Yiya Wang, Tao Sun, Yinzhu Wang, Hao Wu, Yan Fang, Jiangfeng Ma, Min Jiang

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China

Keywords:Exopolysaccharide Glucansucrase Leuconostoc pseudomesenteroides Insoluble α-glucan α-1,3 glycosidic bond

A B S T R A C T Exopolysaccharides can be produced by various bacteria and have important biological roles in bacterial survival depend on molecular weight,linkage,and conformation.In this study,Leuconostoc pseudomesenteroides G29 was identified and found to produce two types of exopolysaccharides from sucrose including soluble and insoluble α-glucans. By regulation of pH above 5.5, soluble α-glucan production was increased to 38.4 g·L-1 from 101.4 g·L-1 sucrose with fewer accumulation of lactic acid and acetic acid.Simultaneously, the quantity of thick white precipitate, that is insoluble α-glucan, was also increased.Then,α-glucans were prepared by enzymatic reaction with crude glucansucrases from the supernatant of G29 fermentation broth and purified for structure analysis. Based on the integration analysis of FT-IR and NMR, it was observed that soluble α-glucan is a highly linear dextran with α-1,6 glycosidic bonds while the insoluble α-glucan has 93%of α-1,3 and 7%of α-1,6 glycosidic bond. The results extend our understanding of exopolysaccharides production by L.pseudomesenteroides,and this water insoluble α-1,3-glucan might have potential application as biomaterials and/or biochemicals.

1. Introduction

α-glucans are homo-polysaccharides consisted of glucose monomers with α-glycosidic bonds. These polysaccharides with the molecular formula of (C6H10O5)ninclude diverse structures,which can be short or long, branched or unbranched, soluble or insoluble due to the ratio of glycosidic bonds (1,3-, 1,4-, or 1,6-)and their molecular weight. Generally, α-glucans are classified to four main types including dextran (α-1,6 linkage), mutan (α-1,3 linkage), reuteran (α-1,4/1,6 linkage), and alternan (α-1,3/1,6 linkage) [1]. Depending on the bonding pattern, α-glucans have distinct properties and applications [2].

α-glucans mainly with α-1,3 linkage are generally waterinsoluble due to the strong hydrogen bond formation, whereas α-glucans consisting of a high level of α-1,6 linkages are conversely freely soluble in water[3].Leuconostocspecies are the most prevalent lactic acid bacteria (LAB), which can primarily produce exopolysaccharides (EPS) in the form of water soluble α-glucans.Previous studies have reported several EPS-producingLeuconostoc pseudomesenteroides, such as strain RJ-5, XG5, YF32 and YB-2 [4].

Although insoluble α-glucans are not yet fully understood and their utility is limited due to its low water solubility, they have potential application as biomaterials and biochemicals. It exhibits antibacterial and anti-inflammatory effects in addition to their potential applications in biomedicine, such as gel and film use[5,6].Moreover,α-1,3 glucans are crucial components of fungal cell walls. The mutant ofSchizosaccharomyces pombe(with a mutation in the gene responsible for α-glucan biosynthesis) has been found to be sensitive to temperature and its cell wall is lysed at temperatures above 37 °C [7]. Wanget al.reported that α-1,3 glucan was capable of immobilizing enzymes, giving them more stability based on the free hydroxyl groups [8]. The US Patent 2018/0258590A1 published that insoluble α-1,3 glucan could be used as excellent ink receptive layer to coating on paper. In addition, modified α-glucans such as carboxymethylated or sulphated derivatives are soluble in water and have potent prebiotic properties [9].

Glucansucrases, catalyzing the reaction of sucrose to glucans,belong to CAZy glycoside hydrolase family 70 (GH70), and they have been identified mainly from LAB includingLeuconostoc,Streptococcus,WeissellaandLactobacillus [10,11]. Generally, glucansucrases include GTF-S which synthesizes water-soluble α-glucans,and GTF-I which synthesizes water-insoluble α-glucans. The former α-glucans contain mainly α-1, 6 linkages and are considered dextrans.L. mesenteroidesNRR B-512F was reported to synthesize dextran with 95% α-1, 6 linkages in the linear main chains and 5% α-1, 3 linkages in the branching [12]. The latter insoluble α-glucans are due to the long continuous blocks of α-1, 3 linkages in its backbone.TheS.mutans GS5strain produces two similar glucansucrases(GTF-B and GTF-C).GTF-B produced a water-insoluble α-glucan with 88% percentage of α-1, 3 linkages and 12% α-1, 6 linkages, and GTF-C also synthesizes a water-insoluble α-glucan with 85% of α-1, 3 and 15% of α-1, 6 linkages [13].

