Characterization of Water-Soluble Extracellular Polysaccharide from Aeribacillus pallidus IM17

Abstract

The production of the extracellular polysaccharide from the thermophilic bacterium Aeribacillus pallidus was carried out in the study. The polysaccharide was isolated and characterized by means of GC–MS, FT-IR, DSC, and XRD analyses. The rheological, foaming, and emulsifying properties of the polysaccharide were determined. Using a sucrose-rich medium, 27.1 mg dried EPS/100 mL was obtained with 94% carbohydrate and 1.5% protein. The monosaccharide profile of water-soluble EPS-IM17 was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose. The foaming capacity and stability of EPS-IM17 were 26.67% (± 4.71) and 40.01% (± 4.95), respectively, and the foaming stability was not affected by time. The emulsification index of EPS-IM17 was 64.54 (± 8.71) and decreased to 38.47 (± 10.44) after 24 h. EPS-IM17 had a crystalline structure. Solutions at different concentrations (10, 20, 40 mgmL^-1) showed pseudoplastic behavior. In conclusion, this report could be a lead study for the use of Aeribacillus pallidus extracellular polysaccharide for different applications.

Introduction

Thermophilic bacteria naturally occur in marine and terrestrial hot springs and deep-sea thermal vents, tropical soils, manure piles and feces, garbage, etc. [1, 2]. High temperature, high pressure, toxicity, and high inorganic or metal concentrations have been reported to characterize the presence of thermophilic bacteria capable of producing valuable metabolites from various carbon sources in these environments [3–5]. Although the high temperature increases the rate of most types of chemical reactions and improves the cumulative production of microbial metabolites, thermophilic bacteria have a commercial value and are attracting the attention of many industrial sectors. The short cultivation time and production process make thermophilic bacteria commercially important producers [3–6].

The physicochemical differences in thermophilic environments induce thermophilic microorganisms to synthesize different compounds with unusual properties. Such compounds are microbial polymers or exopolysaccharides. High molecular weight microbial exopolysaccharides (EPS) are released into the environment and allow microorganisms to adhere to surfaces, as biofilm formation plays an important role in the adaptation of bacteria to the physicochemical conditions of the environment. In addition, the heteropolysaccharides help extremophiles to adapt to extreme conditions [7, 8].

Bacterial polysaccharides generally exhibit higher water solubility and better thickening, stabilizing, and gelling activities than other commercially used plant polymers. EPSs produced by different species have been shown to have stable rheological and emulsifying properties due to their high safety, low side effects, and physical properties, allowing their use in different food products under different conditions. The use of EPS as emulsifying and stabilizing agents has been justified by their potential applications as replacements for synthetic emulsifiers in the food and cosmetic industries. There are few reports on the emulsifying properties of some high-molecular-weight emulsifiers produced by bacteria [9, 10]. Microbial exopolysaccharides have also been used in the petroleum, chemical, and pharmaceutical industries for their emulsifying, gelling, thickening, and film-forming properties [9–11].

The market for microbial products is expected to grow to USD 1910 million by 2030 (Market Research Future-Industry Analysis Report, Business Consulting and Research). Currently, the EPSs produced by microorganisms most commonly used in the market include xanthan from Xanthomonas campestris [12], bacterial alginates secreted by Pseudomonas species [13] and Azotobacter chrococcum [14], bacterial cellulose from Acetobacter xylinum [15], hyaluronic acid from Streptococcus equi [16], and succinoglycan from Rhizobium sp. [17].

The genus Aeribacillus is aerobic, thermophilic, alkali-tolerant, motile, gram-positive, and rod-shaped. They have elliptical and cylindrical endospores. Catalase and oxidase reactions are positive, and the precursor polar lipids are phosphatidylglycerol and di-phosphatidylglycerol. The DNA G + C ratio is between 39 and 41%. Aeribacillus genus has been separated from the genus Geobacillus according to the G + C content of DNA, fatty acid, and polar lipid profiles and Aeribacillus pallidus is the type strain of this genus [18].

