Views: 37 Author: Site Editor Publish Time: 2023-05-12 Origin: Site
Molecular modification can change the polysaccharide dimensional structure, molecular weight and the substituent group types, numbers, and positions, with a profound impact on bioactivity. To date, dozens of molecular modification methods have been tried including carboxymethylation, sulfation, selenylation, phosphorylation, ultrasonic disruption, and the degradation of polysaccharides, which are generally classified as chemical, physical and biological modifications. Molecular modifications remarkably affect the physicochemical properties of polysaccharides and changes their physical and chemical properties including solubility, relative molecular mass, and intrinsic viscosity. Generally the modified polysaccharides display better biological properties because of changes in their physicochemical properties.
The physical method solubilizes and functionalizes the polysaccharide by breaking the main chain of the glucan macromolecule through mechanical action without destroying the basic molecular structure of yeast β-glucan. After physical treatment, lower molecular weight fractions of β-glucans can be obtained, which not only show improved water solubility and the physiological function of β-glucans, but also exhibit conformation change in solution. The operation is simple, no chemical reagent is used and the environment is not polluted. It is mainly used for the modification of polysaccharides that already have bioactivity before treatment in order to reduce their molecular weight and increase their bioactivity efficacy; it can be used to improve the function of some medical materials. However, the yield of soluble yeast glucan is low if prepared by physical methods alone. The most commonly used methods are ultrasonic disruption, irradiation-induced reactions, microwave exposure.
The thermal degradation method refers to the degradation process of the polymer when it is heated, and is an important approach to degrade polymers. The method is simple and the product is easy to obtain with uniformity. It is a solubilization method suitable for functional foods, and by controlling the thermal degradation cycle, products of different molecular weights can be prepared. In 2009, Zhang et al.studied the effects of heat treatment on the transformation of β-glucan solution of Pleurotus ostreatus. It was found that heat treatment of Pleurotus ostreatus β-glucan changed the solution conformation. The first approach was at a temperature of 8~12.5 °C, when the solution conformation changed from a polymer bundle to a small helical cluster. When the temperature was raised to 40–60° C, a second transition occurred, transforming the molecule from a helical cluster into a single polysaccharide chain. In 2018, Ishimoto et al.applied the heat degradation method to solubilize yeast β-glucan and found that it was effectively solubilized by treatment in an aqueous suspension at 135 °C for several hours. Baker’s yeast cell wall β-glucan (BBG) was resuspended in water for injection, and heat-treated at 100, 121, or 135 °C. The solubilization ratio of BBG was examined after several reaction periods at three heating temperatures. As a result, the insoluble BBG first swelled at ∼1 h, and the insoluble component was reduced by continued heating; solubilization at 135 °C was much greater and the solubilized ratio increased with the heating duration. Continued heating also increased the percentage of the lower molecular weight fragments. The molecular weight distribution was clearly shifted to the lower end of the spectrum by heat treatment for 4 h or more.
Irradiation refers to a physical modification method that induces physicochemical changes of β-glucan by ionizing radiation such as gamma rays, X-rays, and electron beams. The average molecular weight of irradiated β-glucan significantly decreased as the irradiation dose increased. In addition, irradiation improved the solubility and decreased the viscosity of β-glucan by the radiolysis of the glycosidic bonds, and this effect was dependent upon the absorbed dose. Radiation can break down β-glucan and change the primary structure, so that cleaved fragments of polysaccharides can be formed. Research conducted by Methacanon et al.with various doses (0–100 kGy) of γ-irradiation was shown to be a clean method to produce different low molecular weight molecules. The results showed that the average molecular weight of the irradiated molecules significantly decreased as the radiation dose increased, whereas the functional groups before and after irradiation, detected by FTIR and 13C-NMR, were identical. It is worth noting that the molecular weight decreased rapidly at first and subsequently leveled off to a much slower rate at dosages > 5 kGy, and subsequently the molecular weight remained almost unchanged at the oligomer level. Khan et al. evaluated the changes in structure, function, antioxidant and antibacterial properties of yeast β-d-glucan after different irradiation intensities and found that γ-irradiation had a great influence on the functional properties of yeast β-d-glucan. The viscosity and swelling power of irradiated samples significantly decreased as the irradiation dose increased. In contrast, the binding strength, emulsifying properties, foaming properties and bile acid binding strength all increased. All antioxidant properties examined using six different analytical methods were significantly increased in a dose-dependent manner. As the irradiation dose increased from 5 kGy to 50 kGy, the antibacterial activity of yeast β-d-glucan also exhibited an upward trend.