Recently,Streptococcus mutansGS5,S. mutansMT8148,L. citreum ABK-1andS. sobrinushave been reported to have both GTF-S and GTF-I, producing water soluble and insoluble α-glucans simultaneously [3]. In this study, we identified a LAB strainL.pseudomesenteroidesG29,and found it produces two types of exopolysaccharides from sucrose including soluble and insoluble α-glucans. Hence, these two α-glucans were prepared by enzymatic reaction with crude glucansucrases from G29 fermentation broth, and purified for molecular weight and structural analysis by GPC, FT-IR and NMR.

2. Materials and Methods

2.1. Materials

Sucrose, fructose, and mannitol (analytical grade) was purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). The Taq DNA polymerase, dNTP mixtures, and Taq reaction buffer for PCR were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China).All other chemicals were reagent grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Man-Rogosa-Sharpe (MRS) medium was adopted for the fermentation of G29, and it is composed of 10 g·L-1peptone,5 g·L-1yeast extract, 2 g·L-1ammonium citrate, 0.1 g·L-1magnesium sulfate,0.05 g·L-1manganese sulfate,2 g·L-1disodium phosphate [14].

2.2. Strain and growth conditions

Previously,more than 20 strains with high glucansucrase activity were screened from homemade kimchi fermentation broth and stored in our laboratory.Here, strain G29,which produces viscous exopolysaccharides and quantity of thick white precipitate from sucrose, was identified and used in the present study.

2 ml of strain stored in the glycerol was inoculated to 200 ml anaerobic bottles containing MRS medium as seed.Approximately 20 g·L-1sucrose was supplemented separately as the carbon source. Anaerobic conditions were obtained by bubbling nitrogen gas through the medium for 2 minutes.The fermentation was carried out at 30°C on a rotary shaker(200 r·min-1)for 12 h and then used as a seed culture.

Batch fermentation was performed in a 3L fermenter with sucrose as carbon source and substrate.The modified MRS medium was composed of 10 g·L-1peptone, 5 g·L-1yeast extract, 4 g·L-1ammonium citrate, 0.2 g·L-1magnesium sulfate, 0.1 g·L-1manganese sulfate and 4 g·L-1disodium phosphate. A concentrated sucrose solution was added to the final concentration of 100 g·L-1. The initial pH was set at 7.0, and the pH was adjusted to 5.0 or 5.5 or without adjustment. The fermentations were carried out at 30°C with an agitation speed of 200 r·min-1.The anaerobic conditions were obtained by bubbling nitrogen gas at 0.1 L·min-1.

2.3. Phylogenetic analysis

Strain G29 was cultivated in MRS medium and the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers 27F and 1492R [15]. DNA sequencing was performed at the Shanghai Personal Biotechnology Co., Ltd. The sequence results were compared with the NCBI database using a BLAST algorithm. The phylogenetic analyses were performed by neighbor-joining with MEGA Software [16,17]. The resulting tree topology was evaluated using the bootstrap method with 1000 iterations.

2.4. Glucansucrase activity analysis

The 3,5-dinitrosalicylic acid method was used to measure the total and supernatant enzyme activity of the fermentation broth to determine whether the α-glucans was produced extracellularly by glucansucrases[18].One unit of enzyme activity was defined as the amount of enzyme that can release 1 μmole of fructose within 1 minute, and fructose was used as a standard for the calibration curve [1].

