**1. Introduction**

Exopolysaccharides (EPSs) are natural biopolymers synthesized by microorganisms and are secreted for various respective functions, such as defense, biofilm formation, pathogenicity, structure, adhesion, etc. [1]. EPSs are long-chain biomolecules with molecular weights ranging from 10 to 30 kD, and are produced during the late exponential and or stationary phase of microbial growth. EPSs are produced in response to environmental stress conditions, such as pH and temperature, and exposure to heavy metals or inhibitors, etc. [2,3]. Biochemically, EPSs are carbohydrate polymers composed of glucose,

**Citation:** Ahuja, V.; Bhatt, A.K.; Banu, J.R.; Kumar, V.; Kumar, G.; Yang, Y.-H.; Bhatia, S.K. Microbial Exopolysaccharide Composites in Biomedicine and Healthcare: Trends and Advances. *Polymers* **2023**, *15*, 1801. https://doi.org/10.3390/ polym15071801

Academic Editor: Alexander Malkin

Received: 24 February 2023 Revised: 27 March 2023 Accepted: 3 April 2023 Published: 6 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

galactose, and rhamnose, accompanied by non-carbohydrate moieties such as proteins, enzymes, nucleic acid, etc. The exact composition may vary with microorganisms and growth conditions.

Based on the biochemical structure and composition, EPSs can be categorized into homo-exopolysaccharides (HOEPSs) and hetero-exopolysaccharides (HEEPSs) [4]. HOEPSs are composed of one type of monosaccharide, such as α-D-glucans, β-D-glucans, fructans, and polygalactans, interlinked with α-1-6, α-1-3, β-1-2, β-1-3, β-2-6, and β-2-1 linkage among the subunits, depending upon the monomeric units. Dextran is one of the wellknown examples of HOEPSs, which is made of glucose interlinked with α-1-6 glucoside linkage. In contrast, HEEPSs are comprised of different sugar monomers, along with their respective derivatives and non-carbohydrate moieties. Succinoglycan is one of HEEPSs present in bacterial biofilm. It is comprised of saccharide-based oligomers in which sugar molecules are derivatized with acyl, pyruvate, and succinic acid [4–6]. Both classes are further recognized as subgroups based on the dominant sugar residues [4]. In recent years, EPSs have gained wide attention for their cost-effective production and diverse applications as pharma and healthcare products, cosmeceutical, nutraceutical, functional food, and biocontrol agents in agriculture [7], oil recovery in petroleum industries [8], heavy metal removal [9], drug delivery, and tissue regeneration and repair [10]. Microbial exopolysaccharides have applications in various sectors, as depicted in Figure 1.

**Figure 1.** Exopolysaccharide composites with natural and synthetic polymers and their application in the health sector.

Most exopolysaccharides have gained attention in healthcare due to their nontoxicity, and biocompatibility, but dextran is the only known exopolysaccharide that has been commercialized in healthcare as a plasma volume compensator [6,11]. Alongside that, the majority of applications in healthcare are still under trial. Prasher et al. [12] used a dextran derivative, i.e., acetylated dextran, as a drug delivery vehicle for the treatment of respiratory disease due to biodegradability, pH sensitivity, high encapsulation efficacy, and the ability to cross the mucosal layer. Yahoum et al. [13] encapsulated metformin hydrochloride in xanthan gum microspheres and found a sustainable release of metformin hydrochloride from the microsphere. The eyes are one of the most sensitive parts of the

body that need special care. EPSs have proven safe and biocompatible for biological systems, which allows their application in ophthalmic formulations. Khare et al. [14] evaluated an ophthalmic solution comprising gellan-gum-based nanosuspension with posaconazole for fungal keratitis. The main bottleneck of using native EPS molecules in commercial products is solubility in different media, bioavailability, degradation, etc. Xanthan, a HEEPS comprising a glucose backbone along with trisaccharide side chains, has poor thermal stability and electrical conductivity and is prone to microbial contamination [15,16]. Curdlan has high immunomodulatory potential, good gelling ability, and thermal stability, but is disadvantaged by the issue of solubility in water [17]. Hyaluronic acid has high water retention, but poor mechanical stability [18,19]. EPS has been blended with other natural biomolecules or synthetic polymers to improve and control the functional features of base EPS molecules, such as solubility, antimicrobial potential, mechanical strength, water retention, etc. The approach has proven beneficial, as a hyaluronic acid (HA) composite has higher mechanical strength and stability. HA composites with poly(Ne-acryloyl L-lysine) have shown a double network structure and is used for the fabrication of a more physiologically relevant 3D in vitro model for breast cancer [20,21]. Similarly, a HA composite with a self-assembling peptide carrying an IKVAV adhesive motif was used for the fabrication of a scaffold for breast cancer [22], and composites with silk fibroin–gelatin and heparan sulfate was used for the scaffold fabrication for cholangiocarcinoma [23]. A chitosan–curdlan composite is characterized by important features of both EPS components, as chitosan forms a fibrillary scaffold, and curdlan provides support to mesenchymal cell adhesion and promotes bone growth [24].