The theory of this method is that low frequency and high-intensity ultrasonic energy can break some of the chemical bonds of pyran dextran by increasing the vibratory energy of molecular particles. It generally leads to depolymerization and side chain breakage within the molecules producing oligo-sugars, making them an effective and efficient method for polysaccharide modification. Future oligosaccharide and nano scale molecule processing will mostly probably be carried out using a sonication process. Following ultrasound treatment, the molecular weight distribution curves gradually shift to the low molecular weight region with a narrower distribution and the chain degradation reaction follows a random scission model. The viscosity of modified glucan solution drops remarkably and the glucan after ultrasonication is better dissolved in water. Hunter et al. successfully obtained an immunologically active, homogeneous, non-aggregated, micro-particulate 1–2 µm diameter β-glucan-containing material from the budding yeast Saccharomyces cerevisiae by using a combination of sonication and spray-drying. This microparticulate β-glucan could remain in suspension longer than the aggregated form. Wang et al. sonicated the large-sized yeast β-glucan (SCG) at a working frequency of 18 to 21 kHz at 400 W, with a processing time of 24 s per cycle and an interval of 6 s for a total of 20 cycles. The average particle size of the SCG was changed from the initial 56.49 µm to 2.33 µm, and the ultrasonic modification effect was remarkable. The modified SCG (C-SCG) showed a uniform distribution of single particles, and the particle size distribution bandwidth was narrowed. Chen et al. proved that ultrasonic treatment is an effective physical method for improving the solution properties of water-insoluble homoglycans such as β-glucan without changing its primary chemical structure, and such modified polysaccharides had enhanced antitumor activity. The results showed that ultrasonic treatment could open up the compact structure of Poria cocos polysaccharide (PCS) into a linear chain.
The high-pressure micro-jet homogenizer (HPM) is a new type of nano-scale homogenization device, a physical method for transferring mechanical energy to a fluid particle under high pressure using ultrasonic radiation and mechanical shear to changes structure and control the molecular weight of polymers such as proteins. A number of polysaccharide studies involving high-pressure micro-jets have demonstrated the ability of micro-jets to cause molecular hydrogen bond cleavage and reorganization, side chain breakage and thus change the microscopic morphology, and even increase solubility and changes in functional properties. Huang et al. found that the molecular weight and monosaccharide composition of Mesona chinensis Benth polysaccharide (MP) were changed after DHPM treatment. They treated MP (1 mg/mL) using a laboratory scale dynamic high-pressure microfluidizer at 120 MPa for six cycles and dialyzed the sample treated with DHPM (MW cut-off 10 kDa) vs. distilled water for 24 h, then freeze-dried it to obtain polysaccharide DMP. Gao et al. modified the solubilized yeast β-glucan with a high-pressure micro-jet combined with ionic liquid to prepare soluble β-glucan and studied the modification effects and structure of the circulating ionic liquid. The ionic liquid 1-ethyl-3-methylimidazolium acetate was combined with high-pressure microfluidic modified solubilized yeast β-glucan and treated at 175 Mpa in 200 mL of ionic liquid for 9 min, concentration 0.5%; the total sugar content of the modified dextran obtained was 97.22 ± 0.54% and the yield was 79.25%. The structure of the new and old ionic liquids was compared by FTIR and 1H NMR techniques. The results revealed that the high-pressure microfluid treatment did not destroy the structure of the ionic liquid.