The aim of the study was to carry out the molecular identification of Aeribacillus pallidus IM17 for the ability to produce and characterize polysaccharides. The importance of the research lies in the fact that it is one of the few studies about the detailed characterization of EPS from A. pallidus.

Materials and Methods

Screening of Microorganism

The sludge samples from different thermal resources of Turkey were collected and screened to determine the ability of exopolysaccharide production. The collected samples were purified with serial dilutions (dilution factors between 10^–1 and 10^–8) and propagated on the screening medium. The screening medium was composed of sucrose (50 g/L), yeast extract (10 g/L) and agarose (15 g/L). All the incubations throughout the study were conducted at 55 °C 120 rpm for 48 h and the isolate which had a mucoid structure on an agar plate was selected as the producer.

The identification of the isolate that was chosen as the producer was performed following the protocol of Genc et al. [19]. Briefly, the genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). The PCR amplification of the 16S rRNA gene was performed using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCA-3′) and 1492R (5′- GGTTACCTTGTTACGACTT-3′). The PCR products were cloned into pGEM-T Easy Vector (Promega UK) and then sequenced (Macrogen-Netherlands). Catalase and oxidase assays were also performed [20].

Exopolysaccharide Production

The exopolysaccharide production by the isolate was carried out in a 250 mL flask containing 100 mL of the screening medium (broth) without agarose (pH 7.2). A full loop of culture from the agar plate was transferred to the broth. The optical density (OD) while growing was monitored at 600 nm to prepare the inoculum. When the OD₆₀₀ value reached 0.1, 1 mL of the inoculum was inoculated into a 250 mL flask containing 100 mL of screening media and the incubation was conducted.

After cultivation, the cell pellet was removed by centrifugation (4500 rpm, 10 min), and the crude extract was obtained. To remove protein impurities from the crude extract, 4% (w/v) trichloroacetic acid was added and the mixture was stirred for 4 h. After the removal of the precipitates, two volumes of cooled (− 20 °C) ethanol were added. The mixture was then allowed to settle overnight in a refrigerator (+ 4 °C) to precipitate the polysaccharide [21, 22]. The polysaccharide was lyophilized and designated EPS-IM17.

Characterization

Total Carbohydrate and Protein Amount

Total carbohydrate analysis of EPS-IM17 was performed using the phenol–sulfuric acid method and total protein was determined using the Bradford method according to Gan et al. [23].

Monosaccharide Composition

The monosaccharide composition of EPS-IM17 was analyzed using the gas chromatography method [24] with minor modifications. The method was based on the derivation of monosaccharides to obtain alditol acetates in gas chromatography (GC) coupled with mass spectroscopy (MS). EPS-IM17 was hydrolyzed with trifluoroacetic acid (2 M, 2 h, 120 °C), and the partially methylated monosaccharides obtained were reduced with sodium borohydride (NaBH₄). The reduced and methylated monosaccharides were neutralized and the access of boric acid was removed by co-evaporation with methanol. The next step was acetylation with a pyridine-acetic anhydride mixture (1:1, 30 min, 120 °C) and the mixture of partially methylated alditol acetates was analyzed by GC–MS. Glucose, fructose, galactose, rhamnose, arabinose, xylose, and mannose were used as standard sugars and the same procedure was applied to all standards.

Functional Group Analysis

The functional groups of EPS-IM17 were analyzed by Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectra were obtained in solid form by applying infrared light to the dry polysaccharide. The EPS-IM17 (2 mg) was mixed with 100 mg potassium bromide, then ground and added to a 1 mm pellet to perform a Fourier Transform Infrared spectrum scan (PerkinElmer Spectrum Version 10.5.2) at a frequency range of 400–4000 cm⁻¹.

