Isidra Recio

A standardised static in vitro digestion method suitable for food-an international consensus

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Antihypertensive peptides: Production, bioavailability and incorporation into foods

Bioactive food peptides are encrypted within the sequence of food proteins but can be released during food processing (by enzymatic hydrolysis or fermentation) or during gastrointestinal transit. Among bioactive food peptides, those with antihypertensive activity are receiving special attention due to the high prevalence of hypertension in the Western countries and its role in cardiovascular diseases. This paper reviews the current literature on antihypertensive food peptides, focusing on the main methodologies for their production, such as enzymatic hydrolysis, fermentation and the use of recombinant bacteria. This paper also describes the structure/activity relationship of angiotensin-converting enzyme (ACE)-inhibitory peptides, as well as their bioavailability, physiological effects demonstrated by both in vitro and in vivo assays, and the contribution of mechanisms of action other than ACE inhibition. Finally, current reported strategies for incorporation of antihypertensive peptides into foods and their effects on both availability and activity are revised in this manuscript.

Antibacterial and Antiviral Effects of Milk Proteins and Derivatives Thereof

Milk forms a rich source of biologically interesting components. In particular, its protein fraction is known to encompass many kinds of biological functions. In this review we focus on antibacterial and antiviral properties of milk proteins and milk protein derivatives. The latter include chemically modified proteins and enzymatically induced peptides. If such peptides are released by enzymes present within the digestive tract (e.g. trypsin or pepsin), it is likely that they play a role in the health defense system. This is especially the case when the active fragments can survive the intestinal conditions long enough to arrive at the right place to exert their beneficial function. In the first part of this paper attention is paid to the antibacterial proteins lactoferrin, lactoperoxidase, and lysozyme. Furthermore, antibacterial peptides originating from caseins and whey proteins are described. The second part reports on studies of antiviral effects of milk proteins and derivatives thereof. Special focus is directed to the antiviral action towards the human immunodeficiency virus (HIV) and the human cytomegalovirus (HCMV). Unmodified milk proteins are generally not active against these viruses. An exception is lactoferrin, which shows significant antiviral activity against both HIV and HCMV. Several other milk proteins tested showed strong antiviral effects only after chemical modification, i.e. by making them polyanionic (for anti-HIV activity) or polycationic (for anti-HCMV activity). In a number of cases, conclusions are drawn concerning possible relationships between antibacterial/antiviral activity and molecular structure of the components described.

Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis.

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 microg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 microg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 < 40 microg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 microM, respectively.

β-Lactoglobulin as source of bioactive peptides

Summary.β-Lactoglobulin (β-Lg) is currently an important source of biologically active peptides. These peptides are inactive within the sequence of the precursor protein, but they can be released by in vivo or in vitro enzymatic proteolysis. Once released, these peptides play important roles in the human health, including antihypertensive, antioxidant and antimicrobial activities as well as opioid-like features and ability to decrease the body-cholesterol levels. Bioactive peptides derived from β-Lg are currently a point of intensive research. Their structure, biological significance and mechanism of action are briefly presented and discussed in this review.

Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease involved in the degradation and inactivation of incretin hormones that act by stimulating glucose-dependent insulin secretion after meal ingestion. DPP-IV inhibitors have emerged as new and promising oral agents for the treatment of type 2 diabetes. The purpose of this study was to investigate the potential of β-lactoglobulin as natural source of DPP-IV inhibitory peptides. A whey protein concentrate rich in β-lactoglobulin was hydrolysed with trypsin and fractionated using a chromatographic separation at semipreparative scale. Two of the six collected fractions showed notable DPP-IV inhibitory activity. These fractions were analysed by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) to identify peptides responsible for the observed activity. The most potent fragment (IPAVF) corresponded to β-lactoglobulin f(78-82) which IC50 value was 44.7μM. The results suggest that peptides derived from β-lactoglobulin would be beneficial ingredients of foods against type 2 diabetes.

Identification of two distinct antibacterial domains within the sequence of bovine alpha(s2)-casein.

