Structural analysis and properties of dextran produced by Weissella confusa and the effect of different cereals on its rheological characteristics

International Journal of Biological Macromolecules 143 (2020) 305–313

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Structural analysis and properties of dextran produced by Weissella confusa and the effect of different cereals on its rheological characteristics Z. Dilek Heperkan a,⁎, Meltem Bolluk b, Sedef Bülbül c a b c

Istanbul Aydın University, Faculty of Engineering, Food Engineering Department, Turkey Istanbul Technical University, Metallurgical and Materials Engineering Department, Turkey Istanbul Technical University, Graduate School of Science Engineering and Technology, Turkey

a r t i c l e

i n f o

Article history: Received 12 September 2019 Received in revised form 15 November 2019 Accepted 5 December 2019 Available online 06 December 2019 Keywords: Weissella confusa Dextran Boza FTIR Exopolysaccharide

a b s t r a c t Weissella confusa is a commonly found species in boza, a highly viscous beverage obtained from fermented cereals. Exopolysaccharide (EPS) produced by Weissella confusa C19 was characterized and the role of different cereals on its rheological characteristics were studied. Thus, the effect of the type of cereals on textural characteristics of boza for standard boza production and the fate of W. confusa during fermentation were assessed. W. confusa C19 EPS consisted of glucose, sucrose and 1.7 mg/kg protein. Structural characterization of water-soluble dextran was determined by 1H NMR, functional groups were identified by Fourier transform infrared spectroscopy (FT-IR) and the crystal structure was determined by X-ray diffraction (XRD). After EPS characterization, different cereal-based media including maize, oat, rice and wheat were used for the growth and EPS production of the bacterium. The rheologial properties of EPS obtained from different cereal-based media showed that the steady shear behavior of EPS was pseudoplastic. W. confusa C19 is a unique strain that can adapt to the environment containing high sugar to produce high amounts of polysaccharides. Besides new information on EPS from W. confusa origin, this study showed that the amount of dextran increased using solid media fortified with different cereals. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Microorganisms are able to transfer different carbon sources from the environment and accumulate them as storage compounds. Some fungi and microalgae accumulate lipids whereas bacteria accumulate mainly sugars. Glycogen and polyhydroxyalkanoate (PHA) are well known storage compounds in bacteria. However, some bacteria synthesize large amounts of polysaccharides; the amounts are so large that they are excreted from the cells [1]. Excreted or extracellular polysaccharides (EPS) are general terms that refer to both cell-bound polysaccharide and excreted polysaccharide in the medium. Most lactic acid bacteria (LAB) excrete their EPS into the medium [2]. Exopolysaccharides of LAB origin show considerable variation in composition and structure. These differences can be seen even in the same species, e.g. different types of exopolysaccharides were determined from W. confusa such as dextran; dextran and levan; dextran and capsular polysaccharide [3]. W. confusa (previously known as Lactobacillus confusus) is a Gram-positive, catalase negative, facultative anaerobic, ⁎ Correspondıng author at: Istanbul Aydın University, Faculty of Engineering, Food Engineering Department, 34295 Istanbul, Turkey. E-mail addresses: [email protected] (Z.D. Heperkan), [email protected] (M. Bolluk), [email protected] (S. Bülbül).

https://doi.org/10.1016/j.ijbiomac.2019.12.036 0141-8130/© 2019 Elsevier B.V. All rights reserved.

heterofermentative, coccobacilli or rod-shaped morphology, producing D- or DL-isomers of lactic acid [4,5]. Since the bacteria have complex nutritional requirements, can grow in cereal based fermented products such as fura [6] and boza [7] as indigenous species. W. confusa has been used in the production of a variety of fermented food, due to its diverse biotechnological applications [8,9]. It is an important species for efficient in situ production of dextrans and isomaltooligosaccharides in sourdoughs without strong acidification [9]. Indeed, the ability to produce dextran is one of the distinctive phenotypic features of the genus Weissella [5,10]. W. confusa was also present in the gut microbiota of healthy humans, the breast milk and the feces of both mothers and infants [11,12] and has been described as a potential probiotic species [13,14]. However, W. confusa, has also been isolated from human clinical specimens and is considered as an opportunistic pathogen [12]. It was recently reported that the use of W. confusa as a probiotic should be approached with caution because its incidence as an opportunistic pathogen is not clearly known [15]. The dextrans produced by Weissella spp. have similar structures with mainly α-(1–6) linkages [16,17], which can be used in the industry for different purposes. It has been shown to retain moisture, improve rheological and processing characteristics of dough and bread [18,19]. However, there are a few limited studies focusing on the rheological properties of dextran produced by W. confusa in cereal based products.

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In this study, structural characterization of dextran produced by W. confusa, grown on different cereal-based solid media and its functional groups were aimed. The influence of different cereals on the physico-chemical and rheological properties of dextran were also investigated. Since, boza is produced using different cereals as raw material, the study also contributes to the production of cereal-based, LABfermented functional beverages, using W. confusa as an adjunct culture.

thin film layer and dried naturally at room temperature. Then the petri dish was sealed with parafilm and stored in a refrigerator for further use. The dreid dextran was dissolved in distilled water (0.8 mg/mL) and the concentration of dextran was determined by the phenol-sulfuric acid method (Shimadzu UV-1700 spectrophotometer), using glucose as standard [7,20,21]. Analyses were carried out with triplicate samples and the results were reported as the avarage concentration of the three repeated experiments.

