Bioprospecting of Microorganism-Based Industrial Molecules. Группа авторов

aspects of microbial gums like their biosynthesis, production, structure, and functional properties. In this chapter important examples of microbial gums like xanthan, gellan, and pullulan have been highlighted. Further, various applications of microbial gums in food, cosmetics, medical, and nanotechnology have been discussed.

3.2 Biosynthesis of Microbial Gums

Microbial gums are produced in two different ways: (i) extracellularly by means of glycosyltransferase enzymes secreted from the microbial cells, or (ii) intracellularly, where a cascade of enzymes produced inside the cells is responsible for precursor synthesis, glycosyltransferase, polymerization, and export of the microbial biopolymer (Figure 3.1). HoPS, such as dextran, alternan, and levan, are produced by extracellular glycosylsucrase enzymes that hydrolyses sucrose to produce long‐chain biopolymers. Well‐known microbial gums such as pullulan, a HoPS of α‐(1 → 6)‐linked polymer of maltotriose subunits, is produced by fungus Aureobasidium pullulans using enzyme glucosyltransferase. UDP‐glucose acts as a precursor for pullulan synthesis. The exact biosynthesis pathway for pullulan is still not known [5]. Curdlan, a (1→3)‐β‐glucan produced by Alcaligenes faecalis, is synthesized by the action of an operon containing four genes, namely crdA, crdS, crdC, and crdR. crdS is curdlan synthase, a glucosyltransferase enzyme, which uses UDP‐glucose as a precursor. The crdC gene is responsible for polymerization of curdlan and subsequent translocation of the biopolymer across the cytoplasmic membrane [6].

Figure 3.1 Schematics of biosynthesis of microbial gums.

      Source: Based on [3].

      HePS are synthesized intracellularly and subsequently exported out from the cells. HePS shows a similar biosynthesis pathway to HoPS‐like pullulan. This pathway is controlled by a collection of genes called EPS gene cluster, which is responsible for regulation, chain‐length determination, polymerization, and export of HePS. The precursors for HePS biosynthesis are UDP and dTDP sugars, and in rare occurrences, non‐sugar groups like acetyl group or pyruvate group could be attached to the biopolymer chain [7, 8].

      Biosynthesis of microbial gums is highly strain‐dependent, and these variability can be seen as differences in biopolymer yield, linkages present between moieties and molecular weight. These differences also can change the functional properties of these microbial gums. Modification of genes involved in the biosynthesis of microbial gums through metabolic engineering of the strains can yield modified biopolymers or produce a higher quantity of microbial gums [9].

3.3 Production of Microbial Gums

      Microbial gums are of great interest across many industries since they can be produced in large quantities, and research into several variations in the methods of production has led to economically viable alternatives. Xanthan gum, a widely used microbial gum, is industrially produced using glucose or invert sugars in batch fermentation mode. The culture conditions for xanthan production include maintaining a temperature between 28 and 30 °C, pH~7.0, and aeration rate higher than 0.3 (v/v), which is carried out for 100 hours [10]. Industrial grade xanthan is obtained by downstream processing that involves sterilization of the fermentation broth to kill the bacterial cells and to deactivate any enzymes. Cells are separated from the broth by centrifugation, and the cell‐free supernatant (CFS) is subjected to alcohol precipitation. The precipitated xanthan gum is either reprecipitated or spray‐dried and subsequently milled to the desired size [11]. Pullulan, another important microbial gum, is industrially produced similarly to xanthan gum. For the production of pullulan, liquefied starch is used as carbon source, and culture conditions include a temperature of 30 °C, pH~6.5 in aerated conditions for 100 hours to obtain the maximum pullulan yield of around 70% of the substrate used [5]. Other microbial gums like dextran can also be purified from CFS using ethanol precipitation. However, an alternate method for dextran production is by using either CFS‐containing enzyme dextransucrase or separately purified dextransucrase. Dextransucrase is added to sucrose in a controlled reaction condition, which results in dextran of high purity [12].

      Centrifugal packed‐bed reactor (CPBR) was used for the production of xanthan gum, where the bacterial cells were immobilized in the rotating fibrous matrix. The pumping and circulation of the media through the matrix ensured the cells having good liquid and gas transfer. This process resulted in good separation of xanthan and cells, and could lead to less energy‐consuming downstream processing [13].

The use of alternative substrates for microbial gum production has seen interest among researchers. Xanthan gum production by using hydrolyzed starch from rice, barley, and corn starch, as well as the use of acid whey, sugarcane molasses, waste sugar beet pulp, and peach pulp, have resulted in competitive yields but less than that of glucose [14]. Coconut milk as a substrate was used to produce pullulan, where 54g/l pullulan was produced in 36 hours of fermentation at 30 °C [15]. Cane molasses have been used to produce welan gum using sulfuric acid hydrolysis as pretreatment, which resulted in a high welan gum yield of 41 g/l. The resulted welan gum was found to have acceptable molecular weight and rheological properties with better thermal stability with control substrate glucose [16].

3.4 Structure and Properties of Microbial Gums

      Microbial gums possess the ability to form “gels” or increase the viscosity of a liquid. These properties are due to the specific macromolecular structural organization seen in these gums. Structural properties such as monomer composition, linkages among the residues, branched or unbranched structure, and neutral or charged properties are essential factors that are analyzed during characterization of these gums [2]. Techniques such as gas chromatography (GC) or high‐performance anion‐exchange chromatography (HPAEC) are used for monomer determination. Fourier‐transformed infrared spectroscopy is used for the analysis of functional groups present in microbial gums. Linkages present are determined by nuclear magnetic resonance like ¹H and ¹³C spectroscopy or by methylation analysis using GC [17].

      Physicochemical properties of microbial gums are another critical factor, that when determined, can establish possible functional applications of microbial gums in various industries. Important physicochemical properties commonly analyzed include molecular weight determination, microstructure analysis, and determination of thermal properties. High‐performance size‐exclusion chromatography, accompanied by refractive index (RI) detector or for higher accuracy with multi‐angle laser light‐scattering detector, is generally used for molecular determination [18]. Microstructure analysis can be determined by different techniques such as scanning electron microscope and atomic force microscope, while the thermal analysis is carried out using thermal gravimetric analyzer and differential scanning calorimetry.

      Different combinations of structural and physicochemical properties of these biopolymers can result in desirable properties for industrial applications. EPS produced from Streptococcus thermophilus CRL1190 has a high molecular weight (1782 kDa), and a porous microstructure, displaying high water holding capacity and oil holding capacity. EPS CRL1190 also possessed antioxidative, emulsifying, and flocculating activities [19].

      Microbial gums have been utilized for the synthesis of hydrogels. Hydrogels can be described as hydrophilic polymeric networks that can imbibe large quantities of liquid. Hydrogels can be used to replace tissue as implants or scaffolds, and those made using microbial gums are nontoxic and biodegradable [1].

3.5 Types of Microbial Gums

In this section, we will be briefly describing different microbial gums that are important in various industries due to their properties. The different types of microbial gums and their producer organisms have been mention in Table