Foaming Activity

The foaming capacity of EPS-IM17 was measured according to the method of Gan et al. [23]. 5 mL of EPS solution (1%, w/v) was prepared. The volume of the mixture was recorded as the volume before homogenization. It was then homogenized using a high-speed blender (Ultra-Turrax T18 basic, IKA, Staufen, Germany) for 2 min and the volume was recorded. Foaming capacity was determined as follows;

$$\text{FC}\left(\%\right)=\frac{\text{volume after whipping}\left(\text{ml}\right)-\text{volume before whipping}\left(\text{ml}\right)}{\text{volume before whipping}\left(\text{ml}\right)}\times 100$$

To test foam stability, the above mixture was allowed to stand for 1, 10, 30, and 60 min. The residual foam layer volume for each time interval was then read and the foam stability (FS) was expressed as follows:

$$\text{FS}\left(\%\right)=\frac{\text{residual foam layer volume}(\text{mL})}{\text{initial foam layer volume}(\text{mL})}\times 100$$

Emulsifying Activity

The emulsifying activity of EPS-IM17 was determined by the method of Ktari et al. [25] with some modifications. 5 mL sunflower oil and 5 mL (1%, w/v) EPS solution were vortexed in a test tube for 5 min. After 15 min of duration, 1, 2, 3, 4, 5, 6, and 24 h, the emulsifying activity was calculated as follows;

$$E_t=\frac{h_e}{h_t}$$

where h_e(mm) is the emulsion layer height, h_t (mm) is the mixture overall height after t time.

Rheological Properties

Flow behavior, oscillation, and temperature sweep tests of EPS-IM17 were measured using a rheometer (Anton Paar MCR 102, Thermo Scientific, Germany) equipped with a parallel plate (diameter 35 mm, gap 1000 mm). Aqueous EPS solution (40 mgmL⁻¹) was placed between a parallel plate with a gap size of 1 mm and allowed to relax for 5 min before rheological testing. For apparent viscosity, the shear rate was ramped from 0.1 to 100 s⁻¹ at 25 °C. The frequency sweep test was performed within the linear viscoelastic range over a frequency range of 0.1–100 Hz at 25 °C. The frequency tests were used to determine the storage modulus (G′) and loss modulus (G′′) values of the samples. The temperature effect on EPS was measured at 5–90 °C with a heating rate of 5.67 °C/minute. The Hershel–Bulkley method was used to analyze the results [12, 26]. The relationship between shear stress and shear rate was described by the Hershel–Bulkley model as follows.

$$\mathbf{\tau }={\mathit{\tau }}_0+\mathit{K}.{\dot{\mathit{\gamma }}}^{\mathit{n}}$$

where $\mathrm{\tau }$ is the shear stress (Pa), ${\tau }_0$ is the yield stress (Pa), γ is the shear rate (1/s), K is the consistency coefficient, and n is the flow behavior index. All rheological experiments were performed in triplicate.

Differential Scanning Calorimeter

Thermal analysis of EPS-IM17 was performed using differential scanning calorimetry (DSC 4000, Perkin Elmer, USA). Firstly, 5 mg of the sample was weighed in aluminum containers. Samples were heated at a rate of 10 °C/min between 40 °C and 300 °C with a nitrogen flow of 20 mL/min. An empty aluminum dish was used as a reference. The temperature at which the endothermic peak occurred was defined as the melting point (T_m).

X-ray Diffraction Analysis

The structure of EPS-IM17 was investigated by X-ray diffraction. For this purpose, a lyophilized sample (~ 100 mg) was analyzed in a Bruker D8 DISCOVER XRD instrument depending on the angle of incidence of the rays between 30° and 100° at an increasing rate of 2°/min.

Results and Discussion

The EPS producer isolate was identified as Aeribacillus pallidus IM17 and deposited in GenBank (accession number MW617894). The isolate was Gram-positive, catalase, and oxidase-positive, as reclassified by Minana-Galbis et al. [18]. The ability of A. pallidus IM17 to produce EPS was tested on sucrose-rich media to induce polysaccharide production, and the amount of EPS was 27.1 mg dried EPS/100 mL, consisting of 94% carbohydrate and 1.5% protein. The exopolysaccharide from A. pallidus IM17 (EPS-IM17) was only water-soluble [27, 28]. There were no DNA or RNA peaks as a result of spectrum scanning in this study, but Radchenkova et al. [27] produced 18 mg EPS/100 mL from A. pallidus 418 with DNA. Zheng et al. [28] isolated an emulsifier from A. pallidus YM-1, which was a complex of carbohydrate (41%) and protein (11.3%). G. stearothermophilus DG1 produced a polysaccharide with carbohydrate (60%) and protein (12%) [29].