2. Material and methods 2.1. Bacterium and media preparation

2.4. Carbohydrate composition of dextran

W. confusa C19 (accesion number is JQ805675.1) was isolated from boza, identified by polymerase chain reaction (PCR) with partial 16S rRNA gene sequencing and its morphological characteristics and carbohydrate fermentation profiles were determined previously [7]. Dextran production and dextran production rate by W. confusa for kinetic experiments were performed in growth media with different cereals. Maize, oat, rice and wheat grains were used to prepare the solid media. Each medium was prepared separately by boiling coarsely ground cereals in water for 2 h. The ratio of cereal and water is approximately 1:10. The slurry with light soup-like consistency was then filtered through cheesecloth two times. 50 mL eluate containing 5 g sucrose was mixed with 50 mL previously boiled Man, Rogosa and Sharpe (MRS) agar (Merck, Germany). The pH of the media was adjusted to 6.4, distributed to falcon tubes (20 mL for each) and test tubes (5 mL for each) and then autoclaved at 121 °C for 15 min under 1 Atm. After sterilization, the media in the falcon tubes were cooled to 45–50 °C and then poured into sterile petri dishes. These media were used for dextran production by inoculation of W. confusa. MRS agar with 5% sucrose were prepared separately, sterilized at 121 °C for 15 min under 1 Atm. and were named modified MRS (mMRS) agar. The test tubes containing mMRS and cereal-based media were also prepared. After sterilization in an autoclave they were tilted to make agar slants. For the enumeration of dextran producing colonies, the agar slants were inoculated with 0.1 mL cultures of W. confusa grown overnight at 37 °C on MRS broth and then incubated at 37 °C for 72 h dextran yield and viable cell count were then evaluated.

The carbohydrate composition (mono and disaccharide) of dextran was determined by High Performance Liquid Chromatography (HPLC) system. The HPLC system (Waters Assoc., Milford, MA, USA) consists of a model 2690 binary pump, a Waters 2410 refractive index detector, and carbohydrate column (125 Å, 10 μm, 3.9 mm × 300 mm). The column temperature was set at 30 °C. The mobile phase was composed of acetonitrile and water (75:25, v/v), the flow rate was 1.0 mL/min and the analysis time was 35 min. Two milliliters acetonitrile:water (50:50, v / v) was added to 0.5 g of the sample and vortexed for 15 s, and then agitated for 30 min. It was then centrifuged at 13200 rpm for 5 min, filtered through a 0.45 μm filter and then injected to the HPLC system. The carbohydrate composition was determined by comparing the retention times of glucose and sucrose standards. The experiments were carried out in three replicates and the results were reported by averaging.

2.2. Reagents and chemicals Tetramethyl silane, glucose, sucrose, phenol, sulfuric acid, Folin– Ciocalteu reagent, sodium carbonate, asetonitrile, methanol, phenylisothiocyanate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dihydrate and triethylamine were all purchased from Merck, Oxoid and Sigma Chemical. 2.3. Isolation and purification of dextran Petri dishes containing MRS agar with 5% sucrose (mMRS) were inoculated with 0.1 mL cultures of W. confusa grown overnight at 37 °C on MRS broth and then incubated at 24 h at 37 °C. This cells was named sugar adapted cells (SAC). Petri dishes and test tubes (agar slant) containing media with different cereals (maize, oat, rice and wheat) and 5% sucrose were then inoculated with these SAC and incubated for 3 days at 37 °C. Modified MRS (mMRS) was used as control The crude dextran on the surface of the media was washed with 10 mL of distilled water from the surface of the solid media and collected into falcon tubes. The crude dextran was centrifuged at 6000 rpm (Hettich, Universal 16A) at 4 °C, for 20 min and then filtered from 0.22 μm membranes (Whatman FP 30/0.2 CA-S, Dassel, Germany). Three volumes of cold absloute ethanol (Riedel-de Häen) were added to supernatant and then kept for 24 h at 4 °C for precipitation. After centrifugation (6000 rpm, 20 min, 4 °C) the pellet was air-dried, dissolved in distilled water and dialysed (cellulose dialysis tube, Sigma D-9652) for 3 days against ultrapure water. The EPS was dispersed into the petri dish as a

2.5. Amino acid composition of dextran The amino acid composition of dextran was determined according to Heinrikson and Meredith [22]. Phenylisothiocyanate (PITC) was used for quantitative precolumn derivatization of amino acids and reverse-phase ultra fast liquid chromatography systems (Shimadzu-20A prominence UFLC) were used for the separation and quantitation of the resulting derivatives. The preparation of the mobile phase A is as follows: 0.78 g sodium dihydrogen phosphate dihydrate and 0.88 g disodium hydrogen phosphate dihydrate were dissolved in water, the pH was adjusted to 6.8 and made up to a liter with water. Acetonitrile was used as the mobile phase B. UV detector, 254 nm wavelength, 10 μl injection volume and 1 mL/min flow rate were used for the analyses. Amino acids in the protein of the dextran (0.5 g) was hydrolyzed with 6 M HCl (110 °C, 24 h, under N2 ) and then filtered. The filtrate (0.2 mL) in the test tube was dried under N 2 at 50 °C and then 0.5 mL acetonitrile was added and redried. 0.5 mL from the mixture of asetonitrile:methanol:triethylamine and 0.1 mL of phenylisothiocyanate were added to the residue in the test tube and then derivatized at 40 °C for 30 min. After drying under N2 at 40 °C, 0.2 mL asetonitrile was added, then redried under N2. Finally, 5 mL 0.02 M ammonium acetate was added to the sample, filtered using 0.2 μm filter and were analyzed by UFLC. 2.6. Total phenolic content of dextran The total phenolic content of dextran was determined by the Folin–Ciocalteu method [23]. Briefly, 1 mL of crude extract (2 mg/mL) was made up to 100 mL with distilled water and then filtered. 0.5 mL of the extract was mixed with 2.5 mL of Folin–Ciocalteu reagent and 2 mL of 7.5% (w/v) sodium carbonate was added and vortexed. The mixture was allowed to stand for a further 30 min in the dark, and absorbance was measured at 760 nm (Shimadzu UV1700). The total phenolic content was calculated from the calibration curve, and the results were expressed as mg of gallic acid equivalent per g dry weight.

