇ-fructofuranosidase activity Activities of ␣-amylase were determined using blue starch (Starch Azure, Sigma) as the substrate according to the method of Manitchotpisit et al. [17]. High-molecular-weight material was removed by centrifugation, and the absorbance of the reaction mixture was determined at 595 nm. The reaction mixture, which contained 200 L of 1% (w/v) substrate in 50 mM sodium acetate buffer (pH 5.0), 50 L of supernatant from each culture broth, and 2.5 L of 2% (w/v) sodium azide was incubated at 28 ◦ C for 20 h. Five-hundred microliters of precipitation buffer (4% (w/v) sodium acetate trihydrate, 0.4% (w/v) zinc acetate, 800 mL of 100% ethyl alcohol, and 200 mL of deionized water per liter) were added and
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mixed by briefly vortexing. The reaction mixture was incubated at room temperature for 10 min and then centrifuged at 10,000g for 10 min. Two-hundred microliters of the reaction mixture were applied to a microtiter plate, and the absorbance was measured as described above. The assays were calibrated using commercial enzymes with known activities. One unit of enzyme activity was defined as the amount necessary to release 1 mol of maltose equivalents per minute, as determined by the dinitrosalicylic acid method. A 0.1 mL aliquot of the enzyme solution was added to 0.9 mL sucrose solution [0.1 M in 0.03 M acetate buffer (pH 5.0)], after which the mixture was maintained at 50 ◦ C for 5 min. The reaction was terminated by adding 1 mL 3,5-dinitrosalicylic acid, and the mixture was maintained at 100 ◦ C for 5 min. The absorbance was measured at 540 nm using a spectrophotometer. The mixture containing the enzyme solution, which was heated at 100 ◦ C for 5 min, was used as the control [18]. Enzyme activity was defined as the amount of enzyme required to produce 1 mol glucose per min per mg protein. 2.8. High performance gel filtration chromatography (HPGFC) The molecular weight (MW) of the pullulan sample was determined via High Performance Gel Filtration Chromatography (HPGFC) using an Ultrahydrogel Size Exclusion Column (300 mm × 7.8 mm). Commercially available dextran standards (5.0 mg/mL, Sigma) were used to construct a calibration curve. For the mobile phase, NaNO3 (0.1 M) was used at a flow rate of 0.9 mL/min. The sample concentration and injection volume were 5.0 mg/mL and 20 L, respectively. 2.9. Determining the purity of pullulan samples The pullulan content was determined using the coupledenzyme assay technique described by Israilides et al. [19]. 2.10. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra of the pullulan sample were obtained via KBr pellets on an infrared spectrometer (Perkin-Elmer 16 PC spectrometer, Boston, USA) over a wavelength range of 650–4000 cm−1 . Commercial pullulan (PI20, MW 200000 Da), which was purchased from Hayashibara Biochemical Laboratories (Okayama, Japan) was used as a reference. 3. Results and discussion 3.1. Effect of different carbon sources on pullulan fermentation We investigated the effects of several kinds of representative carbon sources on pullulan fermentation. As shown in Fig. 2, several carbon sources are utilized by A. pullulans for cell growth. However, the biomass and exopolysaccharide accumulation obtained differed among different carbon sources. Although the fungus could grow in medium containing galactose, almost no pullulan was synthesized in its presence. It is possible that galactose cannot transform and participate in the structure of the pullulan chain. It was reported that Aureobasidium pullulans could utilize xylose, rhamnose, galactose, sucrose, maltose, cellobiose, lactose, inulin, soluble starch, glycerol and acetate for cell growth [20]. Among these, glucose, fructose, xylose, maltose, and sucrose were examined with regard to exopolysaccharide production [21]. Wang et al. also found that only minute quantities of pullulan (approximately 2 g/L) could be produced from galactose [22].
Fig. 2. Effects of various carbon sources on growth and pullulan production by A. pullulans CGMCC1234. Values are given as the means ± standard deviations (n = 3).