2.5. Purification and collection of the α-glucan for structure analysis

Anaerobic fermentations with approximately 80 g·L-1sucrose were carried out with G29 and the supernatant was collected to recycle the glucansucrases after 12 h fermentation. Crude glucansucrases were obtained by ammonium sulphate precipitation followed with dialysis. Then, 100 g·L-1sucrose was incubated with crude glucansucrases in 50 mmol·L-1acetate buffer pH 5.5 containing 1 mmol·L-1CaCl2in 100 ml total volume at 30 °C overnight, using 0.5 units of crude enzyme per ml of reaction mixture. Reactions were halted by 30 minutes of incubation at 100 °C. After that, the solution was centrifuged at 6000 r·min-1for 5 minutes to recover the insoluble α-glucan. And the precipitate was subsequently washed with 50 ml deionised water for 3 times and the pellet was lyophilized. The soluble α-glucan was then extracted from the supernatant. The soluble α-glucan was precipitated by adding 300 ml ethanol and the pellet was then collected by centrifugation at 8000 r·min-1for 10 minutes. Finally,the precipitate was subjected to freeze drying to obtain purified soluble α-glucan. The purified glucans were stored in the desiccator at room temperature for further analysis.

2.6. Analytical methods of substrate and products

The sugars and products were analyzed by high performance liquid chromatography(HPLC)[19]. The samples were centrifuged at 12,000 r·min-1for 1 minute to collect the supernatant and then filtered by a 0.2 μm nylon filter prior to analysis.Sucrose,fructose and mannitol were measured with a BP-100 Pb2+column.The temperature was maintained at 80°C,and ultra-pure water with a flow rate of 0.6 ml·min-1was used as an eluent. The organic acids and ethanol were determined using HPLC with an ion exclusion column(BIO-RAD,Aminx,HPX-87H,USA)with UV and refractive index(RI)detectors. The mobile phase used was 0.5 g·L-1sulfuric acid and the flow rate was 0.6 ml·min-1at 55 °C.

For analyzing the concentration of soluble α-glucan, the purified soluble α-glucan from enzymatic reaction was dried to constant weight as the α-glucan standard. Gel permeation chromatography (GPC)was used to analyze the α-glucan samples,and we have constructed the curve(Fig.S1).The concentration(X)of α-glucan can be calculated by the peak area (Y) with the formula:Y= 0.17X+ 0.0029.

As the molecular weight (Mw) range of α-glucan samples were unknown, these two kinds of α-glucans were analyzed through a third party(Shiyanjia Lab,www.shiyanjia.com).Waters 1525 with 2414 detector was used for GPC analyzed by PL aquqgel-OH MIXED 8 μm column at 30 °C, and the mobile phase was 0.2 mol·L-1NaNO3and 0.01 mol·L-1NaH2PO4(pH 7.0)at a rate of 1 ml·min-1.

2.7. Spectroscopy methods

The FT-IR spectra was measured using a Thermo Fisher IS5 Test,and specimens of polysaccharide film were prepared by the KBr-disk method.32 scans at a resolution of 4 cm-1were collected.NMR spectroscopy analysis was performed using 20 mg of sample dissolved in D2O (water-soluble α-glucan) or 1 mol·L-1NaOD in D2O (water-insoluble α-glucan) and mixed until dissolved. The type and ratio of the glycosidic bonds were analyzed according to the1H and13C NMR spectra [14].

3. Results and Discussion

3.1. Phylogenetic analyses of strain G29

Firstly,phylogenetic analyses of strain G29 was carried out,and its 16S rRNA gene was sequenced(Table S1)and submitted to NCBI(MN809372.1). Although 16S rRNA gene sequence should not be used as the sole criterium for species delineation, it could provide the first evidence for a potential novel species [20]. The 16S rRNA gene sequence revealed that G29 exhibited the highest similarity withL. pseudomesenteroidesstrain FDAARGOS 1003. The phylogenetic relationship of G29 and otherLeuconostoscspecies is shown in Fig. 1. Thus, we certified that strain G29 belongs toL. pseudomesenteroides.

