3.2.5. Pullulan Composites with Synthetic Polymers

Drug-delivery-related applications required hydrophobic carriers and functional groups that can offer a site for attachments. Exopolysaccharides such as pullulan are soluble in an aqueous environment. However, these are sensitive to modification by hydrophobic functional groups, including the cholesterol functional group, which make them amphiphilic and ensure the drug release.

Cholesteryl-modified aminated pullulan polymers were prepared with cholesterol succinate and pullulan. Succinic anhydride cholesterol (0.2–0.6 g), 0.18 g dimethylaminoaniline, 0.35 g 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt, and acid salt were dissolved in DMSO at room temperature, which activated succinic anhydride cholesterol. The activated solution was added to 5.6% amino pullulan solution in DMSO and mixed at 50 ◦C for 48 h, followed by cooling to room temperature. Anhydrous ethanol was added to the reaction liquid that precipitated the composite. The composition of different components affects the properties of the composite. With respect to different concentrations of cholesteryl substitution, particle size reduced from 178.0, 144.4, and 97.8 nm, with an increase in the extent of substitution. With an increase in substitution, the hydrophobicity of the pullulan derivative increased and the particle size reduced. Hydrophobicity also influenced the drug release, as derivatives with maximum hydrophobicity have the slowest drug release, i.e., 57.8%; and the lowest hydrophobicity have maximum drug release, i.e., 72.7% after 48 h. In contrast, the efficacy against lung cancer cells increased with a reduction in hydrophobicity [102]. Mommer et al. [103] have functionalized the pullulan by thiolactone-based activation that adds amines. Modified pullulan offered the possibility of forming a composite with amine-containing biological substrates. With respect to thiolactone substitution (2.5/5.0 mol%), hydrogels have different mesh sizes, i.e., 27.8 and 49.1 nm respectively. Cell proliferation studies were conducted with different cell lines (normal human dermal fibroblasts and hepatocytes) and it was reported that gelatin and H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) support cell proliferation, while H-Gly-Arg-Gly-Asp-Ser-OH as well as H-Gly-His-Lys-OH acetate salt improved the proliferation of hepatocytes (HepG2) by up to 10 folds over gelatin. A thermosensitive composite was prepared with pullulan, carrying pendant carboxymethyl groups and amphiphilic triblock copolymer, poloxamer 407, by grafting. The final composite has about 83.8% poloxamer grafted on the copolymer. The composite was highly flexible and elastic, which allowed for the copolymer to regain and recover the native structure after the removal of external force/stimuli. The gel sustained

amoxicillin release over a period of 168 h. The composite can be used as a hydrogel for scaffold fabrication, as well as for drug delivery [104].

High water holding and moisture retention with mechanical properties are the main strengths of bacterial nanocellulose suitable for the preparation of wound dressing and fabrication of biomedical device production, but cellulose lacks antimicrobial and woundhealing capacity. On the other hand, pullulan can contribute to wound healing, and zinc oxide nanoparticles offered antibacterial properties. Aminoalkylsilane grafted bacterial cellulose membrane and established an interconnection between cellulose with pullulan–ZnO nanoparticle hybrid electrospun nanofibers. Dressing materials prepared from composites have better blood clotting performance over the control, i.e., BNC. The composite released ZnO and offered 5 logs of higher antibacterial activity than cellulose. Cytotoxicity analysis with L929 fibroblast cells suggested that the composite was safe for fibroblast cell proliferation. In the animal (rat) model, the composite material offered rapid healing and re-epithelialization rate with the formation of a small blood vessel and synthesis of collagen [105].

**Table 2.** Exopolysaccharides composites in biomedical applications.



#### **Table 2.** *Cont.*


#### **Table 2.** *Cont.*


#### **Table 2.** *Cont.*

The composite preparation and applications in different fields are still not very well developed. Some of them are under screening and others have crossed trials in cell lines. The efforts have received wide attention, and for outstanding findings, patents have been awarded (Table 3).