Duan et al. [23] reported that both biomass and pullulan production were low when A. pullulans was grown in a medium containing xylose. Interestingly, we found that the biomass obtained with xylose was higher than that obtained with other carbon sources. Duan et al. speculated that A. pullulans would require more energy to synthesize pullulan from xylose because of the complex metabolic pathway involved in converting pentose to hexose [23]. Similarly, Ravella et al. [12] also obtained 16.5 g/L of wet biomass but just 1.0 g/L pullulan from xylose. This phenomenon may be attributed to differences in the types of strains used. With regard to starch as a substrate, before it can be utilized, the soluble starch must first be hydrolyzed by amylase secreted from A. pullulans. Therefore, both biomass and pullulan yields were lower in medium containing soluble starch. The ␣-amylase activities at 24 h were 0.04 U/mL and 48 h were 0.05 U/mL. Manitchotpisit et al. [17] also found that low levels of ␣-amylase (<0.04 U/mL) in starch-grown cultures. Wu et al. [24] used raw potato starch hydrolysates as the substrates for pullulan production and extra ␣-amylase (60 U) was added to the reactor containing 500 mL of raw potato starch suspension. Among the carbon sources tested, the highest pullulan yield was obtained when cells were grown in a sucrose-containing medium. Because of the high conversion rate achieved with sucrose, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234. This result agrees with others, as most earlier reports have suggested that sucrose is a better substrate than glucose for EPS production [7–12]. A number of studies support sucrose as the best substrate for pullulan production by A. pullulans; nevertheless, some studies demonstrated that glucose was conducive to pullulan synthesis [25,26]. A possible reason for this contradiction may be the differences in A. pullulan strains and fermentation conditions. 3.2. Effects of mixed carbon sources on pullulan fermentation It has been reported that sucrose degrades into glucose and fructose in the pullulan fermentation broth and that fructose is isomerized into glucose [27,28]. In addition, pullulan is a single polysaccharide consisting of glucose units. It is presumed that sucrose, a disaccharide composed of glucose and fructose, must proceed through additional metabolic processes to generate pullulan [29]. Nonetheless, sucrose has proven more effective than
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Table 1 Effects of different carbon sources on pullulan fermentation by Aureobasidium pullulans. Carbon source
Sucrose
DCW (g/L) Pullulan (g/L) Residual sugar (g/L) YX/S (g/g) YP/S (g/g) YP/X (g/g)
5.59 39.02 12.54 0.083 0.578 6.964
Glucose ± ± ± ± ± ±
0.25 2.54 1.56 0.004 0.034 0.076
5.89 33.57 18.43 0.096 0.545 5.677
Fructose ± ± ± ± ± ±
0.15 3.11 2.35 0.003 0.021 0.046
6.39 31.01 20.43 0.107 0.521 4.869
± ± ± ± ± ±
Glucose + Fructose 0.31 1.74 3.35 0.006 0.031 0.049
6.05 34.98 17.42 0.097 0.559 5.763
± ± ± ± ± ±
0.21 2.97 2.98 0.003 0.052 0.065
Values are given as the means ± standard deviations (n = 3). Table 2 Comparisons of qualities of pullulan purified from fermentation broth containing different carbon sources. Carbon source Sucrose Glucose Fructose Glucose + Fructose
Pullulan purity(%, w/w) 95.39 94.83 95.24 94.89
± ± ± ±
0.86 0.74 0.92 0.83
Molecular weight( × 105 ) 9.32 10.17 11.63 10.73
± ± ± ±
0.44 0.57 0.73 0.75
Values are given as the means ± standard deviations (n = 3).