3.2. Batch fermentation with sucrose by strain G29

AsL. pseudomesenteroideshas been reported to produce EPS,mainly dextran, from sucrose [9]. Anaerobic fermentations with approximately 100 g·L-1sucrose were carried out to investigate the ability of α-glucan synthesis from sucrose by strain G29. As shown in Fig. 2, sucrose could be exhausted within 8 hours with concomitant production of fructose and soluble α-glucan which lead to the increased viscosity of broth. In addition, the produced fructose could be further metabolized to mannitol, which may be due to the catalytic activity of mannitol dehydrogenase. The fructose was exhausted at 8 h, and 29.8 g·L-1mannitol and 30.5 g·L-1soluble α-glucan were obtained at the end of the fermentation. Since pH was not adjusted, it dropped gradually from pH 7.0 to pH 4.1. Therefore, the low pH would obviously inhibit the growth of cell, notably when pH was lower than 4.5.

At the end of fermentation, the total glucansucrase activity of the broth after ultrasonication and the extracellular glucansucrase activity were determined, and the results showed that they were(1.45 ± 0.02) and (1.43 ± 0.03) U·ml-1, respectively. It indicated that glucansucrases produced by strain G29 were exoenzymes and thus the α-glucans were synthesized extracellularly.

In addition, it was interesting to note that a large quantity of thick white precipitate was present in the broth when pH decreased below 6.0. As reported, it should not appear quantity of water insoluble product since mannitol and majority of the α-glucans are water soluble [21]. Therefore, we speculated that strain G29 might synthesize one type of glucan with different structure leading to water insolubility.

3.3. Fermentation by pH adjustment to enhance the production of αglucans

When the strain G29 were fermented without pH adjustment,the pH dropped gradually to 4.1, leading to the inhibition of cell growth. Therefore, the batch fermentations were carried out by adjusting the pH above pH 5.5 and 5.0. As shown in Fig. 2B, 3B and 3D, the cell density (OD600) was both increased when the pH was regulated,and the maximum OD600(11.1)was obtained when the pH was adjusted above 5.5. In addition, the sucrose consumption rates were improved compared with that without pH adjustment. Sucrose were depleted within 6 and 7 h under pH above 5.5 and 5.0, respectively.

Fig. 1. Phylogenetic tree of L. pseudomesenteroides G29 using the neighborjoining method based on its 16S rRNA gene sequence.

When the pH was above 5.5,the produced fructose could be further metabolized and exhausted at 10 h, and 38.4 g·L-1soluble α-glucan and 42.4 g·L-1mannitol were obtained at the end of the fermentation (Fig. 3A). In contrast, cell growth and sucrose consumption rate were not significantly enhanced when pH was above 5.0, and only 29.6 g·L-1mannitol and 32.1 g·L-1soluble α-glucan were obtained from 99.1 g·L-1sucrose (Fig. 3C).Therefore, the yield of mannitol plus soluble α-glucan to the consumed sucrose was significantly decreased under pH ＞5.0 compared to that under pH ＞5.5. This might be due to the increased production of lactic acid and/or acetic acid to enhance the energy supplement[22].Thus,the organic acids production was analyzed,showing that higher concentration of lactic acid (18.14 g·L-1) and acetic acid (5.92 g·L-1) were accumulated without pH adjustment compared to those with pH regulation (Table 1). Moreover, when the pH was maintained above 5.5, only 3.11 g·L-1lactic acid and 1.29 g·L-1acetic acid were accumulated, which were lower than those when pH was regulated above 5.0. Fig. 4 is the metabolic pathways ofL. pseudomesenteroides, showing that sucrose could be catalyzed extracellularly by glucansucrase without energy consumption, and the released fructose was transported into cell for metabolism and mannitol production. On the other hand, sucrose could be transported and metabolized for cell growth and organic acids production. Theoretically, the reactions from sucrose to αglucan, mannitol/ethanol consume energy, while the reactions from sucrose to lactic acid and acetic acid produce ATP and NADH.Combined with that the lower pH lead to higher accumulation of lactic and acetic acid, we speculated that more ATP was produced to help the strain resisting to acidic stress. This is consistent with our previous work, which demonstrated that intracellular ATP production was enhanced when the bacteria suffered with acidic condition [23]. In conclusion, maintaining the pH above 5.5 was the optimal condition for production of soluble α-glucan and mannitol.

