## 4 Protein Quality Evaluation Methods
### In Vitro Models
#### 4.3.2 Static Methods  
Static and dynamic models are the two primary types of in vitro digestion methods that are typically utilized. In vitro digestion methods are frequently employed to investigate food behavior under simulated upper gastrointestinal tract physiological conditions, encompassing the oral, gastric, and small intestinal phases [89]. Dynamic models have been demonstrated as effective tools for simulating food and pharmaceutical product digestion across various groups and purposes [28]. However, their complexity and the cost of implementation and maintenance limit access to these models for many food researchers. In contrast, static in vitro digestion models have shown great potential in predicting in vivo digestion outcomes [9; 90], making them valuable tools in the field. Static models have been extensively employed for food, animal feed, and pharmaceutical purposes due to their simplicity. The static in vitro models offer several significant advantages, including excellent outstanding intra- and inter-laboratory repeatability, sturminess, easiness, low cost, and the ability to easily assess each phase of digestion. Static models employ a fixed ratio of food, enzymes, and electrolytes, as well as a consistent pH corresponding to each stage of digestion. In vitro digestion models have demonstrated significant efficacy in predicting in vivo digestion outcomes [89]. Standardized static models of varying complexity are available to simulate the gastrointestinal performance of pharmaceutical products, as outlined in United States Pharmacopeia protocols [9]. However, various static models exhibit different modifications in variables such as pH, time, the activity and concentration of enzyme, and simulated digestive fluid composition. To address the issue of diverse static models with varying parameters, the international INFOGEST network of multidisciplinary experts was established. Their first goal was to establish a standardized set of digestion parameters for static in vitro simulation of adult digestion, suitable for food. By doing so, INFOGEST aimed to harmonize these methods and facilitate their use in food and nutritional research [91]. For static digestion methods, foods are exposed to three consecutive phases, oral, gastric, and intestinal, with experimental conditions remaining constant throughout each phase. During the oral phase, the food is diluted 1:1 (\(w/w\)) with simulated salivary fluid (SSF), with or without the inclusion of salivary amylase. For solids or semisolids, simulated mastication of the food is also performed [92]. If salivary amylase is used, the food is exposed to it for a maximum of 2 min at pH 7. The oral phase is a mandatory component of all simulated digestion procedures, irrespective of the food's state (liquid or solid), to ensure consistent dilution. This phase plays a vital role in the initial breakdown of food and the hydrolysis of macronutrients, especially carbohydrates, by salivary enzymes, thereby influencing the subsequent phases of protein digestion. After completion of the oral phase, the resulting bolus is diluted with simulated gastric fluid (SGF) and gastric enzymes, including pepsin and gastric lipase, in a 1:1 (\(v/v\)) ratio. The mixture is then subjected to agitation and incubated at pH 3.0 for 2 h to simulate the gastric phase of protein digestion. Subsequently, the gastric chyme is diluted into simulated intestinal fluid (SIF), bile salts, and pancreatic enzymes (pancreatin, based on trypsin activity or each enzyme) in a 1:1 (\(v/v\)) ratio, followed by incubation at pH 7 for another 2 h to mimic the intestinal phase of protein digestion [93]. The final step in protein digestion includes the sampling, treatment, storage, and analysis of the digested samples. It is crucial to carefully consider this step before commencing digestion as it may vary depending on the purpose of the experiment, the type of analysis, and the desired endpoints [89].