Monosaccharide Composition

The monomeric units of EPS-IM17 were determined by derivation of sugars to alditol acetates using GC–MS apparatus. The chromatogram is shown in Fig. 1. The EPS of A. pallidus IM17 produced in a sucrose-rich medium was heteropolysaccharide and the polysaccharide structure was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose in the proportion of 17.8%, 7.8%, 1.1%, 54.7%, 15.0%, and 3.6%, respectively. The major component in EPS-IM17 was mannose, while xylose was the minor sugar. Almost the same monosaccharide profile was also found in a capsular polysaccharide from a thermophilic cyanobacterium [30]. These results were also similar to the results of polysaccharides from two different Geobacillus sp. [31].

Fig. 1.

GC–MS chromatogram of EPS-IM17 for monosaccharide composition 1: Rhamnose (10.3 min.), 2: Arabinose (10.6 min.), 3: Xylose (11 min.), 4: Mannose (17.9 min.), 5: Glucose (18.2 min.) 6: Galactose (18.4 min.)

Functional Group Analysis (FT-IR)

Nine different peaks were obtained when the FT-IR spectra of EPS-IM17 were examined after TCA precipitation (Fig. 2). The vibrations were 3253 cm⁻¹, 2925 cm⁻¹, 1624 cm⁻¹, 1526 cm⁻¹, 1396 cm⁻¹, 1069 cm⁻¹, 967 cm⁻¹, 856 cm⁻¹, and 526 cm⁻¹ with transmissions of 96%, 96%, 84%, 86%, 89%, 77%, 81%, 80% and 70%, respectively. Although the transmissions were different, the majority of the vibrations were common in the polysaccharides. The vibration at 3253 cm⁻¹ indicated intracellular H-bonds or –OH groups in carbohydrates [32] and 2925 cm⁻¹ indicated C–O carboxyl stretching by aliphatic hydrocarbons [21, 33]. The peak at 1624 cm⁻¹ was related to the amide groups of proteins [34] and the peak at 1526 cm⁻¹ was related to carboxyl groups [33]. According to Alvarez et al. [35], the peaks at 2930 cm⁻¹ and 1599 cm⁻¹ were related to vibrations of C–H and C=O stretches of amide groups in polysaccharide structure. The peak observed at 1396 cm⁻¹ was assigned to C–C stretching and the spectral band at 1069 cm⁻¹ was based on C–O–C vibrations and showed the character of polysaccharides or polysaccharide-like substances [36]. The bands in the 1000–700 cm⁻¹ range (967 cm⁻¹ and 856 cm⁻¹) showed the α and β conformations within the polysaccharide structure [37, 38], while the vibrations at lower spectra (526 cm⁻¹) indicated glycoside linkages between glycosyl groups in EPS [21].

Fig. 2.

FT-IR diagram of EPS-IM17

Foaming Activity

The foam was a dispersion of water and air phases and was important for the food and cosmetic industries to achieve better consistency and sensory quality [39]. Polymers were used as stabilizers and thickeners in colloidal systems due to their hydrophilic properties [39, 40]. Polysaccharides, which are surface-active macromolecules, could form stable films at the interfacial level and this contributed to the foaming stability [39]. The foaming capacity and stability of EPS-IM17 are shown in Table 1 and Fig. 3.

Table 1.

