• Delivery of β-carotene to the in vitro intestinal barrier using nanoemulsions with lecithin or sodium caseinate as emulsifiers

      Gasa-Falcon, Ariadna; Arranz, Elena; Odriozola-Serrano, Isabel; Martín-Belloso, Olga; Giblin, Linda; Ministerio de Economía y Competitividad; Enterprise Ireland; European Union; Science Foundation Ireland; Agencia de Gestio d’Ajuts Universitaris I de Recerca; et al. (Elsevier BV, 2021-01)
      To increase the intestinal delivery of dietary β-carotene, there is a need to develop nanostructured food systems to encapsulate this fat soluble bioactive. The aim of this study was to evaluate the bioacessibility and bioavailability across the intestinal barrier of β-carotene-enriched nanoemulsions stabilised with two emulsifiers (lecithin or sodium caseinate) by coupling an in vitro gastrointestinal digestion with two in vitro cell culture models (Caco-2 or co-culture of Caco-2/HT29-MTX). Nanoemulsions stabilised with lecithin had significantly higher β-carotene in the gastrointestinal digested micellar fraction, lower β-carotene in the Caco-2 (and Caco-2/HT29-MTX) apical compartment and significantly higher β-carotene in Caco-2 cellular content compared to β-carotene-enriched nanoemulsions stabilised with sodium caseinate. Finally, to assess anti-inflammatory activity of digested nanoemulsions, lipopolysaccharide stimulated macrophages were exposed to Caco- 2 basolateral samples with levels of TNF-α and IL-β, subsequently quantified. A TNF-α response from stimulated THP-1 macrophages was elicited by basolateral samples, regardless the emulsifier used to formulate nanoemulsions. This study demonstrated that β-carotene permeability is influenced by the food derived emulsifier used for stabilising nanoemulsions, indicating that composition may be a critical factor for β-carotene delivery.
    • Delivery of β-carotene to the in vitro intestinal barrier using nanoemulsions with lecithin or sodium caseinate as emulsifiers

      Gasa-Falcon, Ariadna; Arranz, Elena; Odriozola-Serrano, Isabel; Martín-Belloso, Olga; Giblin, Linda; Fondo Europeo de Desarrollo Regional; Ministerio de Economía y Competitividad; European Union; Enterprise Ireland; Science Foundation Ireland; et al. (Elsevier, 2021-01-13)
      To increase the intestinal delivery of dietary β-carotene, there is a need to develop nanostructured food systems to encapsulate this fat soluble bioactive. The aim of this study was to evaluate the bioacessibility and bioavailability across the intestinal barrier of β-carotene-enriched nanoemulsions stabilised with two emulsifiers (lecithin or sodium caseinate) by coupling an in vitro gastrointestinal digestion with two in vitro cell culture models (Caco-2 or co-culture of Caco-2/HT29-MTX). Nanoemulsions stabilised with lecithin had significantly higher β-carotene in the gastrointestinal digested micellar fraction, lower β-carotene in the Caco-2 (and Caco-2/HT29-MTX) apical compartment and significantly higher β-carotene in Caco-2 cellular content compared to β-carotene-enriched nanoemulsions stabilised with sodium caseinate. Finally, to assess anti-inflammatory activity of digested nanoemulsions, lipopolysaccharide stimulated macrophages were exposed to Caco- 2 basolateral samples with levels of TNF-α and IL-β, subsequently quantified. A TNF-α response from stimulated THP-1 macrophages was elicited by basolateral samples, regardless the emulsifier used to formulate nanoemulsions. This study demonstrated that β-carotene permeability is influenced by the food derived emulsifier used for stabilising nanoemulsions, indicating that composition may be a critical factor for β-carotene delivery.
    • The harmonized INFOGEST in vitro digestion method: From knowledge to action