**Table 3.** Patents awarded in microbial exopolysaccharide-related research.


Microbe-based polysaccharides are biocompatible, nonimmunogenic, biodegradable and most of these are USA FDA-approved and regarded as safe for human consumption [135]. According to GRAS notice 000099, the use of pullulan is allowed as an ingredient in tablets and capsules for dietary supplements [136]. The commercialization of such products still needs a long way to cover especially in the context of their behavior in various conditions to support their candidature.

#### **4. Challenges and Future Prospective**

Microbial EPS and associated composites have offered several advantages such as biocompatibility, biodegradability, and nontoxicity; however, there is still no product available at the commercial level. The commercialization of products prepared from EPS composites needs to address some of the common challenges, summarized below.

#### *4.1. Strain Selection*

The major fraction of EPS composites is microbial exopolysaccharides, which are produced by fermentation at various scales. Strain selection is one of the major problems associated with the large-scale production and commercialization of EPS and associated products. Several microorganisms have been screened for the production of EPS and some of them are either pathogenic or have low yield [137]. However, some of the microorganisms have offered compatible productivity without any pathogenicity, such as probiotic bacteria, including *Lactobacillus*, *Bifidobacterium*, and *Lactococcus*. In addition to yield, the tolerance to high salt concentrations (bile salt) and extremely acidic pH are other advantages that aided in the commercialization of products. Recent research has also proven that lactic acid bacteria also improved immunity and wound healing [49,138].

Genetic engineering of microorganisms is another possible strategy to improve productivity. However, the production of polysaccharides needs a combinatorial synthesis approach, as it is governed by multiple pathways. Cumulatively the pathways have the following three stages: nucleotide sugar precursors synthesis, extension, and synthesis of oligosaccharide units by glycosyltransferases and assembly of structural units to the final EPS, followed by export [139]. Some of the strategies have been summarized by Schmid et al. [140], but no report is available on the large-scale production of EPS with genetically engineered microorganisms. This may be due to the complex biosynthetic pathway governed by multiple gene systems. There is a need to work on this issue to increase EPS productivity with unique properties.

Production of exopolysaccharides, yield, and cost: Any microorganism can offer a higher possible production under optimal growth and production conditions. Before moving to higher-scale production, the identification of exopolysaccharides, their nature, and optimal conditions for maximum productivity are required [141]. As mentioned above, EPS production relies upon commercial-grade substrates with a higher cost, which also affects the product cost. A possible way out for the substrate is the utilization of low-cost lignocellulosic biomass from agricultural, as well as industrial sources, such as raw material. Previous research has used various agricultural biomass for pullulan production by *Aureobasidium pullulans*, i.e., hazelnut husk [142], sesame seed oil cake [143], corn cobs, and straw [144]. The process can be optimized via one factor or a statistical optimization approach. However, lignocellulosic materials, such as raw materials, need preprocessing such as hydrolysis, followed by detoxification. Acid hydrolysis is one of the most common methods used for sugar recovery in hydrolysate, but some byproducts, including furfurals and hydroxymethyl furfurals (from pentose and hexose), were also produced along with phenolics from lignin. These byproducts have shown an inhibitory effect on microbial growth and hence hydrolysate was detoxified by activated charcoal and membrane separation [33,145,146]. These additional steps for biomass processing and hydrolysate preparation increased the process, as well as product cost. The use of tolerant microorganisms might help overcome the challenges associated with inhibitors.

#### *4.2. Downstream Processing*

Downstream processing and product recovery is one of the major challenges, as it contributes to the majority of the product cost. Therefore, an efficient and low-cost downstream process is a prerequisite for the commercialization of exopolysaccharides and associated products [141]. The chemical nature and characteristics of exopolysaccharides, such as high viscosity and gel-forming ability, hinder their extraction. Some of the recovery approaches are trichloroacetic acid and proteases-assisted protein removal, dialysis, chromatography, and solvent-based precipitation used for EPS recovery. The ultra-grade of purity of a product is necessary for biomedical applications, hence the selection of the recovery process should be done as per the target product, as well as the physicochemical properties of EPS. Proteins are removed by protease/trichloroacetic acid to prevent contamination and cross-reaction. However, heat or use of concentrated acid damage the native EPS structure and change the integrity of bonds, branching, and sugar monomers. [147].
