Human Gastrointestinal Enzymes Research Articles

Dietary fibre and metabolic health: A clinical primer.

Dietary fibres consist of a heterogeneous group of carbohydrate polymers which resist digestion by human gastrointestinal enzymes. Consumption of dietary fibre has been linked with innumerable health benefits encompassing the foundational pillars of metabolic health from obesity to hypertension, dyslipidaemia, and type 2 diabetes mellitus (T2DM), with many of these benefits linked with metabolites produced by the fermentation of fibre by gut microbes [1]. The potential of dietary fibre to provide a safe and effective nonpharmacologic complementary tool with which to combat the evolving consequences of the obesity epidemic has recently garnered tremendous attention in both medical literature and lay media alike. HIGHLIGHTS: Consumption of dietary fibre has been linked with innumerable health benefits encompassing the foundational pillars of metabolic health with many of these benefits linked with metabolites produced by the fermentation of fibre by gut microbes At the present time clinicians are faced with an impossible task in which there is rapidly mounting evidence for dietary fibres advancing metabolic health, but little practical options for healthcare providers other than to simply recommend patients consume more fibre. Benefits of fibre intake may perhaps be maximised in an individual by matching specific fibre consumption with existing microbial functional characteristics If dietary fibres could be demonstrated to act as successful adjuncts to sustain or improve standard of care therapies or even alleviate common gastrointestinal side effects associated with current treatments, they would be an invaluable tool in our metabolic health armamentarium. Neither Dr. Madsen or I have any financial conflicts of interest pertinent to the contents of this manuscript.

Bioactive peptides identified in pea and faba bean after in vitro digestion with human gastrointestinal enzymes

We investigated the digestive peptide profiles of pea and faba bean proteins to evaluate possible bioactive peptides (BAPs), and to test immune-modulating properties of selected peptides using in vitro cell model. More than 400 unique peptides were released upon gastrointestinal digestion of pea and faba bean proteins and identified by high-resolution mass spectrometry (HPLC-ESI-MS/MS). Among these, 24 peptides were potentially bioactive as predicted by in silico analysis. Also, up to 275 recognized BAPs and eight known allergens were identified within the peptide sequences of the digests. Four selected peptides significantly decreased the IL-8 response in Caco-2 cells, with a specific pea peptide, DKPWWPK, reducing the IL-8 response up to 40 % during IL-1β induction. Combined with other nutritional benefits, the immune-modulatory properties of legume proteins point to a regular introduction of legumes in the diet, as they represent a still scarcely exploited food category, especially in the Nordic countries.

Study of the sorptiondesorption of ecdysterone (20 E) as part of adaptogenic compositions with inulin and functional food ingredients based on spinach and quinoa

The main principle in the enrichment of food with minor bioactive compounds is the prediction and evaluation of possible chemical interactions of the components included in the matrix of the food. These interactions have a impact on the bioavailability of minor bioactive compounds. In our work, we studied the processes of sorption and desorption (release), the main processes affecting the bioavailability of the minor bioactive compound ecdysterone (20 E) in the composition of functional food ingredients obtained from spinach leaves (FFI-1) and quinoa grains (FFI-2) on hydrocolloid matrix - inulin. The objective of the research was to study the completeness of sorption-desorption processes of 20 E in adaptogenic compositions with inulin and functional food ingredients based on spinach and quinoa under the influence of hydrolytic enzymes of the gastrointestinal tract (GIT) in vitro. Material and methods. To obtain experimental compositions, containing FFI-1 and FFI-2 and the polysaccharide (inulin), a mechanical mixing method was used. To study the sorption properties, model solutions of the compositions were prepared. Using an in vitro enzymatic model, the ability of 20 E to be released from the matrix of the compositions was studied. The content of 20 E was determined by HPLC-MS/MS. Results. 6 compositions with different ratios of polysaccharide/FFI were obtained. At the first stage of the study, the maximum sorption of 20E in the model solution was observed for 4 compositions with the ratio of inulin : FFI = 2.50 or 3.75 g : 189.19 mg FFI-1 or 68.40 mg FFI-2. At the second stage of the study, when assessing the desorption of 20 E on the enzymatic GIT model, it was found that 20 E almost completely released only from 2 compositions, in other cases about 25% of 20 E remained in a bound state. Conclusion. The formulation of two compositions with the ratio of inulin (2.50 g) : FFI-1 (189.19 mg)/FFI-2 (68.40 mg) were obtained, which have the most optimal sorption / release parameters of 20 E under the influence of human gastrointestinal enzymes. These compositions can be considered promising for inclusion in the formulation of fortified foods.