EPS composites have shown a way to overcome the bottlenecks of native EPS molecules by offering 3D-stable and porous architecture, offering more support for cell adhesion and proliferation, and an overexpression of enzymes governing wound healing and mineralization. However, there are a lot of factors impeding the commercialization of composite materials. Among the major challenges, stability and activity after prolonged storage must be addressed for commercialization apart from the cost of the product. The current review summarizes the major applications of EPS composites in healthcare, as well as research challenges.

#### **2. Microbes Producing Exopolysaccharides**

Exopolysaccharides are produced by diverse microorganisms, including yeasts, fungi, and bacteria, utilizing various raw materials (Figure 2). Besides the native physiological role, microbial EPSs also have diverse applications in many industries. Several efforts have been made by various researchers for the production of EPSs (Table 1). In comparison to yeasts and other fungi, probiotic bacteria are commonly used due to non-pathogenicity and categorization of generally regarded as safe (GRAS) [25]. It has been found that carbon source has a direct relation with EPS production and composition. A study on 20 strains of *Lactobacillus paracasei* revealed that with a change in carbon source, not only does EPS yield, but it also influences the monosaccharide composition. The yield of EPS was increased by 115% with optimized carbon sources, including fructose, glucose, galactose, lactose, mannose, and trehalose [26]. Among different carbon sources, including sucrose, maltose, lactose, glycerol, and sorbitol, maximum EPS production has been achieved with maltose by *Candida guilliermondii* and *Candida famata* (0.505 and 0.321, respectively) [27]. Among 156 lactic acid bacteria isolated from healthy young children's feces, the maximum EPS production, i.e., 59.99 g/L, was reported from *Weissella confusa* VP30 after 48 h in growth media containing 10% sucrose [28].

The use of commercial-grade sugars/substrates has a direct impact on product cost, and is one of the factors responsible for high production costs. Hence low-cost waste materials, such as lignocellulosic residues and wastewater, are preferred as raw materials for EPS production. The carbohydrate and organic fractions present in the waste can be used by microorganisms. It opens up the opportunity to reduce product costs, along with waste management. Da Silva et al. [29] have compared coconut shells, cocoa husks, and

sucrose for xanthan gum production by *Xanthomonas campestris* pv. *campestris* IBSBF 1866 and 1867. The study revealed that xanthan gum yields were higher, i.e., 4.48 g/L and 3.89 g/L, in the case of cocoa husk by *Xanthomonas* strains 1866 and 1867, respectively, but the apparent viscosity was higher than sucrose, i.e., 181.88 mPas over cocoa husk, with a viscosity of 112.06 mPas.

**Figure 2.** Microbial production of EPS using waste, and its recovery.

Choi et al. [30] used spent media wastewater (originated from kimchi fermentation) for EPS production using *Leuconostoc mesenteroides* WiKim32. Under optimal conditions, the maximum EPS productivity was 7.7–9.0 g/L, with a conversion of 38.6–45.1%. The EPSs were nontoxic and exhibited thermal tolerance and antioxidant activity. Pan et al. [31] optimized dextran production from *Leuconostoc pseudomesenteroides* XG5 using an L9 (33) orthogonal test. Under optimal conditions (sucrose 100 g/L, pH 7.0, 25 ◦C, at 100 rpm for 36 h), the maximum dextran yield of 26.02 g/L and 40.07 g/L were recorded at a laboratory scale and fed-batch fermentation, respectively. The EPS was also exhibiting water-holding capacity and antioxidant activity. It reduced the chewiness and hardness of yogurt, but the resilience increased during the 14 days of storage. Product cost is one of the main obstacles in commercialization, hence process economics is one of the major factors that must be assessed. Integration of multiple processes might improve process economics, as well as environmental adaptability. *Sphingobium yanoikuyae* was evaluated for the coproduction of EPS and polyhydroxyalkanoates (PHAs) using lignocellulosic hydrolysate. Hydroxymethyl furfural (HMF), one of the hydrolysis byproducts, improved the consumption of glucose and xylose during fermentation. The optimum C/N ratio of 5 resulted in the maximum EPS production of 3.24 ± 0.05 g/L, however, a further increase in the C/N ratio (30) favored PHB accumulation (38.7 ± 0.08% *w*/*w*) [32]. Biomass hydrolysate is usually accompanied by phenolics, furfurals, and HMF, which also act as fermentation inhibitors and hinder microbial action. Some of the methods, including activated carbon-based adsorption and membrane filtration, have been proposed for hydrolysate detoxification [33,34]. Removal of inhibitors might improve microbial action and product yield. Bhatia et al. [32] have compared the potential of various raw and detoxified hydrolysates for EPS production. In comparison to raw hydrolysate, detoxified biomass hydrolysate showed increased EPS production and maximum EPS production was reported with detoxified pine biomass hydrolysate, i.e., 2.83 ± 0.03 g/L. Table 1 summarizes the recent efforts for EPS production using various microbes (yeasts, bacteria, and fungi), utilizing various pure carbon and organic waste materials.



