Protein is an essential macronutrient in our diet, source of nitrogen and essential amino acids, but the biological utilization of dietary protein depends on its digestibility and the absorption of amino acids and peptides in the gastrointestinal tract. The methods to define the amount and the quality of protein to meet human nutritional needs, such as the Digestible Indispensable Amino Acid Score (DIAAS), require the use of animal models or human studies. These in vivo methods are the reference in protein quality evaluation, but they are expensive and long-lasting procedures with significant ethical restrictions. Therefore, the development of rapid, reproducible and in vitro digestion methods validated with in vivo data is an old demand. This review describes the challenges of the in vitro digestion methods in the evaluation of the protein nutritional quality. In addition to the technical difficulties to simulate the complex and adaptable processes of digestion and absorption, these methods are affected by similar limitations as the in vivo procedures, i.e., analytical techniques to accurately determine bioavailable amino acids and the contribution of the endogenous nitrogen. The in vitro methods used for the evaluation of protein digestibility, with special attention on those showing comparative data, are revised, emphasizing their pros and cons. The internationally harmonized digestion protocol proposed by the INFOGEST network is being adapted to evaluate protein and amino acid digestibility. The inter-laboratory reproducibility of this protocol was demonstrated for dairy products. The in vivo/in vitro comparability results obtained to date with this protocol for several plant and animal sources are promising, but it requires an extensive validation with a wider range of foods and substrates with known in vivo digestibility. These in vitro methods will probably not be applicable to all foods, and therefore, it is important to identify their limitations, not to elude their use, but to apply them within the limits, by using the appropriate standards and references, and always as a complementary tool to in vivo tests to reduce their number.

Tea plants have a long cultivation history in the world, but there are few studies on polysaccharides from fresh tea leaves. In this study, tea polysaccharides (TPSs) were isolated from fresh tea leaves. Then, we investigated the characteristics of TPSs during in vitro simulated digestion and fermentation; moreover, the effects of TPSs on gut microbiota were explored. The results revealed that saliva did not significantly affect TPSs\' molecular weight, monosaccharide composition, and reducing sugar content, indicating that TPSs cannot be digested in the oral cavity. However, TPSs were partially decomposed in the gastrointestinal tract after gastric and intestinal digestion, resulting in the release of a small amount of free glucose monosaccharides. Our in vitro fermentation experiments demonstrated that TPSs are degraded by gut microbiota, leading to short-chain fatty acid (SCFA) production and pH reduction. Moreover, TPSs increased the abundance of Bacteroides, Lactobacillus, and Bifidobacterium but reduced that of Escherichia, Shigella, and Enterococcus, demonstrating that TPSs can regulate the gut microbiome. In conclusion, TPSs are partially decomposed by gut microbiota, resulting in the production of SCFAs and the regulation of gut microbiota composition and function. Therefore, TPSs may be used to develop a prebiotic supplement to regulate the gut microbiome and improve host health.

Quercetin (Q) dietary supplements exhibit poor oral bioavailability because of degradation throughout gastrointestinal digestion (GD), which may be overcome using mesoporous silica particles (MSPs) as an oral delivery system (ODS). This study aimed to elucidate the effect of the functionalization of MSPs with amine-(A-MSP), carboxyl-(C-MSP), or thiol-(T-MSP) groups on their efficiency as a quercetin ODS (QODS). The type and degree of functionalization (DF) were used as factors in an experimental design. The Q-loaded F-MSP (F-MSP/Q) was characterized by gas physisorption analysis, loading capacity (LC), and dynamic light scattering and kinetics of Q release at gastric and intestinal pHs. Antioxidant capacity and Q concentration of media containing F-MSP/Q were evaluated after simulated GD. A-MSP showed the highest LC (19.79 ± 2.42%). C-MSP showed the lowest particle size at pH 1.5 or 7.4 (≈200 nm). T-MSP exhibited the maximum Q release at pH 7.4 (11.43%). High DF of A-MSP increased Q retention, regardless of pH. A-MSP preserved antioxidant capacity of Q-released gastric media (58.95 ± 3.34%). Nonetheless, MSP and F-MSP did not protect antioxidant properties of Q released in intestinal conditions. C-MSP and T-MSP showed essential features for cellular uptake and Q release within cells that need to be assessed.

The bioaccessibility and bioavailability of phenolic compounds (PC) influence directly their role in disease prevention/control. Studies have evaluated this ability through complex plant and food matrices, which may reflect more a synergistic effect of the matrix than the ability of the PCs, hindering their individual exploitation in nutraceutical or pharmaceutical applications. In the present study ten pure PCs representing major classes were evaluated for their bioaccessibility and intestinal absorption in an in vitro simulated gastrointestinal digestion (SGD). This is the first study concerning the bioaccessibility evaluation of pure phloretin, phloroglucinol, naringin, naringenin and daidzein, while no in vitro SGD has been performed before for the other compounds considered here. PCs were analyzed through ultra-high-performance liquid chromatography coupled with diode-array detection and tandem mass spectrometry (UHPLC-DAD-MSn). Most of the compounds remained present along the gastrointestinal tract, and the bioaccessibility was in general higher than 50%, except for quercetin, epigallocatechin gallate, and ellagic acid. All compounds were highly absorbed in the intestine, with phloretin showing the lowest percentage at about 82%. The study findings provide new knowledge on the bioaccessibility and intestinal absorption of different PCs classes.