Dietary Melanoidins from Biscuits and Bread Crust Alter the Structure and Short-Chain Fatty Acid Production of Human Gut Microbiota.

Melanoidins are the products of the Maillard reaction between carbonyl and amino groups of macromolecules and are readily formed in foods, especially during heat treatment. In this study we utilized the three-stage Human Gut Simulator system to assess the effect of providing melanoidins extracted from either biscuits or bread crust to the human gut microbiota. Addition of melanoidins to the growth medium led to statistically significant alterations in the microbial community composition, and it increased short-chain fatty acid and antioxidant production by the microbiota. The magnitude of these changes was much higher for cultures grown with biscuit melanoidins. Several lines of evidence indicate that such differences between these melanoidin sources might be due to the presence of lipid components in biscuit melanoidin structures. Because melanoidins are largely not degraded by human gastrointestinal enzymes, they provide an additional source of microbiota-accessible nutrients to our gut microbes.

Determination of characteristic species-specific protein zones of fish fermentolysates using the method of electrophoretic analysis

The article presents studies on the justification of the most effective technological modes of enzymatic hydrolysis of secondary fish cutting products by the enzyme preparation Collagenase. The optimal duration of enzymatic hydrolysis was established, which is 16 hours for skin fermentolysates, 24 hours for scales fermentolysates and 36 hours for fins fermentolysates. The molecular weights of the protein zones of the obtained fermentolysates are determined, which are mainly represented by low molecular weight digestible proteins with a molecular weight of 13 to 33 kD, which indicates the high availability and degree of protein destruction by human gastrointestinal tract enzymes (from 92.39 to 96.87%).

Influences of freeze‐ and spray‐drying vs. encapsulation with soy and whey proteins on gastrointestinal stability and antioxidant activity of Mediterranean aromatic herbs

SummaryThe aim of the present research was to study the chemical profile of volatile and non‐volatile compounds in freeze‐ and spray‐dried oregano, thyme, sage, and rosemary as well as to study the gastrointestinal stability and the biological activity of the samples after encapsulation using two proteins (soy and whey). Chemical analysis was performed by HS‐SPME/GC‐MS and HPLC‐PDA techniques. The antioxidant activity (FRAP, DPPH and ORAC assays) and HPLC‐PDA analyses were performed before and after simulated two‐phase digestion process (gastric and duodenal) using human gastrointestinal enzymes. The effect of protein carriers was depended on the plant matrix and the class of compound. Soy protein is useful for the protection of thymol and carvacrol content in thyme and the increase of the antioxidant activity of sage. Whey protein is useful for flavanols stability in all studied herbs before and after the duodenal digestive phase.

Stability of blueberry polyphenols subjected to in vitro digestion with human gastrointestinal enzymes