**Table1.**MicrobialproductionofEPSfromvarioussubstrates.

*Polymers* **2023**, *15*, 1801


Preparation of composites for healthcare applications needs high purity, therefore the downstream processing becomes an inseparable part of processing after fermentation. After production recovery, the identification of structural and chemical characteristics is necessary for further applications. The characterization of EPSs is quantitative, as well as qualitative. EPSs are mainly comprised of carbohydrates conjugated with other biomolecules, hence the basic characterization techniques employed are a colorimetric estimation and use spectrophotometry [45]. For carbohydrate estimation 'Dinitrosalicylic Acid Reagent' is one of the common methods which quantify the reducing sugars [46]. Similarly, for protein, Bradford's dye-binding method [47] and Lowry's method [48] are used. Besides basic characterization with colorimetric methods, Fourier transform infrared spectroscopy (FTIR) is employed to detect the available functional groups and structural functionalities of EPSs. For the detailed structures of EPSs, nuclear magnetic resonance (NMR) and mass spectra are used. In NMR, the sample is dissolved in deuterated solvents for quantification with respect to internal standards [49,50]. The mass spectrum of EPS provides a monosaccharide composition. For analysis, EPSs are hydrolyzed with acid hydrolysis, followed by silylation derivatization. The derivatives are detected and identified by gas chromatography-mass spectrometry [49]. The biological potential of EPS has followed the general procedure for antimicrobial, antioxidant, anti-inflammatory and other activities [45].

#### **3. Microbial Exopolysaccharide Composites and Their Applications**

Exopolysaccharides are natural biopolymers exhibiting diverse applications, variation in structure, adaptability, and the presence of different functional groups. These polymers have shown their suitability as drug carriers and medical sealants. Even with polymeric nature and biocompatibility, these polymers have also shown some weak points. Rapid degradation, hydrophilic nature, low mechanical and tensile strength, and stress tolerance, particularly for scaffold preparation, restrict their application. Some EPSs lacks bioactivity itself, which suggests the addition of multiple drug compounds to add bioactivity for in vivo application. Exopolysaccharide composites have shown a higher potential than native EPSs due to the presence of secondary polymer molecules that offer hybrid characteristics of both (Table 2). The higher mechanical strength with different functional characteristics contributes to diverse functional groups, and is responsible for physiological and chemical properties, contributed to by the members of the composite. To date, EPS composites have been prepared with natural, as well as synthetic, polymers. The selection of secondary polymers relies upon the type of application such as drug loading and release, tissue support and engineering, or ex vivo applications such as a sealant (Figure 3).

Piola et al. [51] prepared a composite hydrogel with gelatin and xanthan gum to support the growth of human skin cells. The composite was printed with CellInk Inkredible 3D printer, using glutaraldehyde solution as a crosslinker. The printed hydrogel was compatible and suitable for the growth of human keratinocytes, as well as fibroblast. Alvel et al. [52] also prepared a composite hydrogel of xanthan with Konjac glucomannan, which also focused on wound healing. On the other hand, a 3D scaffold for tissue engineering was prepared with alginate–gellan gum [53] and methacrylated gellan gum [54].

The applications of EPS composites with various polymers have been summarized below:

#### *3.1. Exopolysaccharide Composites with Natural Materials*

EPS itself is a natural biopolymer that is combined with other natural biomolecules, including proteins, enzymes, lipids, carbohydrates, etc., and thus in most cases, the composite has healthcare-associated applications, including medical sealants and scaffolds in tissue recovery and repair. The major advantages of EPS composites with natural polymers are bioactivity, biodegradability, and biocompatibility. As both the components are natural in origin, there will be a negligible possibility for inflammation and rejection. Alongside that, these composites will not have any toxic effect on the host, but may also act as an antimicrobial formulation for targeting microorganisms. Most biological materials can also be produced in bulk amounts using microorganisms, hence the cost of the products is also

reduced along with the higher availability. The addition of other polymers may also add a specific type of bioactivity, such as antimicrobial, anti-inflammatory, and even catalytic activity to the composite, which widens the possible application in different fields [55–57].

**Figure 3.** Composite scaffold prepared from different exopolysaccharides (**A**). Xanthan gum– gelatin [51]; (**B**). Xanthan gum–Konjac glucomannan [52]; (**C**). Alginate–gellan gum composites [53] and (**D**). Composite methacrylated gellan gum [54].