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2.7. Fourier transform infrared (FT-IR) analysis of dextran

2.11. Density of dextran

The functional groups of dextran were identified by Fourier transform infrared spectroscopy (FT-IR, Perkin Elmer Spectrum 65 FT-IR spectrometer, Massachusetts, USA) equipped with attenuated total reflectance (ATR) sampling accessory. Four scans between 1400 cm−1 and 800 cm−1 with a resolution of 4 cm−1 were taken in absorbance mode for each sample.

The density of the dextran was measured with helium gas pycnometer (micromeritics ™ AccuPyc II 1340). The sample and measurement conditions are given below. Sample mass was 1.0062 g, temperature was 24 °C and the number of purges was 10. The density of dextran was determined by averaging. 2.12. Rheological properties of dextran

2.8. Nuclear magnetic resonance (NMR) spectroscopy analysis of dextran NMR spectroscopy was used for the assessment of the dextran structure. The anomeric proton resonances for 400 MHz were determined from the 1H NMR (Agilent VNMRS 500 MHz) spectrum. Twenty milligrams of purified dextran was dissolved in 1.0 mL of D2O and was analyzed. Tetramethyl silane (TMS) was used as an internal reference. 1H NMR spectra were recorded. 1H NMR (500 MHz, D2O): ẟ 4.97 (bs, 1H, C1\\H), 3.98 (d, 3JH,H = 6.70, C6-H1), 3.90 (d, 3JH,H = 6.95, C6-H2), 3.73 (d, 3JH,H = 12.25, C5\\H), 3.70 (t, 3JH,H = 9.20, C3\\H), 3.56 (d, 3JH,H = 9.45, C2\\H), 3.51 (distorted t, 3JH,H = 9.20, C4\\H). 2.9. X-ray diffraction (XRD) analysis of dextran The crystal structure of dextran was determined by X-ray diffraction (XRD). The study was performed on the Bruker D8 advance model XRD with Ni filtered CuKα radiation (λ = 1.5406 Å), running conditions of 40 mA, 40 kV, in the 2θ range of 10–90° incremented at a step size of 0.02° at a rate of 2°/min.

The rheological properties of dextran were determined by a rheometer (Rheostress 1, Haake, Germany) fitted with a plate-plate sensor system (diameter 35 mm, gap 1 mm). All measurements were carried out at 25 °C. The shear rate range was in between 0 and 400 s−1. Measurements were made in duplicate. The rheological behavior of dextran was determined according to a power law model as follows: τ ¼ K γ n

ð1Þ

where τ is the shear stress (Pa), K is the consistency index (Pa.sn), γ is the shear rate consistency and n is the flow behavior index (n). The consistency index (K) and flow behavior index (n) values were obtained by using the equipment software (Rheowin, Haake, Germany) and the apparent viscosities of the samples at 10 and 100 s−1 were calculated from the following equation: η ¼ K γ ðn−1Þ

ð2Þ

where η is the apparent viscosity (Pa.sn). 2.10. Molecular weight of dextran 2.13. Enumeration and observation of colonies The molecular weight of dextran can be determined using different methods depending on the material tested and the infrastructure of the laboratory. Cryoscopic method is routinly used in the determination of molecular weight of various materials in our laboratory. The freezing point depression was measured by Advanced Modular Process Calibrator (GE Druck DPI 620 GENii, Australia). Briefly, 5 g of purified dry dextran was dissolved in 100 mL of pure water and kept overnight under gentle stirring at 30 °C. Ten milliliters of dextran solution was added to test tube with cap and the freezing point depression was determined. The molecular weight of dextran was calculated using the following equations. The solute concentration can be expressed in terms of the molality (m) of the dextran utilizing the freezing point depresion of the solution. The molality is determined using Eq. 1. m ¼ molA=kg solvent

ð1Þ

where m is the molality. The freezing point depression (Tf° - Tf) or (ΔTf) in °C is given by Eq. 2. ΔTf ¼ Kf m