glucose or fructose in pullulan production. Therefore, a mixed carbon source (50% glucose and 50% fructose) was used to produce pullulan in the present study. As shown in Table 1, sucrose was the preferred substrate for pullulan biosynthesis, easily resulting in a higher pullulan yield. Compared to sucrose, the mixed carbon source provided no advantages in terms of pullulan synthesis. These results suggest that sucrose may not simply break down into glucose and fructose in the medium. Gibson and Coughlin [7] examined the effects of glucose and sucrose on EPS production by A. pullulans NRRLY-2311-1 in a medium containing 5% glucose and sucrose and found that sucrose was the better substrate in terms of the EPS yield and conversion efficiency of this isolate. Likewise, Ravella et al. [12] compared the effects of five different carbon sources (5% concentrations) on pullulan production and found that sucrose was the optimal carbon source, followed by fructose. Interestingly, glucose supported very little pullulan production with the isolate used in the latter study. Table 2 compares the quality of pullulan extracted from fermentation broth containing different carbon sources. Minimal differences were observed with regard to pullulan purity of the samples obtained from different media. However, using fructose as the carbon source resulted in the highest MW pullulan. Wiley et al. [30] also found that the MW of pullulan produced from fructose was higher than that from sucrose. The MW of pullulan ranges broadly, from hundreds to thousands of kilodaltons, depending on the strain, pH, cultivation techniques and substrates used [31,32]. 3.3. Metabolic pathway of sucrose in the fermentation broth via pullulans The specific types and concentrations of sugars in the medium were determined using HPLC. Interestingly, we found a number of low-molecular-weight sugars such as kestose in the fermentation liquor when sucrose was used as the carbon source (Fig. 3). Kestose is an excellent water-soluble dietary fiber contained in fruits, vegetables and honey. Given its superior physiological functions, kestose has been popular as a functional food in the international market [33]. Many microorganisms, including molds and yeasts (such as Aspergillus niger, Schwanniomyces occidentalis, Cryptococcus laurentii, etc.), can synthesize kestose [34–36]. Our results indicate that A. pullulans CGMCC1234 is also capable of producing kestose. Yoshikawa et al. [37] reported that A. pullulans DSM 2404 could convert sucrose to kestose via -fructofuranosidase. However, no details were provided with regard to how this strain might simultaneously produce pullulan.
As show in Fig. 4d, when sucrose was used as a single carbon source, the sucrose content rapidly decreased at 24 h, and all the sucrose was consumed before 48 h. Meanwhile, kestose accumulated during the early stages of fermentation, and the concentration of fructose was lower than that of glucose. At 24 h, kestose and glucose concentrations in the fermentation liquor reached 22.69 g/L and 30.02 g/L, respectively. After that, kestose and glucose were continuously consumed, and the concentration of fructose increased. These results implied the existence of -fructofuranosidase, which was confirmed by enzyme activity determinations. These observations revealed that sucrose did break down into glucose and fructose, after which fructose reacted (was connected) with the remaining sucrose to form kestose. Another noticeable phenomenon is that, when glucose and fructose simultaneously existed in the medium, the glucose concentration decreased faster than the fructose concentration (Fig. 4c). Therefore, it appears that, compared with fructose, glucose was preferentially utilized by A. pullulans. Compared to the other three kinds of substrates (Fig. 4a–c), the residual sugar content decreased fastest when sucrose was used as the carbon source (Fig. 4d). Due to the different molecular weights of sugars, the osmotic pressure caused by monosaccharides in the extracellular fluid was higher than that resulting from disaccharides and trisaccharides under the same weight. It has been suggested that a source providing excess carbon would exhibit an inhibitory effect on cell growth and pullulan production [38]. Shin et al. [39] found that the inhibitory effects of high sugar concentrations could be overcome and higher pullulan levels (58 g/L) might be achieved using a fed-batch culture system. Therefore, we speculated that, when grown in a medium containing a high concentration of sucrose, A. pullulans cells could hydrolyze sucrose and then synthesize kestose to reduce the extracellular fluid’s osmotic pressure; afterwards, kestose would be hydrolyzed as a carbon source when the residual sugar level decreased to an ineffective concentration. 3.4. Effects of different carbon sources on ˇ-fructofuranosidase activity -Fructofuranosidase is an enzyme responsible for the catalysis of sucrose to glucose and fructose in low-concentration sucrose solutions. This enzyme can also facilitate the generation of kestose by binding fructose to sucrose in high-concentration sucrose solutions [40]. The effects of various carbon sources on -fructofuranosidase activity are shown in Fig. 5. The fructofuranosidase activity was highest when sucrose was the carbon source and was lower when the strain was grown in media containing other carbon sources. Moreover, enzyme activity could not be detected when xylose or soluble starch were the substrates. This indicates that -fructofuranosidase activity is highly dependent on the carbon source. Sucrose was the best inducer of -fructofuranosidase synthesis. Chen et al. [41] and Kim et al. [42] found that -fructofuranosidase activities were highest when Aspergillus japonicas and Bacillus macerans EG6, respectively, were cultivated in media containing sucrose [41,42]. However, Kim
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Fig. 3. HPLC detection of residual sugars within the fermentation broth when sucrose was used as the carbon source.