Supercritical CO2 (SCCO2) drying technology is a new technology for preparing special materials with green safety and controllable product structure. The use of supercritical drying, to remove the solvent contained within the gels without collapsing its three dimensional polymeric network, provides unique highly porous materials known as aerogels. Biopolymer aerogels are a special class of lightweight highly porous structured materials that are of interest for their low densities, high surface areas, low heat conductivities, and mechanical strength. Use of SCCO2 for the preparation of aerogels offers the advantage of eliminating the use of organic solvents and being able to operate at relatively low temperatures to minimize the degradation of bioactives. These aerogels are being investigated for applications such as component separation, absorbents, catalysts, and supports for chemical reactions, drug delivery and scaffolds for tissue engineering. Comin et al. obtained an aerogels using SCCO2 drying with β-glucan at a 5% level and flax mucilage at a 10% level. SCCO2-dried polysaccharide aerogels showed promise for use as a delivery vehicle for nutraceuticals including flax SDG. Additionally, biobased aerogels also possess interesting functionality, chemistry, biodegradability, biocompatibility and sustainability inherent to biobased polymers. Simultaneously, Comin et al. determined the effect of the β-glucan concentration and drying technique on the resulting characteristics of aerogels. A biodegradable, biocompatible, renewable and edible β-glucan aerogel formed using a barley β-glucan concentrate at 5%, 6%, and 7% (w/v) levels was dried using SCCO2. The results showed an increasing β-glucan concentration (5–7%) had very few effects on the aerogel properties. SCCO2 aerogels had lower densities than air-dried gels and a more consistent structure than freeze-dried gels. Supercritical carbon dioxide is also used for the extraction of β-glucan. Sibakov et al.demonstrated that lipid removal with supercritical carbon dioxide enhanced the separation of oat β-glucan, starch, and protein in distinct fractions and obtained higher β-glucan content than existing products produced with dry fractionation techniques. This was probably due to more efficient milling yielding smaller particles, and the release of starchy material from cellular structures during milling of defatted oats.
Chemical modification is the most widely used method as it can significantly increase the water solubility and bioactivity of polysaccharides by ‘grafting on’ other moieties. Although the introduction of the new group imparts some new activity to the dextran, its original structure has been changed, so its application is limited to some extent. Moreover, the treatment method can easily form residues, and some operations are also faced with a series of shortcomings such as a complicated modification process, high modification costs and the need for special modification equipment. Although chemical modification can increase the solubility of β-d-glucan, it will also alter its natural structure, changing its biological activity. Research has shown that the sulfonation of fungal exocellular β-(1→6)-d-glucan can change its original structural characteristics, thus improving its water solubility. Moreover, Williams and co-workers (1991) reported a method to modify micro-particulate β-d-glucan, in which the insoluble glucan was turned into glucan phosphate and the water solubility of the modified glucan was significantly improved. Chemical modification methods include sulfation, alkylation, carboxymethylation, phosphorylation, selenization, and acetylation.
Sulfation modification is one of the most commonly used methods to modify polysaccharides. Specifically, sulfated polysaccharides are synthesized by substituting hydroxyl, carboxyl, or amino terminal groups with sulfate groups, with improvements of biological activities. Among them, the solubility of the modified glucan is increased thanks to the hydrophilicity of the sulfate group. The good aqueous solubility of the resultant polysaccharide was a huge advance to its application especially in pharmaceuticals. A novel glucan sulphation process is proposed to improve polysaccharide polarity and its aqueous solubility. The group of Mizumoto (1988) first introduced sulfate groups to monosaccharide structures and found that the sulfated polysaccharide could inhibit T lymphocyte virus, turned sulfated modification into an important direction for structural modification of a polysaccharide. Degree of substitution (DS) is an important indicator to evaluate whether sulfated modification will be successful or not. It is generally acknowledged that the higher degree of substitution proves that the polysaccharide binds more sulfated groups. At present, structure–activity relationship studies mostly focused on the different substitution degrees of sulfated polysaccharides. In 2019, Guo et al. applied sulfur trioxide-pyridine (SO3-Pyr) method to modify Qingke β-glucans (THB) and obtained a high degree of substitution of sulfated β-glucans as follows: Ratio of SO3-Pyr to THB of 16.88 g/g, reaction time of 2.03 h, and reaction temperature of 57.54 °C. Results showed that sulfated modification significantly affected the physicochemical structures (water solubilities, apparent viscosities, and molecular weights) and especially the sulfated THB with higher degree of substitution has better solubility and lower molecular weight. Zhang et al. produced the water-soluble fractions (S-TM8-1 to S-TM8-6) with MW from 6.0 to 64.8 × 104 obtained from the sclerotia of Pleurotus tuber-regium, with a weight-average molecular mass ranging from 5.76 to 77.4 × 104 (TM8-1 to TM8-6) were sulfated. The modified β-glucans have a more expanded flexible chain in aqueous solution than the native polysaccharides. Sulfonation of the TM8 fractions, of the (1→3)-β-d-glucan, from the sclerotia of P. tuber-regium increased the water solubility and chain stiffness. The higher chain stiffness of sulfated fractions may be attributed to the polyelectrolytic effect caused by the sulfate groups substituted on the backbone.