ð2Þ

where; Tf: the freezing point of the solution. Tf°: the freezing point of the water. Kf: freezing point depression constant (for water 1.86 °C/m). ΔTf was 0.46, and Kf was 1.86, the molality was calculated as 0.2473 (Eq. (2)). Molality, m was sustituted in Eq. 1 and mol A was calculated, to determine the molecular weight of dextran as 204.966725 g/mol. molA ¼ m  kg solvent ¼ 0:2473  100:0626=1000 ¼ 0:02474548

molecular weight ¼ g solute ðdextranÞ=molA ¼ 5072=0:02474548

Kinetic experiments were performed to determine the number of dextran producing W. confusa cells during 3 days of incubation. Petri dishes containing media with different cereals (maize, oat, rice and wheat) and 5% sucrose and mMRS were inoculated with SAC and incubated for 3 days at 37 °C (explained in Section 2.2). Enumeration of colonies was performed after each day. The colonies collected from the surface of the solid media were examined under a microscope (Nikon LV100ND) with 20× and 50× objectives and the cell shape/morphology was observed. 2.14. pH of creal based media The pH of different cereal-based media was determined with a Jenway 3010 type pH meter during 3 days of incubation. 2.15. Statistical analysis The rheological data were analyzed by the Tukey method using Minitab 2017 software to determine if there were statistically significant differences (p b .05) among the means. Each analysis was performed in triplicate and the results were reported as mean value of standard deviation. 3. Result and discussion W. confusa is one of the dextran producing species of the genus Weisella, well-known worldwide [10,17,24]. Recently, other polysaccharides like fructan [25] and levan [3] of W. confusa origin were found, as well. In this study, higher amounts of water-soluble dextran produced from large sugar adapted Weissella confusa C19 cells were observed. The chemical and molecular characterization of water-soluble dextran was conducted by high performance liquid chromatography (HPLC), ultra fast liquid chromatography (UFLC), Fourier transform infrared spectroscopy (FT-IR) and 1H NMR. The crystal structure was

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determined by X-ray diffraction (XRD). The carbohydrate composition of dextran from W.confusa C19 was consisted of glucose and sucrose (Fig. 1A) The amino acid composition showed that the protein (1.7 mg/kg) of the dextran was consisted of 12 amino acids (Fig. 1B). Lysine was the highest (0.4 mg/kg) followed by leucine and proline (0.2 mg/kg). Glutamine, glycine, histidine, arginine, threonine, alanine, valine isoleucine and phenylalanine were found to a lesser extent (0.1 mg/kg). The peak around 1640 cm−1 (amide I) in the FT-IR spectrum indicating and confirmed that the protein was present in W. confusa C19 dextran (Fig. 2). 3.1. FT-IR analysis of dextran The functional groups of dextran were identified by Fourier transform infrared spectroscopy (FT-IR). The spectrum of water-soluble dextran produced from W. confusa C19 is shown in Fig. 2. The broad band at

3262 cm−1 was due to hydroxyl stretching vibration and the band at 2920 cm−1 was assigned to the C\\H stretching vibration in the spectrum [26]. The band in 1640 cm−1 was due to amide I [24]. Peaks at 1413 cm−1 and 1338 cm−1 were characteristics of carboxyl groups, indicating that the dextran was acidic [2]. The functional groups of dextran showed high absorbencies in the region 1200–950 cm−1 with an intense peak at 1000 cm−1 in the FT-IR spectrum. The bands in this region (1200–950 cm−1) is typical for polysaccharides [7,27]. The main characteristic bands found in the spectra of dextran at 1151 cm−1, 1105 cm−1 and 1000 cm−1 are vibrations of C\\O and C\\C bonds and deformational vibrations of the CCH, COH and HCO bonds [28]. The band at 1151 cm−1 is assigned to vibrations of C-O-C bond and glycoside bridge. The absorption peak at 1105 cm−1 is due to the vibration of the C\\O bond at the C4 position of glucose residues [28]. The intense peak at 1000 cm−1 is wide chain flexibility present in dextran around the α (1 → 6) glycosidic bonds [29]. The band near 861 cm−1 is due to

Fig. 1. HPLC chromatograms obtained from the analysis of (A) a carbohydrate composition of Weisella confusa C19 dextran. UFLC chromatograms of the analysis of (B) amino acid composition of Weisella confusa C19 dextran.

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Table 1 The 1H NMR analysis of dextran from W. confusa C19.

Atoms 1

H ( δ , ppm)

1

2

3

4

5

6

4.97 3.56

3.70

3.51

3.73

3.98 3.90

3

JH,H (Hz)

-

9.45

9.20

9.20

12.25

6.70 6.95

Table 1. The proton signals at 3.98 and 3.90 belongs to diastereotopic protons (C6-H2) and these signals can be considered as an evidence for the dextran structure. The proton of anomeric carbon (C1\\H) has resonance at 4.97 ppm. 3.3. X-ray diffraction (XRD) Fig. 2. FT-IR spectrum of dextran produced from Weisella confusa C19.

alpha configuration of sugars [27]. Absence of the peak between 1700 cm−1 and 1775 cm−1, suggesting that neither glucuronic acid nor diacetyl ester was present in W. confusa C19 dextran.

The X-ray diffraction (XRD) pattern is shown in Fig. 4. The XRD pattern (The crystal structure) of dextran produced by W. confusa C19 showed an extremely broad peak that indicated mainly an amorphous nature. 3.4. Total sugar concentration by the phenol sulfuric acid method and phenolic contents of dextran obtained from different media

3.2. 1H NMR spectroscopy analysis The anomeric proton resonances for the 400 MHz 1H NMR spectrum of α (1 → 6) dextran is shown in Fig. 3. The 1H NMR analysis of watersoluble dextran synthesized from W. confusa C19 is summarized in

The dextran concentration obtained from W. confusa C19 grown on different cereal-based solid media (quantified by the phenol sulfuric acid method) is shown in Table 2. Among cereals, the concentration of dextran was the highest in rice-based medium (21.9 g/L) and the least

Fig. 3. 1H NMR (400 MHz, D2O) spectrum of water-soluable dextran from Weisella confusa C19.

