Microbial growth in food significantly impacts its quality, safety, and shelf life. While water activity (a_w) has been a key indicator for predicting microbial growth, it may be insufficient in low-moisture foods due to time-dependent physicochemical properties and nonequilibrium situations. The glass transition temperature (T_g) also plays a role, as glassy states exhibit reduced molecular mobility, hindering microbial growth. However, some xerophytic microbes can still grow even below T_g. This highlights the need to consider water mobility dynamics, which are crucial for nutrient transport and microbial metabolism. NMR techniques can measure water mobility; however, ¹⁷O-NMR is preferred due to fewer complexities. Previous research has shown a correlation between water mobility and microbial growth, but a comprehensive quantification of water mobility in solid foods is still needed. The 'Strength parameter' (S) has been introduced to classify solid-food systems based on their molecular mobility near T_g, but its compositional dependence needs further exploration using classical thermodynamics. This study uses glucose and whey protein isolate (WPI) as a model system to understand the relationships between water sorption, thermodynamic properties, water mobility, and microbial growth, introducing a novel parameter, water usability (U_w), to improve the understanding of water's role in microbial growth in low-moisture foods.

Extensive research has focused on water activity (a_w) as a primary factor influencing microbial growth in food. However, limitations exist, especially in low-moisture foods, where time-dependent factors and non-equilibrium states impact the a_w's predictive power. The glass transition temperature (T_g) is another crucial parameter, indicating the transition from a rigid glassy state to a more mobile rubbery state. Studies have established a link between T_g and microbial growth; however, exceptions exist, particularly with xerophytic microbes. The significance of water mobility dynamics has emerged as a crucial aspect, impacting nutrient diffusion and microbial metabolic activity. NMR spectroscopy, particularly ¹⁷O-NMR, has been employed to quantify water mobility, revealing correlations with microbial growth. The 'Strength parameter' (S), a measure of structural transformation and mobility resistance, provides a more comprehensive analysis of molecular mobility in solid foods. Previous work has highlighted the compositional dependence of S, yet a more thorough understanding incorporating classical thermodynamics is necessary. This review reveals a gap in quantitatively describing water mobility in solid foods while incorporating thermodynamic properties and dynamic mobility data.

This study used glucose and whey protein isolate (WPI) to create model food systems with varying mass ratios (1:0, 7:3, 1:1, 3:7, and 0:1). These were lyophilized to create amorphous solid matrices. Water sorption was measured over a range of water activities (a_w, 0.11–0.76) at 30 °C. The Guggenheim–Anderson–de Boer (GAB) model was used to fit the sorption data. Differential scanning calorimetry (DSC) determined the glass transition temperatures (T_g). Dynamic mechanical analysis (DMA) measured the loss modulus (E”) to determine the α-relaxation time (τ) and the WLF constants (C₁ and C₂) via the William-Landel-Ferry (WLF) equation. The Strength parameter (S) was calculated using the WLF constants. The relationship between water content and S was modeled using a previously established equation with a partition constant (k_sp). A novel parameter, water usability (U_w), was defined as the ratio of the Strength parameter of water in the matrix (S₁) to that of liquid pure water (S₂). *D. hansenii* yeast was used as a model microorganism, inoculated onto the solid matrices at various a_w (0.75–0.92) and incubated at 30 °C for 36 h. Microbial growth was assessed using an ATP fluorescence detector, SEM imaging, and growth rate (µ) and doubling time (g) calculations. Statistical analysis included the GAB and GT equation fitting, analysis of variance (ANOVA), and two-sided t-tests.

Water sorption isotherms showed compositional dependence and crystallization of glucose, influenced by WPI content and a_w. T_g values were composition-dependent, increasing with WPI content. DMA analysis revealed that α-relaxation times decreased with increasing a_w and decreased with increasing WPI content. The Strength parameter (S) increased with WPI content but decreased with a_w. Classical thermodynamic discussion was used to explain the compositional dependence of S, relating k_sp and k_GT to changes in heat capacities. The novel parameter, water usability (U_w), which reflects the difference in mobility between water in the matrix and liquid water, was introduced. Microbial growth of *D. hansenii* was significantly affected by U_w. Higher U_w values correlated with increased growth rates (µ) and decreased doubling times (g). SEM images showed that *D. hansenii* grew both on the surface and within the porous structure of the freeze-dried samples, resulting in structural collapse at high a_w and WPI content.

This study demonstrates that water mobility, as reflected by U_w, is a superior predictor of microbial growth in low-moisture food compared to a_w alone. The findings underscore the importance of considering not only the amount of water but also its dynamic state and mobility in predicting microbial behavior. The classical thermodynamic discussion clarifies the relationship between the empirically determined parameters (k_sp and k_GT) and the thermodynamic properties of the system. The introduction of U_w provides a more comprehensive and nuanced understanding of the water-microorganism interactions in food preservation. Further research should explore the applicability of U_w to other microorganisms and food matrices to validate its broader utility.

This research successfully introduced water usability (U_w) as a novel parameter for predicting microbial growth in low-moisture foods, surpassing the limitations of a_w. The results highlight the critical role of water mobility dynamics in microbial behavior. Future studies should expand the application of U_w to various food systems and microorganisms, explore its correlation with other microbial growth parameters (lag phase, metabolic activity), and investigate its potential applications in developing improved food preservation strategies.

The study focused on a single yeast species (*D. hansenii*) and a specific model food system (glucose/WPI). The generalizability of the findings to other microorganisms and food matrices requires further investigation. The lyophilization process could have introduced artifacts affecting water mobility. Future research should explore additional methodologies to validate the U_w parameter.