Design of an Experimental Viscoelastic Food Model System for Studying Zygosaccharomyces bailii Spoilage in Acidic Sauces

Within the field of predictive microbiology, the number of studies that quantify the effect of food structure on microbial behavior is very limited. This is mainly due to impracticalities related to the use of a nonliquid growth medium. In this study, an experimental food model system for studying yeast spoilage in acid sauces was developed by selecting a suitable thickening/gelling agent. In a first step, a variety of thickening/gelling agents was screened, with respect to the main physicochemical (pH, water activity, and acetic acid and sugar concentrations) and rheological (weak gel viscoelastic behavior and presence of a yield stress) characteristics of acid sauces. Second, the rheological behavior of the selected thickening/gelling agent, Carbopol 980, was extensively studied within the following range of conditions: pH 4.0 to 5.0, acetic acid concentration of 0 to 1.0% (vol/vol), glycerol concentration of 0 to 15% (wt/vol), and Carbopol concentration of 1.0 to 1.5% (wt/vol). Finally, the applicability of the model system was illustrated by performing growth experiments in microtiter plates for Zygosaccharomyces bailii at 0, 0.5, 1.0, and 1.5% (wt/vol) Carbopol, 5% (wt/vol) glycerol, 0% (vol/vol) acetic acid, and pH 5.0. A shift from planktonic growth to growth in colonies was observed when the Carbopol concentration increased from 0.5 to 1.0%. The applicability of the model system was illustrated by estimating μ_max at 0.5% Carbopol from absorbance detection times.Food structure is, next to the chemical composition and storage conditions, one of the key factors that affect microbial behavior in food products. The effects of food structure are mainly related to the mechanical distribution of water, the chemical redistribution of organic acids, and the mobility of microorganisms (55). In the case of a liquid food product, microbial growth is typically planktonic, and transport of nutrients and metabolites occurs by diffusion, resulting in a homogeneous environment. The majority of foods, however, have some degree of structure, causing microorganisms to be immobilized and constrained to grow as colonies. Within the field of predictive microbiology, where mathematical models are developed for describing microbial growth, inactivation, and survival in food (model systems), most models are based on data obtained in liquid broth media. The scarcity of predictive models that incorporate the effect of structure has been recognized as one of the most important shortcomings in this field of research (41).Conducting experiments on a structured culture medium gives rise to several impracticalities due to the nonliquid nature of the culture medium. Starting from a liquid culture medium, a thickening or gelling agent is added to obtain a structured model system that, ideally, mimics the microstructural properties of the target food product. In order to evaluate the effect of structure on a systematic and consistent basis, the experimental setup must enable careful control and sampling methods. A widely used experimental setup is the gel cassette system (Institute of Food Research, Norwich, United Kingdom). The system, described by Brocklehurst et al. (7, 8), consists of a frame sealed with gas-permeable plastic film. This setup has been used to study growth behavior both on the surface (8, 24) and within the gel matrix (7, 10, 30, 45, 51) by applying traditional microbiological methods or noninvasive microscopical techniques (29, 46). In most of these cases, gelatin was used to induce a gelled microstructure. Other experimental setups used agar or gelatin gels in petri dishes (1, 2, 3, 48) or studied bacterial growth in oil-in-water emulsions (9, 38, 39).The existing experimental model systems most often make use of agar or gelatin as these are widely used gelling agents within the field of microbiology. The main reasons for using agar are its stability at sterilization temperatures, high clarity, nontoxic nature, and physiologically inert behavior toward microorganisms. Gelatin has the advantage of a lower melting point (37°C compared to 85°C for agar), which facilitates sampling procedures. Main drawbacks include the possible metabolization by microorganisms and breakdown of structure during autoclaving. Although both agar and gelatin are widely used in food applications, their relevance is limited to food products with a gelled microstructure. Expanding structured food model systems to a wider range of food products, therefore, implies the use of other thickening or gelling agents. In the past, several attempts have been made to use food hydrocolloids as substitutes for agar and gelatin as solidifying agents in microbiological media (4, 19, 25, 35, 44, 53). The functional properties of a food hydrocolloid depend on its origin, preparation method, thermal processing, and environmental conditions, such as salt content, pH, and temperature (20). In choosing a food hydrocolloid for a structured food model system, the physical and chemical nature of the target food product must therefore be taken into account.So far, most studies in the field of predictive microbiology mention only the concentration of the gelling agent as a quantitative measure of food structure. Within our research group, we introduced the use of rheological properties as a more objective way to relate structural characteristics to microbial behavior (51). This allows comparisons between food model systems based on different thickening/gelling agents and accounts implicitly for the variability in structure between different brands or processing methods of the same gelling/thickening agent. Rheology quantifies the relation between stress and flow of materials, but its concepts can also be used to analyze behavior “at rest” (31). Its methods are widely used in the food industry as they are essential tools in product development, quality control, sensory evaluation, and the design of processing equipment (49).Among the wide range of existing foodstuffs, sauces are known for their complex microstructure and typical rheological properties. The group of acid sauces includes both emulsions, such as mayonnaise and salad dressings, and concentrated suspensions, such as ketchup. These sauces are viscoelastic, i.e., they have both viscous and elastic properties, and typically show non-Newtonian flow behavior, characterized by the presence of a yield stress. Yield stress is the minimum shear stress required to initiate flow and is an indication of the suspension abilities of a fluid. Other characteristics of acid sauces are low pH, low water activity (a_w), and the presence of organic acid preservatives. Due to this harsh environment, spoilage is predominantly caused by yeasts and lactic acid bacteria (22). Within this range of microorganisms, Zygosaccharomyces bailii is particularly troublesome because of its high resistance toward organic acid preservatives and its osmophilic behavior (26).The objective of this research is to develop an experimental viscoelastic food model system for acid sauces. In a first step, a set of requirements is formulated, taking into account the main physicochemical properties and viscoelastic characteristics of the target food product. Several thickening and gelling agents are screened and evaluated with respect to these requirements, and a suitable thickening/gelling agent is selected. As a last step, the applicability of the model system is tested by performing growth experiments for the spoilage yeast Z. bailii.