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Z.D. Heperkan et al. / International Journal of Biological Macromolecules 143 (2020) 305–313 Table 3 The rheological parameters (apparent viscosity) of dextran obtained from different cerealbased media. τ = K •γn

K

Maize Oat Rice Wheat mMRS

Fig. 4. XRD pattern of dextran from Weissella confusa C19.

Table 2 Total sugar concentration and total phenolic content of dextran obtained from Weisella confusa C19 on different media. Media

Total sugar concentration (g/L)

Total phenolic content (g/L)

Maize Oat Rice Wheat mMRS

19.1 18.5 21.9 20.9 23.4

0.9 1.1 0.9 1.1 1.1

± ± ± ± ±

0.20 0.15 0.78 0.17 0.25

± ± ± ± ±

0.35 1.12 0.46 1.09 1.10

in oat-based medium (18.5 g/L). Arabinoxylan (AX) is the main polymer of cell walls in cereals. Oat has the greatest concentration of total AX, whereas rice contain the least [30]. Arabinoxylans may encapsulate available nutrients [30]. Besides, of the cereal grains, oat can bind less water and is less soluble compared with rice [30,31]. In addition, carbohydrates present in rice (73.7 g/100 g) are higher than wheat (59.4%), corn (65.0%) and oats (56.2%) [32]. Therefore, the bacteria can make more use of the nutrients in rice and thereby produce more dextran. The amount of dextran was higher in mMRS (23.4 g/L) than cerealbased media. The amount of dextran from W. confusa C19 in mMRS liquid medium was 0.93 (g/L) in our previous study [7]. The amount of dextran was more than 20 times higher in solid media with sugar adapted cells compared to liquid media. The viscosity of the medium

n

0.58 0.34 0.19 0.38 0.08

Viscosity (Pa.sn)

r

0.66 0.71 0.72 0.69 0.85

0.999 0.999 0.999 0.999 0.999

shear stress (Pa)

30 25 20 15 10 5 0 50

100

150

200

250

300

350

400

shear rate (s-1) Wheat

Rice

(at 100 s−1)

0.27 0.17 0.10 0.19 0.06

0.12 0.09 0.05 0.09 0.04

affects the nutrient uptake of bacterium, metabolic activity and ultimately the viability. The viscosity of dextran obtained from rice medium was lower than the dextran viscosity obtained from other cereal media in this study. The results were significantly different (p b .05). Bacteria make more use of nutrients and thereby produce higher amounts of dextran in rice medium. On the other hand, a high sugar concentration around the cell blocks the bacterial specific carrier systems. The reason for the absence of viable bacteria after 72 h in cereal media is that the dense dextran layer around the cells prevent the bacteria from transporting more nutrients. In mMRS, as the dextran viscosity is lower than the viscosity of dextrans obtained from all cereal media, this may lead to a gradual decrease in the number of bacteria (from 2.109 cfu/g to 2.105 cfu/g) in mMRS. Since the medium still contains solid nutrients available to the bacteria for a longer time. Among lactic acid bacteria, Weissella species produce the most EPS, followed by lactobacilli whereas streptococci produce the least. It was reported that most of the Lactobacillus species produce 1–10 g/L EPS [33,34]. Wongsuphachat et al. [35] found that W. confusa NH02 produced 18 g/L EPS in MRS broth with 40 g sucrose at 37 °C. Our results, although slightly higher (23.4 g/L in solid media) were in agreement with the literature [35]. Solid media are easy to use to determine the EPS producing potential of a culture and are a good choice. However, Jin et al. [36] have recently reported that Weissella confusa VP30 from young children's feces produced much higher amounts (59.9 g/L) of dextran in liquid medium. In traditional boza production, the viscosity is modified by adding hot water if necessary by the manufacturer. Therefore, in our previous study, an attempt was made to choose a non-EPS producing species as adjunct culture in boza production [7]. However, the textural and sensory characteristics of boza were weak and the liquid phase separated during storage when non-EPS producing species were used. Thus, in this study we concentrated on the possibility of using either EPS-

35

0

(at 10s−1)

Oat

Maize

mMRS

Fig. 5. The rheological properties of dextrans obtained from different cereal-based media.

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Z.D. Heperkan et al. / International Journal of Biological Macromolecules 143 (2020) 305–313 Table 4 The change of pH on different media inoculated with Weisella confusa C19. Media

Day 1

Day 3

Maize Oat Rice Wheat mMRS

4.0 4.1 4.1 4.1 4.6

4.1 4.1 4.0 4.1 4.5

311

producing species or EPS from certain species to obtain better textural properties in boza. The total phenolic content of dextran from W. confusa C19 was similar in different media ranging from 0.9 to 1.1 g/L (Table 2). Among various natural antioxidants, polysaccharides in general, have strong antioxidant activities and can be explored as novel potential antioxidants [37,38].

Fig. 6. The colony on solid media and microscopical appearences of Weissella confusa C19 cells. A and B: MRS agar at 37 °C for 24 h, C and D: mMRS agar at 37 °C for 24 h, E and F: mMRS agar at 37 °C for 72 h, G and H: wheat-based medium at 37 °C for 72 h.

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3.5. Avarage molecular weight and density

4. Conclusion

The molecular weight of dextran from W. confusa C19 was found to be 204.966 g/mol is considered as high molecular weight dextran. The avarage density of dextran obtained from W. confusa C19 was measured as 1.5185 ± 0.0021 g/cm3.

