Production Of Xanthan Gum With Glycerol And The Study Of Its Properties And Application

Due to the properties of safety, stability, suspensibility, emulsibility, pseudoplasticity and thickening, xanthan gum has become an “industrial aginomoto” and been widely used in many industries, such as food, pharmaceutical, textile, agriculture and oil recovery. Corn starch is generally the raw material in xanthan production, but with the growth of worldwide population and food shortage, more and more scholars have paid attention to finding substitutes from byproducts of industry and agriculture for xanthan production instead of corn starch. Glycerol is an inevitable byproduct of transesterification during biodiesel production, and its output increased with the increasing yield of biodiesel. Based on the sustainability, environmental friendliness and simple operation, application of glycerol in the field of fermentation has attracted more and more attention. If glycerol can be used for xanthan production, which will make a great contribution to the relief of global food crisis. In this work, a domesticated strain, X. campestris CCTCC M2015714, was obtained by adaptive evolution, and genes related to glycerol metabolism in Xanthomonas were investigated. With further adaptive evolution, glycerol tolerant capacity of this domesticated strain increased to 100 g·L-1. With adoption of multi-stage control and feeding glycerol strategies, xanthan yield(33.9 g·L-1) and fermentation time(60 h) had all reached the commercial production level of xanthan gum with corn starch as raw material. At the same time, the molecular characteristic, structure characteristic, rheological properties and potential application of xanthan gum produced from glycerol were analyzed. The main results and conclusions are summarized as follows:(1) With X. campestris NRRL B-1459 as parent strain, a domesticated strain, X. campestris CCTCC M2015714, was obtained through adaptive evolution. From the results of real-time reverse transcription PCR, the transcriptional levels of genes related to glycerol metabolism(glpF, glpK, glpD, and fbp) in the parent strain were all 1.0, but these four genes were all up-regulated in the domesticated strain, and the RT level order of these four genes was glpD(4.76) > glpF(3.36) > glpK(3.05) > fbp(2.53), indicating that the enhanced expression of genes related to glycerol metabolism in Xanthomonas was the possible reason for domesticated strain using glycerol for xanthan gum production. With 5 g·L-1 sucrose or glucose as starter substrate, cell growth time reduced from 36 h to 24 h and xanthan yield increased from 11.0 g·L-1 to 12.5 g·L-1. Moreover, the effect of impurities in crude glycerol used in this work, such as sodium salt, methanol and ash, on the productivity of X. campestris CCTCC M2015714 can be ignored.(2) Through further adaptive evolution, glycerol tolerant capacity of this domesticated strain increaed to 100 g·L-1 and the expression of genes related to glycerol metabolism was further enhanced, which was glpD(8.56) > glpF(7.73) > glpK(6.48) > fbp(5.31). With adoption of multi-stage control and feeding glycerol strategies: low initial glycerol concentration(40 g·L-1), varied aeration and agitation(0~24 h, 0.5 vvm and 200 rpm; 24~60 h, 1.0 vvm and 400 rpm), and varied feeding glycerol(24~34 h, 3 g·L-1·h-1; 34~44 h, 2 g·L-1·h-1; 44~54 h, 1 g·L-1·h-1). The inhibition of substrate on domesticated strain was relieved and the high C/N during the process of xanthan gum synthesis was maintained, which made xanthan yield reached 33.9 g·L-1 and fermentation time decreased to 60 h. These two parameters were close to the current status of commercial production of xanthan with corn strach as raw material, and 33.9 g·L-1 is the maximum yield of xanthan gum produced from glycerol so far.(3) With glycerol as carbon source, the monosaccharide composition of xanthan gum secreted by X. campestris CCTCC M2015714 is glucose: mannose: glucuronic acid=2.0:1.65:1.0, which is close to that of the commercial xanthan(2.0:1.85:1.0). Meanwhile, functional groups and chemical structure of xanthan gum produced from glycerol are similar to those of the commercial xanthan through FT-IR and NMR. Above results indicated that the exopolysaccharide produced from glycerol by X. campestris CCTCC M2015714 is xanthan gum. Remarkably, the molecular weight of xanthan gum produced using our method(3.0×106 Da) is about half that of the commercial one(6.4×106 Da), which lead to the solution viscosity of xanthan gum produced from glycerol is far lower than that of the commercial xanthan. As for the 1.0%(w/v) two xanthan solutions, the consistency index(K) of xanthan gum produced from glycerol is 1.7958, which is less than 1/10 that of the commercial xanthan(21.0842). Measwhile, the behavior index of xanthan gum produced from glycerol(0.235) is lower than 1.0, suggesting that xanthan gum produced from glycerol is still a pseudoplastic fluid. Atomic force microscopy results showed that xanthan gum produced from glycerol forms a disconnected network in aqueous solution when compared to a dense and honeycomb structure of commercial xanthan. In addition, scanning electron microscope and differential scanning calorimeter results suggest that the spatial structure of xanthan gum produced from glycerol is thin and loose, but a rod-like and closely aggregated spatial structure is formed by commercial xanthan.(4) Compared to commercial xanthan, low viscosity of new xanthan can increase the removing rate of pigment, biomass and insoluble impurities in fermentation broth, thus leading to high transparency of xanthan gum produced from glycerol. The transparency of xanthan gum produced from glycerol is 95%, but the transparency of commercial xanthan is about 80%. In comparison with commercial xanthan, the low viscosity and low molecular weight of new xanthan can accelerate its combination speed with water molecules, so the dissolution rate of new xanthan is faster than that of the commercial xanthan. The solution viscosity of new xanthan and commercial xanthan increases with the increase of gum concentration, and they are stable to temperature, pH and salts. When the salt concentration was lower than 0.5 g·L-1, the low viscosity and low molecular weight of new xanthan make salt ions can neutralize the negative charge on the carboxyl of new xanthan’s side chain, reduce the electrostatic repulsion between xanthan molecules and increase the viscosity of xanthan solution. At the same time, bivalent ion can form “salt bridge” between xanthan molecules and make the increasing effect of solution viscosity is higher than that of the monovalent ion. The pyruvate content of new xanthan(5.2%) is higher than that of commercial xanthan(4.1%), which increases the cross-linking of xanthan molecules and xanthan solution viscosity during the treatment of freeze-thaw cycles at-20 ℃, and the viscosity maintained stability after the third cycle.(5) Compared to commercial xanthan’s high viscosity, the low viscosity of new xanthan can increase its additive amount and make it to be a potential dietary fiber microbial exopolysaccharide. From the results of new xanthan’s application in food industry, its absorption capacity to unsaturated fat and saturated fat were 2.15±0.26 g·g-1 and 2.08±0.21 g·g-1; cation exchange capacity was 1.15±0.08 mmol·g-1; the removal rate of Cu, Cd and Pb by this new xanthan within 1 h was more than 50%, and this removal rate was higher than 75% when the absorption time was prolonged to 4 h. The absorption capacity of this new xanthan to cholesterol at the simulated conditions of stomach(pH=2.0) and small intestine(pH=7.0) were 12.36 mg·g-1 and 11.72 mg·g-1, respectively. The absorption capacity of this new xanthan to cholic acid sodium increased with the increase of cholic acid sodium concentration. The removal rate of nitrite ion by this new xanthan were 80%(pH 2.0) and 60%(pH 7.0). At the same time, this new xanthan can retard the glucose diffusion rate in water and the rate of starch digestion by glucoamylase, as well as maintaining the viscosity of starch solution in the process of hydrolysis by α-amylase. Moreover, this new xanthan can compound with soluble fructo-oligosaccharide and insoluble soybean protein at any ratio.