Foaming and emulsification properties and the coefficients of Herschel Bulkley model for forward steady flow curve of EPS-IM17

Foaming and emulsification properties		The coefficients of the Herschel Bulkley model
		Concentration (mgmL−1)	η50−1 (Pas)	K	n	τ0	R2
Foaming capacity (%)	26.67 (± 4.71)	40	1.18 (± 0.02)	0.0001	0.4237	0.0182	0.9366
Foaming stability (%)	40.01 (± 4.95)	20	0.99 (± 0.02)	0.0001	0.4339	0.0166	0.9317
Emulsification index	64.54 (± 8.71)	10	0.98 (± 0.01)	0.0001	0.4799	0.0176	0.9278

η₅₀⁻¹: apparent viscosity at 50 s⁻¹ (Pa.s)

K: Consistency index, n: flow behavior index, τ₀: yield stress (Pa)

Fig. 3.

Foaming capacity of EPS-IM17

The foaming of the polysaccharide solution was still stable after 120 min. However, the foaming stability was negatively affected by time, and the foaming stability did not fall below 40% even after 8 h. Gongi et al. [41] isolated a polymeric substance from a cyanobacterium and investigated its foaming capacity (22.4%) and stability (10.3%). A novel EPS from Lactobacillus sp. showed a foaming capacity of 30%, but the foaming stability was 22% after 60 min [42]. These results suggest that EPS-IM17 could be used in the food industry for enhanced foaming properties.

Emulsifying Activity

The emulsification index of EPS-IM17 solution (1%) was calculated to be 64.54 (± 8.71) at baseline (Fig. 4). The emulsification index decreased by 38.47 (± 10.44) after 24 h. To be considered an emulsifier, its capacity had to be maintained at at least 50% of the initial emulsion volume [43]. EPS-IM17 retained 55% of its emulsion capacity after 5 h. Radchenkova et al. [44] determined the 60% emulsifying activity of 1% EPS by A. pallidus 418 and concluded that the high protein content in the polysaccharide structure caused a higher emulsifying index. Among the thermophilic producers, a highly stable emulsifier isolated from A. pallidus YM-1 with emulsifying properties promising for biotechnological applications has recently been observed by Zheng et al. [28]. A. pallidus YM-1 was a producer of an emulsifier and the emulsification index reached 60% at the 10th hour of cultivation. Similar studies have been reported in other bacterial species. Han et al. [45] investigated the emulsifying properties of the polysaccharide produced by Bacillus amyloliquefaciens LPL06. They showed that 0.1% EPS was the optimum concentration and that there was no significant increase in emulsifying activity at higher concentrations. When they examined the emulsion stability, they calculated the emulsion stability index values to be 68.26% after 24 h. Castellane et al. [46] prepared EPS from the strain R. tropici SEMIA 4080 and the mutant strain and reported that both strains had a lower emulsion stabilizing capacity with 55.85% after 24 h.

Fig. 4.