The ability of producing EPS is an important property of lactic acid bacteria which should be considered in manufacturing fermented beverages. In cereal-based beverages like boza, the rheological properties of the end product are influenced by raw materials and the endogenous microorganisms, therefore the EPS production of a particular bacterium in a particular raw material (type of cereal) should be considered. The textural characteristics and thereby the viscosity of boza can change due to the production of EPS by W. confusa from different cereals as raw materials. Thus, the change in formulation for standard boza production may be necessary depending on the type of raw material and the bacterium. Water-soluble dextran was characterized using the combination of different methods and approaches in this study. EPS production by microorganisms are preferred in food related industries due to several advantages. Mainly liquid media prepared from different raw materials are used in fermenters under controlled conditions both in laboratories and the industry, worldwide. However, problems such as gelling and foaming during fermentation make their commercialization difficult. Thus, new approaches are needed to overcome these hurdles. Using solid media to collect EPS is the challenge in this study. Solid media allow observation, collection and purification of EPS produced during fermentation more easily. Although the aim of the study was not to compare the yield of solid and liquid media, it is worth to highlight that the yield of EPS (dextran) is considerably higher in solid media. Since cereals can be used in EPS production as assessed in this study, cereal-based wastes from bakery, pasta as well as catering and other food sectors can be utilized as cheap raw material for media preparation in EPS production. W. confusa C19 is a unique strain that can adapt to the environment containing high sugar and thereby produces high amount of dextran. Since the dextran from W. confusa C19 has α(1–6) linkages it can be utilized in the industry to improve rheological and processing characteristics of different products.

3.6. The rheological properties of dextran The rheological properties of dextran obtained from different cerealbased media are shown in Fig. 5. The steady shear behavior of dextran was pseudoplastic and was described by a power law model. The rheological parameters, K and n, are presented in Table 3. The calculated apparent viscosities of the samples at 10 s−1 and 100 s−1 are also given in Table 3. The viscosity of dextran obtained from maize medium was significantly higher (p b .05) than the dextran obtained from other cereals. The viscosity of dextran from rice medium was similar to the viscosity from mMRS as shown in Fig. 5. However, the differences between samples were significant (p b .05). 3.7. pH of media and kinetic studies The pH of media inoculated with W. confusa was determined in cereal-based liquid media (similar formulae without agar) during the incubation period (Table 4). The pH of media was 6.4 at the beginning of fermentation, reduced down to 4.0–4.1 after 24 h and did not change afterwards during 3 days of incubation. The pH of cereal-based media used in the study showed similar trends for the final pH. The pH was higher in mMRS than cereal-based media. The weak acidification properties of W. confusa were also reported in foods such as sourdoughs previously [9]. The number of W. confusa on mMRS agar decreased with increasing incubation time e.g. the number of bacteria for the first day was 2.109 cfu/g, the second day was 6.107 cfu/g and the third day was 2.105 cfu/g. The number of bacteria decreased gradually day by day in mMRS agar; however after 72 h of incubation on cereal based media neither colonies nor living cells were determined. The microscopic appearances of W. confusa on MRS, mMRS and on cereal-based media during incubation period were shown in Fig. 6A-H. After 24 h of incubation on MRS agar, W. confusa appeared as typical small, white to creamishcoloured colonies (Fig. 6A). Correspondingly their cells were Grampositive with non-endospore forming, rod-shaped and coccobacilli, appeared in single or short or long chains (Fig. 6B). After transferred to mMRS agar the colonies became larger and fluent (Fig. 6C) but still kept their original cell shape (Fig. 6D). The EPS around the cells appeared as thick clear zones. After 72 h of incubation on mMRS agar, the colonies were fairly large, cream-coloured and irregularly shaped (Fig. 6E). The cells that lost their original shape, vary widely in shape and size, some are very large, whereas others small; the homogeneity in chain was disrupted. After 72 h of incubation on wheat-based medium, the colonies were very large, over 100 mm (Fig. 6G). They were colorless, transparent, not slimy but fluent. The cells lost their shape and the structure of the chain was disrupted. Cell walls between individual cells in a chain were broken and the content were fused or released to the environment; the cells bursted and were completely destroyed (Fig. 6H). High sugar content may be the reason of cells to burst and thus neither colonies nor alive cells were determined on cereal-based media at the end of the incubation period. The decrease in EPS production during stationary phase of growth by different LAB strains in different media were reported previously [39–41]. Correspondingly, this decrease in EPS production by L. plantarum and L. helveticus after prolonged incubation was attributed to the possible presence of glycohydrolases in the culture that catalyzed the degradation of polysaccharides [42,43]. Sugar adapted cells formed a giant colony and thereby high amounts of EPS which is a unique characteristic of W. confusa C19 assessed in this study.