This study evaluated the impact of in vitro gastrointestinal digestion on the digestibility, amino acid release, and antioxidant activity of proteins from amaranth (Amarantus cruentus L.) and cañihua (Chenopodium pallidicaule Aellen). Antioxidant activity was assessed using ORAC, ABTS, DPPH, and cellular antioxidant activity (CAA) assays in human intestinal Caco-2 and hepatic Hep-G2 cell lines. The results showed that amaranth had higher protein digestibility (79.19%) than cañihua (71.22%). In addition, intestinal digestion promoted the release of essential amino acids, such as leucine, lysine, and phenylalanine, in both protein concentrates. Concentrations of amaranth and cañihua proteins, ranging from 0.125 to 1.0 mg mL-1, were non-cytotoxic in both cell lines. At a concentration of 0.750 mg mL-1, simulated gastrointestinal digestion enhanced cellular antioxidant activity. Intestinal digest fractions containing peptides >5 kDa were the principal contributors to CAA in both cell lines. Notably, cañihua proteins exhibited high CAA, reaching values of 85.55% and 82.57% in Caco-2 and HepG2 cells, respectively, compared to amaranth proteins, which reached 84.68% in Caco-2 and 81.06% in HepG2 cells. In conclusion, both amaranth and cañihua proteins, after simulated gastrointestinal digestion, showcased high digestibility and released peptides and amino acids with potent antioxidant properties, underscoring their potential health benefits.

Bioactive compounds in red fruits, such as strawberries, are vulnerable to digestion, and encapsulation has become an alternative for their protection. This study aims at encapsulating strawberry juice (SJ) by freeze-drying with pea protein and okra mucilage (SJPO), pea protein and psyllium mucilage (SJPP), and pea protein, psyllium mucilage, and okra mucilage (SJPPO) and investigating the in vitro release. The highest encapsulation efficiency was observed in capsule SJPPO (95.38%) and the lowest efficiency in SJPO (82.45%). Scanning electron microscopy revealed an amorphous glassy structure for the structure of the strawberry microcapsules, and X-ray diffraction confirmed that observation. However, X-ray diffraction further showed that SJPPO was crystalline, indicating a tighter crosslinking density than the other microcapsules. Fourier transform infrared spectroscopy showed peaks at 3390 and 1650 cm-1, confirming the presence of polyphenols and polysaccharides in the strawberry microcapsules. Thermal stability was higher for SJPPO, and the observed thermal transitions were due to the bonds formed between the polymers and polyphenols. Pelargonidin 3-glucoside, cyanidin 3-glucoside, cyanidin, delphinidin, malvidin 3-glucoside, ellagic acid, chlorogenic acid, catechin, and kaempferol were identified in the strawberry microcapsules. Digestion affected the compounds\' content; the bioaccessibility for SJ was 39.26% and 45.43% for TPC and TAC, respectively. However, encapsulation improved the bioaccessibility of both TPC (SJPP, 51.54%; SJPO, 48.52%; and SJPPO, 54.39%) and TAC (SJPP, 61.08%; SJPO, 55.03%; and SJPPO, 71.93%). Thus, encapsulating pea protein isolate, psyllium mucilage, and okra mucilage is an effective method to facilitate targeted release and preserve the biological activities of fruits.

To explore the effect of different microbial strains on blueberry pomace with solid-state fermentation (SSF), three fungi strains and three lactic acid bacteria (LAB) strains were utilized to investigate with respect to polyphenol profiles, antioxidant capacities, and bioaccessibility. Different strains exhibited different capacities for metabolizing polyphenolic compounds in blueberry pomace. The contents of 10 phenolic acids and 6 flavonoids (except (+)-catechin) were increased in blueberry pomace fermented by Lactobacillus acidophilus (LA). A similar tendency was observed in blueberry pomace fermented by Aspergillus niger (AN) and Lactobacillus plantarum (LP), where the concentration of 8 phenolic acids and 5 flavonoids was enhanced, with the following exceptions: (+)-catechin, ferulic acid, vanillic acid, and quercitrin. Chlorogenic acid and quercetin were the maximum phenolic acids and flavonoids in blueberry pomace with SSF, upgraded at 22.96 and 20.16%, respectively. Contrary to the growth of phenolic acids and flavonoid compounds, all individual anthocyanins showed a decreased trend. Only in the blueberry pomace fermented by AN, all anthocyanidins exhibit a rising trend. After SSF, 2,2\'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenylpicrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) radical scavenging abilities were increased by up to 33.56, 59.89, and 87.82%, respectively. Moreover, the simulated gastrointestinal digestion system revealed that SSF improved the bioaccessibility of polyphenolic compounds. Compared with other strains, LA, LP, and AN showed better excellent capacities for metabolizing polyphenolic compounds, which led to a greater increase in antioxidant activity and bioaccessibility in fermented blueberry pomace.