      Egger, Lotti; Ménard, Olivia; Delgado-Andrade, Cristina; Alvito, Paula; Assunção, Ricardo; Balance, Simon; Barberá, Reyes; Brodkorb, Andre; Cattenoz, Thomas; Clemente, Alfonso; et al. (Elsevier, 2015-12-08)
      Within the active field of in vitro digestion in food research, the COST Action INFOGEST aimed to harmonize in vitro protocols simulating human digestion on the basis of physiologically inferred conditions. A harmonized static in vitro digestion (IVD) method was recently published as a primary output from this network. To validate this protocol, inter-laboratory trials were conducted within the INFOGEST network. A first study was performed using skim milk powder (SMP) as a model food and served to compare the different in-house digestion protocols used among the INFOGEST members. In a second inter-laboratory study applying the harmonized protocol, the degree of consistency in protein hydrolysis was investigated. Analysis of the hydrolyzed proteins, after the gastric and intestinal phases, showed that caseins were mainly hydrolyzed during the gastric phase, whereas β-lactoglobulin was, as previously shown, resistant to pepsin. Moreover, generation of free amino acids occurred mainly during the intestinal phase. The study also showed that a few critical steps were responsible for the remaining inter-laboratory variability. The largest deviations arose from the determination of pepsin activity. Therefore, this step was further clarified, harmonized, and implemented in a third inter-laboratory study. The present work gives an overview of all three inter-laboratory studies, showing that the IVD INFOGEST method has led to an increased consistency that enables a better comparability of in vitro digestion studies in the future.
    • The harmonized INFOGEST in vitro digestion method: From knowledge to action

      Egger, Lotti; Ménard, Olivia; Delgado-Andrade, Cristina; Alvito, Paula; Assunção, Ricardo; Balance, Simon; Barberá, Reyes; Brodkorb, Andre; European Union; Fundação para a Ciência e Tecnologia; et al. (Elsevier BV, 2015-12)
      Within the active field of in vitro digestion in food research, the COST Action INFOGEST aimed to harmonize in vitro protocols simulating human digestion on the basis of physiologically inferred conditions. A harmonized static in vitro digestion (IVD) method was recently published as a primary output from this network. To validate this protocol, inter-laboratory trials were conducted within the INFOGEST network. A first study was performed using skim milk powder (SMP) as a model food and served to compare the different in-house digestion protocols used among the INFOGEST members. In a second inter-laboratory study applying the harmonized protocol, the degree of consistency in protein hydrolysis was investigated. Analysis of the hydrolyzed proteins, after the gastric and intestinal phases, showed that caseins were mainly hydrolyzed during the gastric phase, whereas β-lactoglobulin was, as previously shown, resistant to pepsin. Moreover, generation of free amino acids occurred mainly during the intestinal phase. The study also showed that a few critical steps were responsible for the remaining inter-laboratory variability. The largest deviations arose from the determination of pepsin activity. Therefore, this step was further clarified, harmonized, and implemented in a third inter-laboratory study. The present work gives an overview of all three inter-laboratory studies, showing that the IVD INFOGEST method has led to an increased consistency that enables a better comparability of in vitro digestion studies in the future.
    • In vitro digestion of protein-enriched restructured beef steaks with pea protein isolate, rice protein and lentil flour following sous vide processing

      Baugreet, Sephora; Gomez, Carolina; Auty, Mark; Kerry, Joseph P.; Hamill, Ruth; Brodkorb, Andre; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 11/F/045 (Elsevier, 2019-04-12)
      The effect of plant protein inclusion in cooked meat upon in vitro gastro-intestinal (GI) digestion was investigated. Pea protein isolate, rice protein and lentil flour were used to increase the protein content in a meat model system restructured using two transglutaminase enzymes [Activa®EB (TG) and Transgluseen™-M (TS)]. Restructured beef steaks were subjected to simulated GI digestion using the static INFOGEST method. Samples taken at different digestion times were analysed using SDS-PAGE, size exclusion-HPLC, free amino acid analysis and microscopy. SDS-PAGE analysis revealed significant protein hydrolysis during GI digestion. Most soluble peptides had a molecular weight smaller than 500 Da, corresponding to peptides of <5 amino acids, regardless of food treatment. The amounts of released, free amino acids isoleucine, lysine, phenylalanine and valine were higher (P < 0.05) in lentil-enriched restructured beef steaks following GI digestion. Confocal laser scanning microscopy (CSLM) revealed pronounced aggregation in digested samples. In vitro digestates of protein-enriched restructured beef steaks showed lower production of small molecular weight peptides. This study demonstrated how the bioaccessibility of protein-enriched restructured beef steaks are influenced by formulation and processing.
    • Monitoring molecular composition and digestibility of ripened bresaola through a combined foodomics approach