CRediT authorship contribution statement Z. Dilek Heperkan:Conceptualization, Project administration, Supervision, Writing - original draft, Writing - review & editing, Data curation, Funding acquisition, Visualization, Investigation.Meltem Bolluk:Methodology, Formal analysis, Resources, Visualization, Software.Sedef Bülbül:Methodology, Formal analysis, Validation. Declaration of competing interest Authors declare that there are no conflicts of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements The authors wish to thank Seyma Tufekyapan for supporting NMR analysis and Dr. Fusun Seyma Gungor for her valuable evaluation of the NMR analysis. References [1] J.P. Wynn, A.J. Anderson, Microbial polysaccharides and single cell oils, in: C. Ratledge, B. Kristiansen (Eds.), Basic Biotechnology, Cambridge University Press, London 2006, pp. 381–384. [2] W. Li, X. Xia, W. Tang, J. Ji, X. Rui, X. Chen, M. Jiang, J. Zhou, Q.M. Zhang, J. Dong, Structural characterization and anticancer activity of cell-bound exopolysaccharide from Lactobacillus helveticus MB2-1, Agric. Food Chem. 63 (2015) 3454–3463. [3] S.K. Tinzl-Malang, P. Rast, F. Grattepanche, J. Sych, C. Lacroix, Exopolysaccharides from co-cultures of Weissella confusa 11GU-1 and Propionibacterium freudenreichii JS15 act synergistically on wheat dough and bread texture, Int. J. Food Microbiol. 214 (2015) 91–101. [4] J.A. Björkroth, W.H. Holzapfel, Genera Leuconostoc, Oenococcus and Weissella, in: M. Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, E. Stackebrandt (Eds.), The Prokaryotes, Springer, New York 2006, pp. 267–319.

Z.D. Heperkan et al. / International Journal of Biological Macromolecules 143 (2020) 305–313 [5] M.D. Collins, J. Samelis, J. Metaxopolous, S. Wallbanks, Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella from the Leuconostoc paramesenteroides group of species, J. Appl. Bacteriol. 49 (1993) 405–413. [6] J. Owusu-Kwarteng, F. Akabanda, D.S. Nielsen, K. Tano-Debrah, R.L.K. Glover, L. Jespersen, Identification of lactic acid bacteria isolated during traditional fura processing in Ghana, Food Microbiol. 32 (2012) 72–78. [7] D. Heperkan, C. Daskaya Dikmen, B. Bayram, Evaluation of lactic acid bacterial strains of boza for their exopolysaccharide and enzyme production as a potential adjunct culture, Process Biochem. 49 (2014) 1587–1594. [8] V. Fusco, G.M. Quero, G. Stea, M. Morea, A. Visconti, Novel PCR-based identification of Weissella confusa using an AFLP-derived marker, Int. J. Food Microbiol. 145 (2011) 437–443. [9] K. Katina, N.H. Maina, R. Juvonen, L. Flander, L. Johansson, L. Virkki, M. Tenkanen, A. Laitila, In situ production and analysis of Weissella confusa dextran in wheat sourdough, Food Microbiol. 26 (2009) 734–743. [10] J.A. Björkroth, L.M.T.D. Dicks, A. Endo, The genus Weissella, in: W.H. Holzapfel, B.J.B. Wood (Eds.), Lactic Acid Bacteria, Biodiversity and Taxonomy, Wiley Blackwell, Chichester 2014, pp. 418–428. [11] R. Albesharat, M.A. Ehrmann, M. Korakli, S. Yazaji, R.F. Vogel, Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies, Syst. Appl. Microbiol. 34 (2011) 148–155. [12] V. Fusco, G.M. Quero, G.S. Cho, J. Kabisch, D. Meske, H. Neve, W. Bockelmann, C.M.A.P. Franz, The genus Weissella: taxonomy, ecology and biotechnological potential, Front. Microbiol. 155 (2015) 1–22. [13] M. Amari, S. Laguerre, M. Vuillemin, H. Robert, V. Loux, C. Klopp, S. Morel, B. Gabriel, M. Remaud-Siméon, V. Gabriel, C. Moulis, C. Fontagné-Faucher, Genome sequence of Weissella confusa LBAE C39-2, isolated from a wheat sourdough, J. Bacteriol. 12 (2011) 1608–1609. [14] J. Walter, C. Hertel, G.W. Tannock, C.M. Lis, K. Munro, W.P. Hammes, Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis, Appl. Environ. Microbiol. 67 (2001) 2578–2585. [15] M.R. Fairfax, P.R. Lephart, H. Salimnia, Weissella confusa: problems with identification of an opportunistic pathogen that has been found in fermented foods and proposed as a probiotic, Front. Microbiol. 5 (2014) 254. [16] R.Z. Ahmed, K. Siddiqui, M. Arman, N. Ahmed, Characterization of high molecular weight dextran produced by Weissella cibaria CMGDEX3, Carbohydr. Polym. 90 (2012) 441–446. [17] N.H. Maina, M. Tenkanen, H. Maaheimo, R. Juvonen, L. Virkki, NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confuse, Carbohydr. Res. 343 (2008) 1446–1455. [18] P. Decock, S. Cappelle, Bread technology and sourdough technology, Trends Food Sci. Tech. 16 (2005) 113–120. [19] C. Ruehmkorf, H. Ruebsam, T. Becker, C. Bork, K. Voiges, P. Mischnick, M.J. Brandt, R.F. Vogel, Effect of structurally different microbial homoexopolysaccharides on the quality of gluten-free bread, Eur. Food Res. Technol. 235 (2012) 139–146. [20] T. Smitinont, C. Tansakul, S. Tanasupawat, S. Keeratipibul, L. Navarini, M. Bosco, P. Cescutti, Exopolysaccharide producing lactic acid bacteria strains from traditional Thai fermented foods: isolation, identification and exopolysaccharide characterization, Int. J. Food Microbiol. 51 (1999) 105–111. [21] G.H. Van Geel-Schutten, F. Flesch, B. ten Brink, M.R. Smith, L. Dijkhuizen, Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides, Appl. Microbiol. Biotechnol. 50 (1998) 697–703. [22] R.L. Heinrikson, S.C. Meredith, Amino acid analysis by reverse-phase highperformance liquid chromatography: precolumn derivatization with phenylisothiocyanate, Anal. Biochem. 136 (1984) 65–74. [23] N. Miceli, A. Trovato, A. Marino, V. Bellinghieri, A. Melchini, P. Dugo, F. Cacciola, P. Donato, L. Mondello, A. Güvenç, R. De Pasquale, M.F. Taviano, Phenolic composition