Some macromolecular polysaccharides (cellulose, scleroglucan, and pachymaran) are difficult to induce bioactivities because of poor water solubility. Some current reports related to the successful carboxymethylation of cellulose, scleroglucan, and chitin which the water solubility and bioactivities are obviously increased were proved, in comparison with the native polysaccharides that were used in the derivatization. The preparation of carboxymethylated polysaccharides is generally carried out by using chloroacetic acid or sodium mono-chloroacetate as a substrate and reacting with polysaccharides in basic 2-propanone. It has been found that carboxymethylation improves the solubility of polysaccharides, and also changes the natural conformation of dextran molecules, together with its physiological activity. The structure of the CM glucans in the solution depends upon the degree of substitution and with the increase of the DS, a transition from triple-helical structure through single helical to random structure takes place. Kogan et al. suspended 10 g of the glucan in a mixture of 12.4 mL of aqueous NaOH (300 g/L) and 125 mL of iso-propyl alcohol and stirred the suspension vigorously at 10 °C for 1 h. Subsequently, sodium salt of monochloroacetic acid was added (7.9 g for achievement of the substitution degree 0.5) in 14 mL of water, and the mixture was stirred at 70 °C for 2 h. The excess of NaOH is neutralized with 6 N HCl and the salts removed by dialysis. Finally, the water-soluble yeast polysaccharide derivative—carboxymethylated (1→3)-β-d-glucan (CMG)—a well-known macrophage-stimulator was obtained. Tang et al. prepared and characterized respectively six (1→3)-d-glucan derivatives prepared from yeast cell wall. It indicated that sulfated glucan (S-PJ) had the significant reduction capacity, phosphorylated glucan (P-PJ) had the obvious hydroxyl radical/superoxide anion scavenging activities and anti-lipid peroxidation effect.
It has been shown that fructose, glucose, and some other monosaccharides have no natural bioactivities which could be activated after phosphorylated modification. Phosphorylation is a modification method that introduces a phosphate group into a polysaccharide. Common phosphating agents are phosphorus oxychloride, phosphoric anhydride, phosphoric acid or a salt thereof. The reaction of phosphorylation requires strong acid as a catalyst, which often leads to the degradation of the polysaccharide and complex compositions. This material has been demonstrated to possess anti-infective, anti-inflammatory, cardio-protective, and immunomodulating properties. Most of the researchers have focused on the artificial synthesis of phosphorylated polysaccharides and some of their analogs. Williams et al. dissolved Saccharomyces cerevisiae β-glucan in a mixed system of dimethyl sulfoxide and urea, and added phosphoric acid for phosphorylation to prepare phosphorylated β-glucan. The prepared yeast glucan phosphate has two molecular mass peaks by high-performance size exclusion chromatography, on-line multi-angle laser light dispersion and differential viscosity measurement, and one is MW = 3.57 × 106, accounting for about 2%, the other is MW = 1.10 × 105, accounting for about 98% of the total. Through the Congo red experiment, it was found that the phosphorylated yeast β-glucan can be aggregated into a triple helix configuration in solution. Several studies have shown that both natural and artificially modified phosphorylated polysaccharides have specified medicinal values, due to the fact that the existence of charged phosphate groups can improve water solubility, change molecular weight, and modify chain conformation of polysaccharides. Chen et al. and Huang et al. studied the chain conformation and antitumor activity of phosphate modified Tuckahoe β-glucan and Tuckahoe hyphae α-glucan, respectively. In 0.15 mol/L NaCl solution, β-glucan phosphate showed a relatively extended compliant chain conformation, and Tuckahoe hyphae α-glucan phosphate showed a more extended semi-rigid chain conformation. The introduction of phosphate groups leads to steric hindrance between molecules, increases significantly