Table 1 Organic acids and alcohol production under different pH strategies

Fig. 2. Batch fermentation with L. pseudomesenteroides G29 from sucrose without pH adjustment. (A) Consumption of sucrose and production of mannitol, fructose and glucan; (B) pH and cell growth.

Fig. 3. Batch fermentation with L. pseudomesenteroides G29 from sucrose with pH adjustment (A) Consumption of sucrose and production of mannitol, fructose and glucan above pH 5.5;(B)pH and cell growth above pH 5.5;(C)Consumption of sucrose and production of mannitol,fructose and glucan above pH 5.0;(D)pH and cell growth above pH 5.0.

In addition, the quantity of thick white precipitate was further increased in the broth when the pH was regulated,especially when the pH was controlled above 5.5.Recently,some glucan-type insoluble exopolysaccharides have been synthesized by microorganisms, such as pullulan byAureobasidium pullulans, curdlan byAgrobacterium spp.and α-1,3 glucans byLeuconostoc spporStreptococcus spp[24]. Thus, we speculate the thick white precipitate might be one type of glucan synthesized from sucrose.

3.4. Preparation and analysis of α-glucan by enzymatic synthesis

3.4.1. Preparation and purification

As the insoluble α-glucan was difficult to separate from the cells,anaerobic fermentations with approximately 80 g·L-1sucrose were carried out by strain G29 with pH adjustment above 5.5,and the supernatant was collected to recycle the glucansucrases after 12 h fermentation. Enzymatic reactions were performed at 30 °C with 100 g·L-1sucrose and 1 mmol·L-1CaCl2in 50 mmol·L-1sodium acetate buffer at pH 5.5 overnight, using 0.5 units of enzyme per milliliter of reaction mixture, and halted by 30 minutes of incubation at 100 °C. Similarly, both soluble and insoluble α-glucan could be obtained by enzymatic reaction (Fig. S2), and the concentration of soluble α-glucan reached 38.55 g·L-1. Then soluble and insoluble α-glucan were separated and collected for further analysis. Finally, 2.64 g soluble α-glucan and 0.98 g insoluble α-glucan were obtained from 100 ml enzymatic reaction mixture. Thus, the yield of insoluble α-glucan was higher than 9.8%.

3.4.2. Molecular weight estimation

The GPC results showed a single, symmetrical, narrow peak,confirming the homogeneity of the soluble α-glucan (Fig. 5).According to the calibration curve from Shiyanjia Lab, theMwof soluble α-glucan is estimated to be 552,466 Da. Since insoluble α-glucan is hardly soluble in water, the molecular weight is measured by dissolving with DMSO. TheMwof insoluble α-glucan is estimated to be 3337 Da (Fig. 5).

3.4.3. FT-IR analysis

The FT-IR spectra of the water-soluble α-glucan and waterinsoluble α-glucan in the range of 4000-500 cm-1resembled those of dextran and mutan are shown in Fig. 6 [25,26]. The spectra showed absorption band of O-H stretching, C-H stretching and adsorbed water,at 3400 cm-1, 2939 cm-1and 1640 cm-1, respectively. FTIR absorption bands in the region 1200-700 cm-1corresponded to of C-O-C and C-O stretching and C-C-H, C-O-H,and H-C-O deformation, which is unique to sugar rings and glycosidic linkage [14,27].

Thus, several observed differences were associated with the linkage fingerprint. Dextran with mainly α-1,6-linkages showed unique bands at 1102 cm-1, 915 cm-1, and 761 cm-1while α-1,3 linked mutan showed specific bands at 928 cm-1, 901 cm-1and 819 cm-1which was the signals of contiguous α-1,3 linkage[12,26]. As shown in Fig. 6, the spectrum indicated that watersoluble α-glucan was similar to the fingerprint bands of dextran while those of water-insoluble α-glucan and mutan were similar.This was further confirmed by1H and13C NMR analysis.

Fig. 4. Metabolic pathways of L. pseudomesenteroides G29 with sucrose as sole carbon source.