Fig. 4. Types and concentrations of residual sugars within the fermentation broth by A. pullulans CGMCC1234 using the following different carbon sources: (a) glucose, (b) fructose, (c) glucose and fructose, and (d) sucrose. Values are given as the means ± standard deviations (n = 3).
et al. [42] reported that -fructofuranosidase production by Bacillus macerans EG6 could be improved with the use of soluble starch as well. This result is not consistent with our findings, which might be due to the differences in the organism. The results revealed that, in sucrose media, A. pullulans cells had the ability to secrete substantial amounts of -fructofuranosidase to decrease the extracellular fluid’s osmotic pressure. Therefore, A. pullulans cells grew faster during the initial stages of fermentation and produced more pullulan.
3.5. Kinetics of ˇ-fructofuranosidase activity and pullulan fermentation of Aureobasidium pullulans The time courses of -fructofuranosidase activity and pullulan fermentation by A. pullulans CGMCC1234 are shown in Fig. 6. There was a concomitant increase in the production of pullulan in the fermentation medium. The maximum pullulan yield was obtained at 96 h; thereafter, the pullulan yield slightly decreased. Similarly, the MW of pullulan showed the same trend, with a tendency to increase initially and then decrease later. These results agree with
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Fig. 5. Effects of various carbon sources on -fructofuranosidase activity by A. pullulans CGMCC1234. Values are given as the means ± standard deviations (n = 3).
Fig. 6. The time course of -fructofuranosidase activity and pullulan production by A. pullulans CGMCC1234 in fermentation broth containing 80 g/L sucrose. Values are given as the means ± standard deviations (n = 3).
our previous work, and Gibson and Coughlin also reported that the molecular weight of pullulan decreased during cultivation [7,43]. It has been reported that, when secreted into the fermentation broth, ␣-amylase attacked the minor maltotetraose subunits of pullulan [17]. This action would reduce the MW of pullulan. The fructofuranosidase activity rapidly increased before 36 h, and the highest enzyme activity (45.3 U/mg) was obtained at 48 h, after which enzyme activity was sustained at a high level for additional 24 h -fructofuranosidase maintained high enzyme activity during the last part of the logarithmic phase and the early stage of the stationary phase. The FT-IR spectra obtained for the sample obtained from the fermentation broth containing sucrose, using commercial pullulan as a reference, are compared in Fig. 7. In the specific range (1000–650 cm−1 ) characterizing the pullulan molecule as a whole, the spectra for commercial pullulan exhibited features that were similar to those observed for the evaluated sample. Absorptions at 930 cm−1 , 850 cm−1 and 755 cm−1 were characteristic of ␣-1,6-d-
Fig. 7. FT-IR spectra of pullulan produced by A. pullulans CGMCC1234 and commercial pullulan.
Fig. 8. Effects of different sucrose concentrations on -fructofuranosidase activity by A. pullulans CGMCC1234. Values are given as the means ± standard deviations (n = 3).