[24]

[25]

[26] [27] [28]

[29]

[30] [31] [32] [33] [34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

313

and biological activities of Juniperus drupacea Labill. Berries from Turkey, Food Chem. Toxicol. 49 (2011) 2600–2608. S. Shukla, Q. Shi, N.H. Maina, M. Juvonen, A. Maijatenkanen, A. Goyal, Weissella confusa Cab3 dextransucrase: properties and in vitro synthesis of dextran and glucooligosaccharides, Carbohydr. Polym. 101 (2014) 554–564. A. Malik, M. Radji, S. Kralj, L. Dijkhuizen, Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two different Weissella confusa strains from soya, FEMS Microbiol. Lett. 300 (2009) 131–138. A. Synytsya, M. Novak, Structural analysis of glucans, Ann. Transl. Med. 2 (2014) 1–14. N. Shang, R. Xu, P. Li, Structure characterization of an exopolysaccharide produced by Bifidobacterium animalis RH, Carbohydr. Polym. 91 (2013) 128–134. R.K. Purama, P. Goswami, A.T. Khan, A. Goyal, Structural analysis and properties of dextran produced by Leuconostoc mesenteroides NRRL B-640, Carbohydr. Polym. 76 (2009) 30–35. K.I. Shingel, Determination of structural peculiarities of dextran, pullulan and cirradiated pullulan by Fourier-transform IR spectroscopy, Carbohydr. Res. 337 (2002) 1445–1451. K.E. Bach Knudsen, Fiber and nonstarch polysaccharide content and variation in common crops used in broiler diets, Poult. Sci. 93 (2014) 2380–2393. D.M.D.L. Navarro, J.J. Abelilla, H.H. Stein, Structures and characteristics of carbohydrates in diets fed to pigs: a review, J Animal Sci Biotechnol 10 (2019) 1–13. P. Koehler, H. Wieser, Chemistry of cereal grains, in: M. Gobbetti, M. Ganzle (Eds.), Handbook of Sourdough Biotechnology, 2013, Springer, New York 2013, pp. 11–45. A. Patel, J.B. Prajapati, Food and health applications of exopolysaccharides produced by lactic acid bacteria, Adv. Dairy Res. 1 (2013) 107–114. A.T. Adesulu-Dahunsi, A.I. Sanni, K. Jeyaram, J.O. Ojediran, A.O. Ogunsakin, K. Banwo, Extracellular polysaccharide from Weissella confusa OF126: production, optimization, and characterization, Int. J. Biol. Macromol. 111 (2018) 514–525. W. Wongsuphachat, A. H-Kittikun, S. Maneerat, Optimization of exopolysaccharides production by Weissella confusa NH 02 isolated from Thai fermented sausages, J. Sci. Technol. 1 (2010) 27–35. H. Jin, Y. Jeong, S. Yoo, T.V. Johnston, S. Ku, G.E. Ji, Isolation and characterization of high exopolysaccharide-producing Weissella confusa VP30 from young children’s feces, Microb. Cell Factories 18 (2019) 110 1–13. S. Guo, W. Mao, Y. Li, Q. Gu, Y. Chen, C. Zhao, N. Lia, C. Wang, T. Guo, X. Liu, Preparation, structural characterization and antioxidant activity of an extracellular polysaccharide produced by the fungus Oidiodendron truncatum GW, Process Biochem. 48 (2013) 539–544. J.L. Wang, J. Zhang, B.T. Zhao, X.F. Wang, Y.Q. Wu, J. Yao, A comparison study on microwave-assisted extraction of Potentilla anserina L. polysaccharides with conventional method: molecule weight and antioxidant activities evaluation, Carbohydr. Polym. 80 (2012) 84–93. W. Li, J. Ji, X. Rui, J. Yu, W. Tang, X. Chen, M. Jiang, M. Dong, Production of exopolysaccharides by Lactobacillus helveticus MB2–1 and its functional characteristics in vitro, LWT-Food Sci. Technol. 59 (2014) 732–739. K. Wang, W. Li, X. Rui, X. Chen, M. Jiang, M. Dong, Characterization of a novel exopolysaccharide with antitumor activity from Lactobacillus plantarum 70810, Int. J. Biol. Macromol. 63 (2014) 133–139. L. Zhang, C. Liu, D. Li, Y. Zhao, X. Zhang, X. Zeng, Z. Yang, S. Li, Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88, Int. J. Biol. Macromol. 54 (2013) 270–275. B. Degeest, F. Mozzi, L. De Vuyst, Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations, Int. J. Food Microbiol. 79 (2002) 161–174. J. Wang, X. Zhao, Z. Tian, C. He, Y. Yang, Z. Yang, Isolation and characterization of exopolysaccharide-producing Lactobacillus plantarum SKT109 from Tibet kefir, Pol. J. Food Nutr. Sci. (4) (2015) 269–279.