AbstractBlueberries (Vaccinium corymbosum, L.) are a valuable source of health-promoting bioactive compounds, particularly polyphenols. The beneficial effects of polyphenols on health depend not only on food sources but also on their stability, which can vary depending on the post-harvest processing methods, the matrix in which they are incorporated, and endogenous factors such as microbiota and digestive enzymes. The aim of this study was to investigate the influence of high-intensity ultrasound treatment on in vitro gastrointestinal stability of the total polyphenol content and antioxidant capacity of blueberries. Blueberry puree was treated with high-intensity ultrasound using different amplitudes (30, 45 and 60μm) and treatment times (3, 6 and 9 min), and a part of sample was used as a control (untreated sample). Total phenolic content (TPC) and antioxidant capacity (by DPPH and FRAP methods) were determined prior to and after two-phase in vitro digestion process (gastric and duodenal) using human gastrointestinal enzymes. The results showed a high stability of total blueberry polyphenols and antioxidant activity during simulated gastric digestion step, both in untreated and ultrasound treated samples. The use of high-intensity ultrasound had a significant effect on the stability of polyphenols and their antioxidant activity after simulated digestion in duodenum. Significant increase in polyphenol content after duodenal digestion step was determined in three ultrasound treated samples with longer treatment time (6 and 9 minutes), and only sample with significant reduction of TPC was untreated sample. Modification of the food matrix as a result of cavitation from ultrasound treatment may be the reason for higher stability of polyphenol during in vitro digestion. After in vitro digestion blueberry polyphenols still possessed antioxidant activity that may be directly related to their beneficial effects on health. The highest antioxidant activity measured with DPPH and FRAP method was determined for sample treated with maximum amplitude (60μm) and longest time (9 minutes). Determination of the stability of phenolic compounds is important as it affects their bioavailability for uptake as well as their possible beneficial effects on intestinal epithelial tissue such as reduction of oxidative stress and cancer prevention. Our results showed that ultrasound treatment can preserve the blueberry phenolics and their antioxidant characteristics after in vitro digestion.

Degradation of β-casomorphin-7 through in vitro gastrointestinal and jejunal brush border membrane digestion

This work aimed to study the opioid peptide β-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

GASTROINTESTINAL STABILITY OF CAROTENOIDS FROM RAW AND FREEZE-DRIED VEGETABLES

In this study, the gastrointestinal stability of carotenoids (lycopene and β - carotene) extracted from raw and freeze - dried samples of tomato, carrot and red pepper was investigated. Extracted carotenoids fractions (lycopene and β - carotene) were submitted t o a two - phase in vitro digestion process using human gastrointestinal enzymes. The use of freeze - drying has a strong effect on the enhancement of the gastrointestinal stability of carotenoid, especially after simulated intestinal phase. In addition, the ef fect of pH on carotenoid stability is much lower in freeze - dried plant material than in raw samples of tomato, carrot and red pepper. The food matrix also plays important role in carotenoids gastrointestinal stability rate , which was found to be the most s table in red pepper.

Impact of Interfacial Composition on Emulsion Digestion Using In Vitro and In Vivo Models

This study explored the influence of different emulsification layers as mono- and bilayers on lipid digestion by using invitro and invivo digestion methods. The monolayer emulsion of rapeseed oil contained whey proteins and the bilayer emulsion, whey proteins and carboxymethyl cellulose. The invitro digestion using human gastrointestinal enzymes showed that the lipid digestion as free fatty acids was slowed down in the bilayer emulsion compared with the monolayer. Droplet size was still low in the gastric phase and pseudoplasticity was well preserved (even though viscosity decreased) during invitro gastrointestinal digestion. The invivo studies confirmed a lower fat bioavailability from bilayer emulsions by a reduction in the triglyceride level in the blood of rats, fed by the bilayer emulsion. The results clearly showed that lipid digestion was slower in the bilayer emulsion than in the monolayer. These results provide bio-relevant information about the behavior of emulsions upon digestion. PRACTICAL APPLICATION: The layer-by-layer production approach that was presented here allows the preparation of emulsions with slower fat bioavailability. Such behavior of the bilayer emulsion made it interesting for the formulation of food products with low fat bioavailability.

Physiopathology and Management of Gluten-Induced Celiac Disease.

Proline- and glutamine-rich gluten proteins are one of the major constituents of cereal dietary proteins, which are largely resistant to complete cleavage by the human gastrointestinal (GI) digestive enzymes. Partial digestion of gluten generates approximately 35 amino acids (aa) immunomodulatory peptides which activate T-cell-mediated immune system, followed by immunological inflammation of mucosa leading to the onset of celiac disease (CD). CD is an autoimmune disease associated with HLA-DQ2/DQ8 polymorphism and dysbiosis of gut microbiota. CD is either diagnosed using duodenal mucosal biopsis or serological testing for transglutaminase 2 (TG2) specific antibodies (IgA and IgG). Current therapy for CD management is gluten-free diet, while other therapies like glutenase, probiotics, immunomodulation, jamming of HLA-DQ2, inhibition of TG2, and gluten tolerance aided by gluten tolerizing vaccines are being developed.