Two distinct domains with antibacterial activity were isolated from a peptic hydrolysate of bovine alpha(s2)-casein. The digested alpha(s2)-casein was fractionated by cation-exchange chromatography, after which the peptides in the two active fractions obtained were separated by high-performance liquid chromatography and sequenced by electrospray-ionization tandem mass spectrometry. The major component in each active fraction, f(183-207) and f(164-179), was further purified and the antibacterial activity of these components was tested against several microorganisms. Depending on the target bacterial strain, these peptides exhibited minimum inhibitory concentrations between 8 and 99 microM. Peptide f(183-207) exhibited a consistently higher antibacterial activity than f(164-179), although both peptides showed a comparable hemolytic effect. A method of in situ enzymatic hydrolysis on a cation-exchange membrane to obtain a fraction enriched in the most active antibacterial domain is presented. The antibacterial and hemolytic activities are discussed in relation to the structure and hydrophobicity of the peptides.

Stability to gastrointestinal enzymes and structure–activity relationship of β-casein-peptides with antihypertensive properties

Physiological digestion plays a key role in the formation and degradation of angiotensin-converting enzyme (ACE)-inhibitory peptides. In this study, we evaluated the impact of a simulated gastrointestinal digestion on the stability of eight peptides previously identified in fermented milk with antihypertensive activity. Two of these identified peptides with sequences LHLPLP and LVYPFPGPIPNSLPQNIPP, possess ACE-inhibitory activity in vitro and antihypertensive activity in vivo. The results showed that LHLPLP was resistant to digestive enzymes. In contrast, LVYPFPGPIPNSLPQNIPP was totally hydrolyzed and its activity decreased after incubation with pepsin and a pancreatic extract. The peptide LHLPLP was incubated with ACE and was found to be a true inhibitor of the enzyme and to exhibit a competitive inhibitor pattern. A structure-activity relationship study of this peptide was carried out by synthesizing several modified peptides related to the sequence LHLPLP. The substitution of amino acid Leu in the penultimate position by Gly improved the ACE-inhibitory activity twofold and the substitution of Pro at C-terminal position by Arg increased the activity twofold, with an IC50 of LHLPLR as low as 1.8 microM.

Potential anti-inflammatory, anti-adhesive, anti/estrogenic, and angiotensin-converting enzyme inhibitory activities of anthocyanins and their gut metabolites

Epidemiological studies have indicated a positive association between the intake of foods rich in anthocyanins and the protection against cardiovascular diseases. Some authors have shown that anthocyanins are degraded by the gut microflora giving rise to the formation of other breakdown metabolites, which could also contribute to anthocyanin health effects. The objective of this study was to evaluate the effects of anthocyanins and their breakdown metabolites, protocatechuic, syringic, gallic, and vanillic acids, on different parameters involved in atherosclerosis, including inflammation, cell adhesion, chemotaxis, endothelial function, estrogenic/anti-estrogenic activity, and angiotensin-converting enzyme (ACE) inhibitory activity. From the assayed metabolites, only protocatechuic acid exhibited a slight inhibitory effect on NO production and TNF-α secretion in LPS-INF-γ-induced macrophages. Gallic acid caused a decrease in the secretion of MCP-1, ICAM-1, and VCAM-1 in endothelial cells. All anthocyanins showed an ACE-inhibitory activity. Delphinidin-3-glucoside, pelargonidin-3-glucoside, and gallic acid showed affinity for ERβ and pelargonidin and peonidin-3-glucosides for ERα. The current data suggest that anthocyanins and their breakdown metabolites may partly provide a protective effect against atherosclerosis that is multi-causal and involves different biochemical pathways. However, the concentrations of anthocyanins and their metabolites, as used in the present cell culture and in vitro assays mediating anti-inflammatory, anti-adhesive, anti-estrogenic, and angiotensin-converting enzyme inhibitory activities, were often manifold higher than those physiologically achievable.

Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin. In situ enzymatic hydrolysis on an ion-exchange membrane.

Two ion-exchange chromatographic methods are reported for the rapid isolation of antibacterial peptides from lactoferrin (LF). Using the first method, a pepsin hydrolysate of LF was fractionated by bead-based cation-exchange chromatography. After removal of weakly bound material by washing with ammonia, highly purified lactoferricin-B (LFcin-B) was obtained in a single step by elution with 2 M NaCl. Some other cationic peptides, copurified as minor components, were also characterised by N-terminal sequencing, mass spectrometry and antibacterial activity determination. With the second method, cheese whey was filtered through a cation-exchange membrane, and the selectively bound LF was directly hydrolysed in situ with pepsin. Inactive LF fragments were washed off the membrane with ammonia, and a fraction enriched in LFcin-B was obtained by further elution with 2 M NaCl. The membrane method is more rapid and offers several economic advantages.