## 2 Evolution of NMR Methodologies
### Solution-State NMR for Polysaccharides
#### 2.1.3 Applications in Complex Mixtures  
NMR in the solution state is very good for analyzing polysaccharides in complex mixtures, like those in food matrices and biological samples. The reason is that it can detect and distinguish molecular components in their natural state without requiring extensive sample preparation. For instance, [PERSON] et al. measured mixtures of food polysaccharides using \({}^{1}\)H NMR, and these measurements can be used for quality control and process optimization [19]. Their research showed that NMR can analyze multiple polysaccharide components at the same time. This provides a reliable way to characterize heterogeneous systems. In the pharmaceutical field, [PERSON] et al. used diffusion-ordered NMR spectroscopy (DOSY) along with methylation and advanced 1D/2D magnetic resonance techniques (like HSQC, HMBC, TOCSY) to analyze the structural characteristics of \(\beta\)-glucan from yeast and mushrooms, as shown in Figure 2[27]. The main interesting thing about this paper is that it uses DOSY to tell different types of polysaccharides in complex mixtures apart by analyzing their diffusion behavior. This method provides useful information about the patterns and structures of macromolecular chains. These patterns and structures are hard to find with traditional techniques. By using this method, DOSY demonstrates its role as a powerful complementary tool for polysaccharide structural analysis. It also has the potential to show details at the molecular level. These details are very important for us to understand the biological functions of polysaccharides. NMR-based metabolomics is also helpful for the study of polysaccharides in biological matrices. [PERSON] et al. studied the polysaccharides from _Polygonatum sibricum_ in traditional medicine using \({}^{1}\)H NMR. They identified the bioactive components and connected them to observed health benefits [20]. This method combines structural analysis and functional studies. It helps us understand how specific polysaccharide components promote biological activity. [PERSON] et al. used \({}^{1}\)H NMR-based metabonomics to investigate the antidiabetic activity of _Entemporpha prolifera_ polysaccharides and proved that they have therapeutic potential [28]. Likewise, [PERSON] et al. developed a general NMR method to analyze the polysaccharide composition, providing a reliable means for structural characterization [29]. In addition, [PERSON] et al. analyzed the polysaccharide part of glycoptides and found the uses of these drug structures [30]. [PERSON] et al. utilized graded acid hydrolysis and NMR to elucidate the structure of uronic-acid-containing polysaccharides from abalone, which demonstrated how sequential hydrolysis can aid structural studies [31]. This research illustrates that NMR-based metabolomics can be a very powerful tool for linking polysaccharide structures to their biological and pharmaceutical functions. This integrative approach not only helps us understand polysaccharides better but also creates new ways to use them in therapeutic innovation and drug design. Figure 2: The structural characteristics of polysaccharides from yeast and mushrooms using diffusion-ordered NMR spectroscopy (DOSY). Copyright (2025) Elsevier. [PERSON] et al. used NMR technology to monitor polysaccharides during wine fermentation. Their research showed the changes in polysaccharide components during fermentation. The study presents a novel approach to improve wine quality by managing polysaccharides [32]. [PERSON] et al. studied how polysaccharides interact with water by dynamic vapor adsorption and NMR to understand hydration kinetics at the molecular level [33]. [PERSON] et al. characterized polysaccharide interactions in bilateral mixtures with low-field NMR. They also pointed out the application of this research in food and polymer research [34]. These examples illustrate NMR's versatility in analyzing polysaccharides in complex and dynamic environments, from food to biological and industrial systems.