#### 3.1.1. Cellulose Composites with Natural Polymers

Cellulose is a linear structural polysaccharide comprised of glucan chains connected with cellobiose residues via β-1,4-glycosidic linkage. These structures are packed in forms of microfibrils, kept together with hydrogen bonds and Van der Waals interactions. Depending upon the packaging, it exhibits different degrees of polymerization. It is usually present in the secondary cell wall of plants, but is also present in some bacteria, including members of the Acetobacter, Agrobacterium, Azotobacter, Alcaligenes, Pseudomonas, Rhizobium, and Sarcina genera [58]. The main function of cellulose is to provide strength to structure [59], however, cellulose does not exhibit antimicrobial or similar bioactivity in itself [60], but its high mechanical strength and stress tolerance nature make it suitable for the fabrication of scaffold and dressing material when used together with some bioactive material. Various polymers have been exploited to form composites with EPSs, which improve their strength and water retention capacity and also aid in antimicrobial properties.

Ojagh et al. [61] produced cellulose with *Gluconacetobacter xylinus* by static fermentation. The cellulose and diethylaminoethyl cellulose were derivatized to carboxymethyl cellulose and carboxymethylated diethylaminoethyl cellulose, respectively, and processed

for composite preparation. Briefly, cellulose is a carbohydrate that is rich in hydroxyl groups, which makes it negatively charged. However, similar charges on both components make interactions repulsive. Derivatization of cellulose to carboxymethylated diethylaminoethyl cellulose (CMDEAEC) has both hydroxyls, as well as amine groups in its structure. In contrast to cellulose, CMDEAEC has a net positive charge, and thus the higher amount of CMDEAEC can be loaded on BC in composite formation. The composite of bacterial cellulose–carboxymethyl cellulose has a higher drug loading capacity and swelling ratio than the composite of bacterial cellulose–carboxymethylated diethylaminoethyl cellulose, and even native bacterial cellulose itself. The drug release follows the Higuchi and Korsmeyer–Peppas models. The model is most suitable to describe the release of drugs from the polymer matrix hydrogel. The model suggests that drug release increases from the carrier with time. Methylene blue is a positively charged molecule and the presence of cellulose supports its binding to the composite. The work has also shown that chemical modification adds unique properties to EPSs, improves functionality, and enhances the interaction with another polymer to form a stable composite. Bacterial cellulose was functionalized by derivatization with two active agents, i.e., glycidyl trimethylammonium chloride and glycidyl hexadecyl ether. These agents act upon hydroxyl functional groups of glucose by a heterogeneous reaction, which simultaneously deprotonates the hydroxyl group, as well as the addition of epoxides. It was observed that mere derivatization reduced the bacterial population of *Staphylococcus aureus* 6538PTM and *Escherichia coli* (Migula) ATCC® 8739TM almost by half (53% and 43%, respectively) within 24 h upon direct contact. However, the derivative did not have any cytotoxic effect in terms of morphology and viability upon keratinocytes (HaCaT cell line) and almost 90–100% viability was recorded after 6 days of direct contact. Modified hydrogel has shown an equitant wound closure rate in an in vitro scratch assay, with complete coverage of the wound area after 5 days [60]. It was suggested that the addition of epoxide to cellulose adds antimicrobial properties to cellulose that are also exhibited by the composite.

Composite hydrogels are made by attaching TEMPO-modified nanocrystalline cellulose to the methacrylated gelatin backbone. The human adipose-derived mesenchymal stem cells were encapsulated within the composite and cultured in normal and osteogenic media for 14 days, and the expression of valve interstitial cell phenotypes was observed. The encapsulated cells have lowered alpha-smooth muscle actin expression, while the expression of vimentin and aggrecan increased. Cells cultured in osteogenic media have a reduced expression of osteogenic genes (Runx2 and osteocalcin) that support resistance to calcification. With the composite, a tall and self-standing tubular structure was constructed with a composite hydrogel that also sustained cell viability for possible application in cardiovascular systems [62]. The addition of cellulose to chitosan has improved the mechanical strength, porosity, cytocompatibility, and drug release rate for use in bone tissue engineering. The ternary complex of alginate, chitosan, and bacterial cellulose was used for the preparation of scaffolds using a hydroxyapatite/D-glucono-δ-lactone complex-based gelling system. The composite-based scaffold can adapt to 3D morphology and is stabilized with extensive cross-linking. The smaller size of pores supports the attachment and growth of tissue, along with the required mechanical integrity. Alginate controls the swelling behaviors of the scaffold by intermolecular hydrogen bonds and prevents the degradation of the composite. It has shown high protein adsorption and release potential, along with commendable cytocompatibility, that supported its application in tissue engineering [63]. The polymer supported the 3D structure and allowed for the proliferation of cells, increasing the possible application opportunities in the reconstruction of complex and large organ structures.