      Picone, Gianfranco; De Noni, Ivano; Ferranti, Pasquale; Nicolai, Maria Adalgisa; Alamprese, Cristina; Trimigno, Alessia; Brodkorb, Andre; Portmann, Reto; Pihlanto, Anne; El, Sedef Nehir; et al. (Elsevier, 2018-11-14)
      In this work, the effects of maturation time and simulated gastrointestinal digestion on the molecular and peptide profiles of “Bresaola Valtellina” were assessed through the foodomics approach, in this case food proteomics and peptidomics combined to other analytical and biological assays, aiming at depicting a holistic food quality. Human digestion of this Italian cured meat product was simulated using an in vitro static protocol and the degree of proteolysis and the in vitro bioactivity of the soluble free compounds in the digestates were evaluated by biochemical assays, e.g. SDS-PAGE, size exclusion HPLC, HPLC/MS, 1H NMR, enzymatic and antioxidant activities. The obtained results demonstrated that in vitro gastrointestinal digestion contributed to a considerable release of myofibrillar proteins by the muscle tissue. Data from SDS-PAGE, peptidomic and size exclusion HPLC assays showed that the in vitro digestion largely degraded proteins of muscle tissue to peptides smaller than 250 Da. The released peptides were likely responsible for the inhibitory activity on amylolytic enzymes and for the antioxidant properties elicited by the gastric digestates of Bresaola. Overall, the results demonstrated the negligible role of ripening in making meat proteins more bioaccessible, whereas they confirmed the highly in vitro digestibility of meat proteins from Bresaola. This study represents a new approach merging proteomics and foodomics to evaluate the effect of ripening and in vitro digestion on the bioactivity and bioaccessibility of proteins and peptides of meat products.
    • Simulated gastrointestinal digestion of nisin and interaction between nisin and bile

      Gough, Ronan; O'Connor, Paula M.; Rea, Mary; Gomez-Sala, Beatriz; Miao, Song; Hill, Colin; Brodkorb, Andre; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 10/RD/TMFRC/701; et al. (Elsevier, 14/08/2017)
      Nisin, an antimicrobial peptide showing activity against many Gram positive bacteria, is widely used as a food preservative. The simulated gastrointestinal digestion of nisin (variant A) was studied using the in vitro INFOGEST digestion method. Following oral, gastric and small intestinal digestion, there was no intact nisin in the system and the nisin was primarily digested by pancreatin. After digestion, six nisin fragments (1–11, 1–12, 1–20, 1–21, 1–29 and 1–32) were identified by reversed phase high performance liquid chromatography and mass spectroscopy and four of these nisin fragments (1–20, 1–21, 1–29 and 1–32) demonstrated low antibacterial activity against Lactococcus lactis HP in agar diffusion activity assays. Additionally, it was observed that bile salts form a complex with nisin. This was examined by atomic force microscopy, turbidity and dynamic light scattering, which showed that this interaction resulted in significantly larger bile salt micelles. The presence of bile salts at physiological levels significantly altered the relative amounts of the nisin fragments 1–12, 1–20 and 1–29 produced during an in vitro digestion. This study highlights the importance of including bile in simulated digestions of antimicrobial peptides in order to obtain a more accurate simulation of the in vivo digestion products and their activity.
    • Simulated gastrointestinal digestion of nisin and interaction between nisin and bile

      Gough, Ronan; O'Connor, Paula M.; Rea, Mary; Gomez-Sala, Beatriz; Miao, Song; Hill, Colin; Brodkorb, Andre; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 10/RD/TMFRC/701 (Elsevier, 2017-08-14)
      Nisin, an antimicrobial peptide showing activity against many Gram positive bacteria, is widely used as a food preservative. The simulated gastrointestinal digestion of nisin (variant A) was studied using the in vitro INFOGEST digestion method. Following oral, gastric and small intestinal digestion, there was no intact nisin in the system and the nisin was primarily digested by pancreatin. After digestion, six nisin fragments (1–11, 1–12, 1–20, 1–21, 1–29 and 1–32) were identified by reversed phase high performance liquid chromatography and mass spectroscopy and four of these nisin fragments (1–20, 1–21, 1–29 and 1–32) demonstrated low antibacterial activity against Lactococcus lactis HP in agar diffusion activity assays. Additionally, it was observed that bile salts form a complex with nisin. This was examined by atomic force microscopy, turbidity and dynamic light scattering, which showed that this interaction resulted in significantly larger bile salt micelles. The presence of bile salts at physiological levels significantly altered the relative amounts of the nisin fragments 1–12, 1–20 and 1–29 produced during an in vitro digestion. This study highlights the importance of including bile in simulated digestions of antimicrobial peptides in order to obtain a more accurate simulation of the in vivo digestion products and their activity.