glucosidic bonds, ␣-configurations and ␣-1,4-d-glucosidic bonds, respectively. These results agree with those of previous reports and confirm the identity of the purified polysaccharide [44,45]. 3.6. Effects of sucrose concentrations on ˇ-fructofuranosidase activity Fig. 8 shows the influence of different sucrose concentrations on -fructofuranosidase activity. Increasing the sucrose content increased the biomass obtained; however, the biomass began to decrease when the sucrose concentration became greater than 120 g/L. A similar phenomenon was also observed using Sphingomonas paucimobilis [46]. Obviously, a high initial sucrose concentration was not beneficial to cell growth. Moreover, A. pullulans cell growth may be inhibited by high osmotic pressure. Whereas -fructofuranosidase activity could be detected regardless of how much sucrose was added to the culture, fructofuranosidase activity sustainably increased as the sucrose content increased. Consequently, -fructofuranosidase production
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did not directly and/or proportionally correlate with the growth of A. pullulans cells. 4. Conclusions Given the high conversion rate achieved using sucrose, sucrose constituted the best source of carbon for pullulan synthesis by A. pullulans CGMCC1234. Sucrose did not simply break down into glucose and fructose in the fermentation broth; instead, kestose was synthesized by the -fructofuranosidase secreted from A. pullulans cells during the early stages of fermentation. The resulting reduced osmotic pressure was beneficial to cell growth and pullulan production. -Fructofuranosidase activity strongly depended on the carbon source but did not correlate directly and/or proportionally with the growth of A. pullulans cells. These results improve our understanding of the physiological responses of A. pullulans and facilitate optimization of industrial pullulan fermentation. Acknowledgement This project was supported by the Fundamental Research Funds for the Central Universities (Program No.2015BQ042). References [1] K.C. Cheng, A. Emirci, J.M. Catchmark, Pullulan: biosynthesis, production, and applications, Appl. Microbiol. Biotechnol. 92 (2011) 29–44. [2] L. Jiang, S. Wu, J.M. Kim, Effect of different nitrogen sources on activities of UDPG-pyrophosphorylase involved in pullulan synthesis and pullulan production by Aureobasidium pullulans, Carbohydr. Polym. 86 (2011) 1085–1088. [3] R.S. Singh, N. Kaur, J.F. Kennedy, Pullulan and pullulan derivatives as promising biomolecules for drug and gene targeting, Carbohydr. Polym. 123 (2015) 190–207. [4] P. Dixit, A. Mehta, G. Gahlawat, G.S. Prasad, A.R. Choudhury, Understanding the effect of interaction among aeration, agitation and impeller positions on mass transfer during pullulan fermentation by Aureobasidium pullulans, RSC Adv. 5 (2015) 38984–38994. [5] K.I. Shingel, Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide pullulan, Carbohydr. Res. 339 (2004) 447–460. [6] R.S. Singh, G.K. Saini, J.F. Kennedy, Pullulan Microbial sources, production and applications, Carbohydr. Polym. 73 (2008) 515–531. [7] L.H. Gibson, R.W. Coughlin, Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations, Biotechnol. Progr. 18 (2002) 675–678. [8] R.S. Singh, H. Singh, G.K. Saini, Response surface optimization of the critical medium components for pullulan production by Aureobasidium pullulans FB-1, Appl. Biochem. Biotechnol. 152 (2009) 42–53. [9] H.P. Seo, K.I. Jo, C.W. Son, Continuous production of pullulan by Aureobasidium pullulans HP-2001 with feeding of high concentration of sucrose, J. Microbiol. Biotechnol. 16 (2006) 374–380. [10] S. Prasongsuk, M.A. Berhow, C.A. Dunlap, D. Weisleder, T.D. Leathers, D.E. Eveleigh, Pullulan production by tropical isolates of Aureobasidium pullulans, J. Ind. Microbiol. Biotechnol. 34 (2007) 55–61. [11] Z. Ma, W. Fu, G. Liu, Z. Wang, Z. Chi, High-level pullulan production by Aureobasidium pullulans var. melanogenium P16 isolated from mangrove system, Appl. Microbiol. Biotechnol. 98 (2014) 4865–4873. [12] S.R. Ravella, T.S. Quinones, A. Retter, M. Heiermann, T. Amon, P.J. Hobbs, Extracellular polysaccharide (EPS) production by a novel strain of yeast-like fungus Aureobasidium pullulans, Carbohydr. Polym. 82 (2010) 728–732. [13] L. Sheng, G. Zhu, Q. Tong, Comparative proteomic analysis of Aureobasidium pullulans in the presence of high and low levels of inorganic nitrogen compound, J. Agric. Food Chem. 43 (2014) 10529–10534. [14] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [15] L. Sheng, G. Zhu, Q. Tong, Mechanism study of Tween 80 enhancing the pullulan production by Aureobasidium pullulans, Carbohydr. Polym. 97 (2013) 121–123. [16] M.M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–253. [17] P. Manitchotpisit, C.D. Skory, T.D. Leathers, alpha-Amylase activity during pullulan production and alpha-amylase gene analyses of Aureobasidium pullulans, J. Ind. Microbiol. Biotechnol. 38 (2011) 1211–1218. [18] C. Aranda, A. Robledo, O. Loera, J.C. Contreras-Esquivel, R. Rodriguez, C.N. Aguilar, Fungal invertase expression in solid-state fermentation, Food Technol. Biotechnol. 44 (2006) 229–233.
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