the solubility of Tuckahoe β-glucan phosphate and Tuckahoe hyphae α-glucan phosphate in water and the rigidity of the chain. Compared with glucan that has not been phosphorylated, water-solubility and chain stiffness of the phosphorylated derivative increased, as a result of the introduction of phosphate groups on main chain. β-glucan phosphate and Tuckahoe hyphae α-glucan phosphate have significant anti-tumor activity for relatively high molecular masses and the extended chain conformation can increase the interaction between polysaccharide and the immune system, thereby improving anti-tumor activity. Furthermore, selection of derivatives will make the study of their structure–activity relationships more effective and systematic.
In general, biological modification of polysaccharides mainly refers to enzymatic modification, which is the degradation of β-glucan due to catalysis by enzymes. Since the biological method is mild but environmentally friendly, it is a promising polysaccharide modification technique. Compared with chemical modification, enzymatic modification has the advantages of high specificity, high efficiency and few side effects. By controlling the enzymatic reaction conditions, polysaccharides with different structures can be produced and their physical and chemical properties could also change. It features high security and controllability.
Enzymatic modification has found widespread industrial use in changing the functional properties of β-glucan both in vivo and in vitro. Depolymerization is the most popular catalytic reaction, which depends on the specific enzyme or enzyme mixture employed and on the particular industrial requirement. In some instances, the aim may be to maintain or, at best, only slightly alter the molecular size of functional polysaccharides and produce specific oligosaccharide fragments from them. The main course of enzymatic degradation is to degrade the backbone of a polysaccharide, before reducing the molecular weight and decreasing its viscosity. The research of Kery et al. showed that β-1,3-glucanase has a highly specific action and acts at the β-l,3-glycosidic bond to randomly cleave β-glucan. The β-1,3-glycosidic bond reduces the molecular weight of β-glucan, causing the β-glucan to degrade into dextrin or oligosaccharide. Duan et al. showed that the yeast β-glucan was hydrolyzed by the production of β-1,3-glucanase by Trichoderma strain, and the components after enzymatic hydrolysis included large molecular weight polysaccharides and oligomerization products. Sugar, small molecule oligosaccharides and monosaccharides were the components after enzymatic hydrolysis with MWs > 30 kDa, and accounted for 66.2% of them. The maximum yield of water soluble β-1,3-glucan was 52% when 20% of substrate with 4 U/mL enzyme was incubated at 55 °C in pH 4.5 buffer for 2 h. Yu et al. used yeast glucan as the raw material to obtain a water-soluble glucan by alkaline protease treatment and obtained water-soluble glucan by hydrolysis. Solubilized yeast glucan has good antioxidant activity and inhibitory activity against angiotensin converting enzyme. The enzymatic modification is promising since it can effectively promote the solubility of polysaccharides without chemical residues or environmental pollution. The use of enzymatic modification is currently limited to only a few kinds of polysaccharides. Research and development of other types of enzymes, such as transferase and synthase, will enrich the application of enzyme technology of structural modification of polysaccharide.
Microbial modification refers to the change for the composition and structure of the polysaccharide by the fermentation of microorganisms, thereby changing the physical properties and biological activity of the polysaccharide. Liang et al. developed an efficient and cost-effective microbial fermentation method for the direct production of water-soluble β-1,3-glucan (w-glucan), a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT). W-glucan production reached 17.31 g/ L, with a degree of polymerization of 19–25. It proved that the addition of 10 g·L−1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis.