European Carp (Cyprinus carpio L.) Protein-Derived Ex Vivo Digests and In Vitro Hydrolysates Differ in the ACE I Inhibitory Activity and Composition of Released ACE Inhibitory Peptides.

Carp muscle tissue is a valuable source of biologically active constituents known to positively influence human health. In this study, carp protein digests/hydrolysates generated by human/ porcine digestive enzymes were analysed for their angiotensin I-converting enzyme inhibitory (ACEi) activity. The ex vivo digests and in vitro hydrolysates were used in a screening for ACEi peptides as well. Carp proteins were hydrolysed more rapidly by human gastrointestinal juices than by porcine enzymes. Sarcoplasmic protein fractions were digested/hydrolysed more easily than the myofibrillar ones. The inhibitory concentrations at 50% (IC50) for ex vivo digested sarcoplasmic and myofibrillar protein fractions were established at 1.50 and 1.04 mg/mL, respectively. While, for in vitro hydrolysed sarcoplasmic and myofibrillar protein fractions, the IC50 values were calculated as 2.57 and 1.12 mg/mL, respectively. The digested/hydrolysed samples of carp sarcoplasmic and myofibrillar protein fractions were separated by RP-HPLC-MS. Amino acid sequences were identified with the use of the LC-MS/MS method coupled with in silico systematic screening for ACEi peptides. ACEi peptides with IVY, IY, VY, ALPHA and VKAGF sequences were found in the carp digests/hydrolysates. In the ex vivo carp digest, an ACEi peptide TVY was also detected, while the ACEi peptide IW was identified in in vitro hydrolysate. Our study showed that different ACEi effects of carp digests and hydrolysates were generated with the use of human gastrointestinal and porcine enzymes.

Stability of Rosmarinic Acid in Aqueous Extracts from Different Lamiaceae Species after in vitro Digestion with Human Gastrointestinal Enzymes.

The present study compares the gastrointestinal stability of rosmarinic acid in aqueous extracts of thyme, winter savory and lemon balm with the stability of pure rosmarinic acid. The stability of rosmarinic acid was detected after two-phase in vitro digestion process (gastric and duodenal) with human gastrointestinal enzymes. The concentration of rosmarinic acid in undigested and digested samples was detected using HPLC-DAD. Results showed that gastrointestinal stability of pure rosmarinic acid was significantly higher than that of rosmarinic acid from plant extracts after both gastric and intestinal phases of digestion. Among plant extracts, rosmarinic acid was the most stable in lemon balm after gastric (14.10%) and intestinal digestion phases (6.5%). The temperature (37 °C) and slightly alkaline medium (pH=7.5) did not affect the stability of rosmarinic acid, while acid medium (pH=2.5) significantly decreased its stability (≥50%). In addition, the stability rate of rosmarinic acid is influenced by the concentration of human gastrointestinal juices.

Ex vivo Digestion of Milk from Red Chittagong Cattle Focusing Proteolysis and Lipolysis.

Ex vivo digestion of proteins and fat in Red Chittagong Cattle milk from Bangladesh was carried out using human gastrointestinal enzymes. This was done to investigate the protein digestion in this bovine breed’s milk with an especial focus on the degradation of the allergenic milk proteins; αs1-casein and β-lactoglobulin and also to record the generation of peptides. Lipolysis of the milk fat and release of fatty acids were also under consideration. After 40 min of gastric digestion, all the αs-caseins were digested completely while β-lactoglobulin remained intact. During 120 min of duodenal digestion β-lactoglobulin was reduced, however, still some intact β-lactoglobulin was observed. The highest number of peptides was identified from β-casein and almost all the peptides from κ-casein and β-lactoglobulin were identified from the gastric and duodenal samples, respectively. No lipolysis was observed in the gastric phase of digestion. After 120 min of duodenal digestion, milk fat showed 48% lipolysis. Medium (C10:0 to C16:0) and long (≥C17:0) chain fatty acids showed 6% to 19% less lipolysis than the short (C6:0 to C8:0) chain fatty acids. Among the unsaturated fatty acids C18:1∑others showed highest lipolysis (81%) which was more than three times of C18:2∑all and all other unsaturated fatty acids showed lipolysis ranging from 32% to 38%. The overall digestion of Bangladeshi Red Cattle milk was more or less similar to the digestion of Nordic bovine milk (Norwegian Red Cattle).