Emulsifying index of EPS-IM17 with sunflower oil

Rheological Properties

The rheological behavior of exopolysaccharides is an important property with implications for the food and pharmaceutical industries [47]. Therefore, the flow behavior, temperature effect, or viscoelastic properties of EPS-IM17 should be investigated. Figure 5 shows the flow curves of EPS solutions at different concentrations (10, 20, 40 mgmL⁻¹). The Herschel–Bulkley model was run to accurately determine the relationship between viscosity and shear rate. The calculated values of the constants (K and n) and the coefficient of determination (R²) for the Herschel–Bulkley model at concentrations of 40, 20, and 10 mgmL⁻¹ are given in Table 1. The high values of the correlation coefficients (R²) indicate that the flow behavior of EPS solutions is adequate for modeling the viscosity curve using the model. Yield stress (τ₀), represents the minimum stress required to initiate flow [48]. Higher concentration showed higher yield stress (τ₀), although the consistency index (K) was not affected by the concentration of EPS solutions. The flow behavior index values (n) expressed that the EPS solutions exhibited shear thinning (pseudoplastic) behavior. As the shear rate increased, the apparent viscosity of the pseudoplastic materials would decrease [49]. This behavior could be explained by the weak forces between the particles of aggregated EPS. The shear treatment caused breakage and formation in the particles and therefore the viscosity of the solutions, which had weaker resistance to flow, decreased [50]. Similar results have been reported by other researchers [45–47, 50]. Although the viscosity increased with increasing concentration of EPS solutions, there was no significant difference between the apparent viscosities (at 50 s⁻¹) at 20 mgmL⁻¹ and 10 mgmL⁻¹. The apparent viscosity value of exopolysaccharide obtained from B. amiloliquefaciens LPL061 at 50 s⁻¹ shear rate was about 1 mPa.s [45], while the apparent viscosity value of exopolysaccharide from A. pushchinoensis G11 is 2.03 mPa.s [19]. Figure 6 shows the viscosity of the EPS solution at temperatures ranging from 5 °C to 90 °C. The apparent viscosity values decreased with increasing temperature. The changes in rheological properties with temperature would support the use of EPS-IM17 in the food industry. Figure 7 shows the elastic modulus (G′) and viscous modulus (G′′) as a function of the frequency of the EPS-IM17 solution at 40 mgmL⁻¹. Both G′ and G′′ values increased with frequency. Similar results were also reported by Abedfar et al. [50]. Although the EPS solution showed liquid-like behavior (G′′ > G′) at low frequencies, the behavior of the solutions shifted from predominantly liquid-like behavior (G′′ > G′) to solid-like behavior (G′ > G′′) at higher frequencies. Ayyash et al. [47] reported that the EPS product from Lactococcus garvieae C47 showed more viscous than elastic behavior at low frequencies. The dependence of G′ and G′′ on frequency indicates that the EPS solution has weak gel properties [50].

Fig. 5.

Viscosity changes versus the shear rate of EPS-IM17 at various concentrations

Fig. 6.

Temperature effect on viscosity of EPS-IM17 at 40mgmL⁻¹

Fig. 7.

Frequency analysis of EPS-IM17 at 40 mgmL⁻¹

Differential Scanning Calorimeter

The thermal behavior of EPS was an important property for its commercial use [51] and glass transition and melting points were determined. As shown in Fig. 8, EPS-IM17 had exothermic peaks with a glass transition temperature at 50 °C, a semi-crystalline region between 45 and 145 °C, and a melting point at 146 °C. Degradation was observed at 200 °C.

Fig. 8.

DSC results of EPS-IM17

There was no result on the thermal properties of EPS from Aeribacillus sp. For comparison, EPS from Bacillus tequilensis-GM [51] had a melting point of 222 °C. G. stearothermophilus DG1 produced EPS with a melting point of 174 °C [29].

XRD Analysis

The high and narrow peaks indicate the crystal structure, while small and wide peaks indicate the presence of amorphous parts of the exopolysaccharide, according to the XRD analysis, which allowed a more detailed examination of the exopolysaccharide structure by measuring its refractive response to X-rays. As a result of the XRD analysis of EPS-IM17, intense and prominent peaks indicative of the crystalline structure were observed in the 25–35° (2θ) spectral region, but no peak indicative of the amorphous region was obtained (Fig. 9). Wang et al. [52] isolated two fractions of polysaccharides from Geobacillus sp. with a crystalline structure.

Fig. 9.

XRD results of EPS-IM17

Conclusion

Although there have been studies on bacterial polysaccharide production, this study is one of the few publications characterizing the polymeric substance from A. pallidus. The results showed that EPS from A. pallidus IM17 had a high carbohydrate concentration with the presence of protein. The considerable properties in terms of rheology, foaming, and emulsification and higher thermal values and crystalline structure of the polysaccharide made it a candidate for use in different applications. The monosaccharide composition and chain structural conformation of polysaccharides could influence their interaction at the oil/water interface of emulsions in different ways, so therefore, a detailed structure determination study of the polysaccharide should be carried out to determine the backbone and branching of the monomer units, as further studies.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Declarations

Conflict of interest

There is no conflict of interest to be declared.

Ethical approval

This article does not contain any studies related to human participants or animals.