3.4.4. NMR analysis

Subsequently, the structural characterization of the α-glucan was performed by NMR spectroscopic analysis, and the1H and13C NMR spectra are shown in Fig. 7.1H and13C NMR spectra of water-soluble glucan showed a single set of signals, and resonances were in good agreement with linear α-1,6-glucans[28,29]. The positions of anomeric signals at 4.89 and 97.85 in1H NMR and13C NMR spectra are characteristic of α-1,6-glucosidic linkages whereas signals at 65.47 in13C NMR spectra are characteristic of 6-substituted glucopyranose residues [30].No additional peaks were observed in the region of 75-85 indicating that the absence of branched linkages [31]. Thus, the soluble α-glucan from the enzymatic reaction is a highly linear dextran with α-1,6 glycosidic bonds.

Fig. 5. The GPC chromatogram of the water insoluble glucan (A) and soluble glucan (B).

Fig. 6. FT-IR spectra of water insoluble glucan (A) and soluble glucan (B). Water insoluble glucan and soluble glucan are shown in black while mutan and dextran are presented in red.

Fig. 7. 1H (A) and 13C (B) NMR spectrum of insoluble glucan in NaOD-D2O and soluble glucan in D2O.

1H NMR spectra of water-insoluble α-glucan had a high intensity peak at 5.31,indicating that the structure contained anomeric α-1,3-linkage.And three low intensity bands were present at 5.01,5.12/5.17 indicating the branching of some glucose units in the polysaccharide backbone [32].13C NMR spectra of waterinsoluble α-glucan contained two sets of signals,including anomeric carbons at 100.02 and 99.65 indicating that two different types of glucose residues were within the polysaccharide chain.Based on previous work on NMR analysis of α-1,3-linked mutan, three signals within the group of minor resonances of water-insoluble α-glucan can be assigned to α-1,3-linked glucose residues:100.02 (C-1). 82.62 (C-3) and 65.83 (C-6). The rest of the carbon signals of the residues (C-2, C-4 and C-5) appeared in a narrow 71.6-69.4 region, which also encompasses C-2, C-4 and C-5 of 1,6- linked residues [33].

The combination of the1H and13C NMR spectra demonstrated that the structure of this water-insoluble polysaccharide was mainly composed of α-1,3 linked d-glucopyranose units. In addition, based on the integration analysis of signals intensity on1H NMR, it was observed that the insoluble α-glucan from the enzymatic reaction has 93% of α-1,3 and 7% of α-1,6 glycosidic bond.

Until now, the highest ratio of the α-1,3 bond was 88% among the reported insoluble α-glucans containing both α-1,3 and α-1,6 glycosidic bonds (Table 2). This was consistent with the previous studies demonstrating that α-glucans with mostly α-1,6 linkages were water-soluble, while the α-glucans with high ratio of α-1,3 linkages lead to water-insoluble [13].

Table 2 Insoluble α-glucans containing both α-1,3 and α-1,6 lycosidic bonds

4. Conclusions

In this study, we identified the strainL. pseudomesenteroidesG29, which produced expolysaccharides including both soluble and insoluble α-glucan. By regulation of pH above 5.5, soluble αglucan production was increased to 38.4 g·L-1from 101.4 g·L-1sucrose with fewer accumulation of lactic acid and acetic acid.Simultaneously, the quantity of thick white precipitate, that is insoluble α-glucan, was further increased. Then, α-glucans were prepared by enzymatic reaction with crude glucansucrases from the supernatant of G29 fermentation broth and purified for structure analysis.Based on the integration analysis of FT-IR and NMR,it was observed that soluble α-glucan is a highly linear dextran with α-1,6 glycosidic bonds while the insoluble α-glucan has 93% of α-1,3 and 7%of α-1,6 glycosidic bond.The results extend our understanding of exopolysaccharides production byL. pseudomesenteroides, and this water insoluble α-1,3-glucan might have potential application as biomaterials and/or biochemicals. Further work is necessary to certify the glucansucrase and its encoding gene to construct recombinant strain so as to establish an economical process for production of this insoluble α-glucan.Also,biological function, mechanical properties, biodegradability,biocompatibility and other properties of this insoluble α-glucan should be further investigated.

Acknowledgements

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals Foundation (JSBEM2016010), Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture of China.

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.06.020.