## 4 Protein Quality Evaluation Methods
### In Vivo Models  
In the assessment of protein digestibility, in vivo methods involve feeding trials on animals or humans. Animal models are critical in designing in vivo and ex vivo experimental studies [32]. Controlled feeding experiments with rodents, chickens, or pigs are preferred for obtaining accurate results [33]. Nonetheless, caution must be exercised when extrapolating findings and inferences from animal trials to humans, given the significant differences between the gastrointestinal tracts of the two groups. The scientific community has widely employed animal models to investigate the impact of diet quality on health. Dietary intervention studies in animals are essential for understanding the biological functions of specific nutrients before human validation [32]. In the past century, rats were the most common model for biochemical research due to their simple ileal digestibility model. However, their popularity has decreased in the last decades due to limitations in performing reverse genetics in rats [34]. The key methodology used in  
Figure 1: Schematic representation of the methods for assessing plant protein digestibility. (*) adapted from [PERSON] and [PERSON] [29]; (**) adapted from [PERSON] et al. [30]; (***) adapted from [PERSON] et al. [31]. these studies is the measurement of the digestive losses of nitrogen and amino acids [35]. However, to minimize the impact of colonic microbiota activity, it is important to assess nitrogen and amino acid losses in the ileum content rather than in the faeces, since amino acids are absorbed in the small intestine. Nonetheless, no standardized rat model method is currently available to evaluate ileal protein digestibility [36]. For instance, [PERSON] et al. [37] evaluated the protein digestibility of microalgae species. For this study, the researchers used a rat model and fed rats with three different microalgae species (_Chlorella vulgaris_, _Chlorella sorokiniana_, and _Acutodesmus obliquus_) for 3 days. After that, faeces were collected from individual rats for 5 days, with a daily recording of food intake. The collected faeces were then pooled and stored at \(-80\)\({}^{\circ}\)C for further analysis. The results revealed that the true protein digestibility of _Chlorella vulgaris_ was 64.7%, 59.3% for _Chlorella sorokiniana_, and 37.9% for _Acutodesmus obliquus_. In a recent study, [PERSON] et al. [34] investigated the digestibility of gluten and pea protein using the cecal digestibility technique in rats as an alternative to the ileal digestibility method. The rationale for this alternative is that the caecum of rodents is large and accumulates digesta from the small intestine before it enters the colon [38]. As a result, a considerable amount of dietary nitrogen and amino acid losses could be retained in the caecum after a sufficient digestion period, potentially eliminating the necessity for an indigestible marker [34]. In this study, the rats were first fed with a standard chow diet for six days to adapt to the experimental conditions. Following that, they were trained to consume a calibrated meal of 4 g dry weight containing 16 mg of Cr\({}_{2}\)O\({}_{3}\) (10.9 mg of chromium) in a short time at the beginning of the dark period. After 10 days of habituation to the diets, the rats were given a calibrated meal of their respective diets and euthanized six hours later using gaseous anesthesia and decapitation. The study's primary finding suggested that true caecal digestibility could be an adequate approximation of true ileal digestibility and eliminate the need for a non-absorbable marker, provided that the nitrogen amount in each digestive tract segment is known. In an ideal scenario, the true ileal protein digestibility of foods should be determined in humans using techniques such as naso- ileal tube sampling [39]. However, due to practical and ethical limitations, this method is not recommended for routine studies. Alternatively, ileum-fistulated growing pigs are used as animal models, as recommended by [PERSON] et al. [40].