Probiotics are known for their health-promoting properties and are recognized as beneficial microorganisms. The current investigation delves into the isolation and comprehensive in vitro characterization of lactic acid bacteria (LAB) obtained from the Indian-origin Theobroma cacao L. Forastero variety to assess their potential as probiotic candidates. Eleven LAB isolates were obtained, and among them, five exhibited classical LAB traits. These five isolates underwent rigorous in vitro characterization to evaluate their suitability as probiotics. The assessments included resilience against acid and bile salts, which are crucial for probiotic viability. Additionally, the isolates were subjected to simulated gastric and pancreatic fluids and lysozyme exposure to assess their survival rates. Auto- aggregation, co-aggregation, hydrophobicity, and exopolysaccharide production were also examined. The inhibitory potential of α-glucosidase, an enzyme related to glucose metabolism, was measured, and antioxidant activity was evaluated using DPPH and ABTS assays. A safety assessment was conducted to confirm the non-pathogenic nature of the isolates. Among the five isolates, CR2 emerged as a standout candidate with maximal bile salt hydrolase activity, phenol resistance, and lysozyme resistance. CR2 and CYF3 exhibited notable survival rates under simulated conditions. The isolates displayed variable degrees of auto-aggregation, co-aggregation, and hydrophobicity. CR2 exhibited the highest exopolysaccharide production (0.66 mg/mL), suggesting diverse applications in the food industry. CR2 also demonstrated the highest inhibition rate against α-glucosidase (56.55%) and substantial antioxidant activity (79.62% DPPH, 83.45% ABTS). Safety assessment confirmed the non- pathogenic nature of the isolates. Molecular characterization identified CR2 as Lactococcus lactis subsp. lactis and CYF3 as Limnosilactobacillus fermentum. Both strains exhibited commendable probiotic and technological attributes, positioning them as promising candidates for functional foods and beyond. This study provides valuable insights into the in vitro characterization of LAB isolated from Indian Theobroma cacao L., highlighting their potential as probiotic candidates with advantageous traits, including survival in hostile conditions, beneficial enzymatic activities, bioactivity, and other essential attributes.

The study aimed to investigate the effects of alcalase, papain, flavourzyme, and neutrase on the structural characteristics and bioactivity stability of Cucumaria frondosa intestines and ovum hydrolysates (CFHs). The findings revealed that flavourzyme exhibited the highest hydrolysis rate (51.88% ± 1.87%). At pH 2.0, the solubility of hydrolysate was the lowest across all treatments, while the solubility at other pH levels was over 60%. The primary structures of hydrolysates of different proteases were similar, whereas the surface hydrophobicity of hydrolysates was influenced by the types of proteases used. The hydrolysates produced by different proteases were also analyzed for their absorption peaks and antioxidant activity. The hydrolysates of flavourzyme had β-fold absorption peaks (1637 cm-1), while the neutrase and papain hydrolysates had N-H bending vibrations. The tertiary structure of CFHs was unfolded by different proteases, exposing the aromatic amino acids and red-shifting of the λ-peak of the hydrolysate. The alcalase hydrolysates showed better antioxidant activity in vitro and better surface hydrophobicity than the other hydrolysates. The flavourzyme hydrolysates displayed excellent antioxidant stability and pancreatic lipase inhibitory activity during gastrointestinal digestion, indicating their potential use as antioxidants in the food and pharmaceutical industries.

Raw bars have become popular among health-conscious consumers due to their nutrient-dense ingredients and lack of additives and preservatives. However, the effect of simulated gastrointestinal digestion on the nutrient content of these bars has yet to be extensively studied. In this study, four different raw bar recipes were subjected to simulated gastrointestinal digestion to evaluate the impact on their nutrient content. The recipes have dates and almond flour as base ingredients and specific ingredients such as Maca root powder, Ginger powder, Aronia powder, Pollen, Propolis extract, Astragalus powder, and Cacao powder. These variations were intended to provide diverse flavors and potential health benefits to cater to different preferences and needs. The in vitro digestion model was designed to mimic the conditions of the human gastrointestinal tract, including the mouth, stomach, and small intestine. The results showed that the simulated gastrointestinal digestion significantly impacted the nutrient content of the bars, with varying degrees of nutrient loss observed depending on the recipe. The highest phenolic content and antioxidant activity were observed in the salivary phase for all samples. Vitamin B content generally decreases from the salivary to the intestinal stage. After digestion, the recovery rates of total phenols, antioxidant capacity, and vitamins B1, B3, and B6 varied across the recipes. The recovery rates of vitamins B1, B3, and B6 were generally high across all recipes, indicating their stability and retention during digestion. The findings suggest that simulated GI digestion provides insights into the nutrient bioavailability of raw bars. These results can inform the formulation and optimization of raw bars to enhance nutrient absorption and nutritional value. Further research is warranted to investigate the effects of different processing techniques and ingredient combinations on nutrient bioavailability.