Ex vivo digestion of omega-3 enriched buffalo skimmed milk

The aim of this study was to investigate whether the protein digestion in skimmed buffalo milk was affected by adding cod liver oil as a source of omega-3 fatty acid to the milk. Human gastrointestinal enzymes were used in ex vivo digestion. After gastric and duodenal digestion, 32% of the neutral lipid underwent lipolysis. Both the omega-3 fatty acids, eicosapentaenoic (C20:5 n-3) and docosahexaenoic (C22:6 n-3) acid showed 23% lipolysis. Most of the caseins were degraded after 20 min gastric digestion; the tiny traces found after 40 min gastric digestion were digested completely after 5 min duodenal digestion. The α-lactalbumin and β-lactoglobulin were resistant to gastric digestion, however, readily digested after 5 min duodenal digestion. Addition of cod liver oil did not affect the milk protein digestion and subsequent peptide generation. Thereby, enrichment of skimmed milk by cod liver oil could be a good nutritional source of omega-3 fatty acids.

Angiotensin I-converting enzyme (ACE) inhibitory activity and ACE inhibitory peptides of salmon (Salmo salar) protein hydrolysates obtained by human and porcine gastrointestinal enzymes.

The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE) inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes) and ex vivo digestion (with human gastrointestinal enzymes). Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50%) of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes.

Reciprocal interacting effects of proteins and lipids during ex vivo digestion of bovine milk

Human gastrointestinal enzymes were used to study the interacting effects of ex vivo protein and lipid digestion of skimmed bovine milk, homogenised full fat bovine milk and ultrafiltrate permeate reconstituted with bovine milk fat (UFMF). The β-lactoglobulin protein was resistant to digestion in homogenised full fat milk, but readily digested in skimmed milk. During digestion, 37% of the neutral lipids in full fat milk and 16% in UFMF underwent lipolysis. Lipolysis of unsaturated fatty acids in both homogenised full fat milk and UFMF was 30% higher than for saturated fatty acids.

Release of EPA and DHA from salmon oil – a comparison ofin vitrodigestion with human and porcine gastrointestinal enzymes

In the present study, we hypothesised whether in vitro digestion of salmon oil would release different amounts of PUFA depending on the origin of the lipolytic enzymes used. For this purpose, in vitro digestion of salmon oil (SO) was performed using human duodenal juice (HDJ) or a commercial enzyme preparation consisting of porcine pancreatin and bile (PB). The lipolytic effect was determined by measuring the release of fatty acids (FA) using solid-phase extraction and GC-flame ionisation detection, withdrawing samples every 20 min during digestion. The amount of FA released indicated that a plateau was reached after 80 min with approximately similar amounts of FA detected using both HDJ and PB (379 (sd 18) and 352 (sd 23) mg/g SO, respectively). However, the release of 18 : 2, EPA (20 : 5) and DHA (22 : 6) was significantly different during in vitro digestion. At 80 min, HDJ and PB released 43 and 33% of 18 : 2, 14 and 9% of EPA and 11 and 9% of DHA, respectively. Both enzyme preparations released approximately the same amounts of the other FA analysed. The effect of the addition of bile salts (BS) was significantly different in the two enzyme systems, where porcine pancreatin highly responded to the increase in BS concentration, in contrast to HDJ.

Microalgae as human food: chemical and nutritional characteristics of the thermo-acidophilic microalga Galdieria sulphuraria

The use of microalgae as a food source is still poorly developed because of the technical difficulties related to their cultivation and the limited knowledge about their chemical composition and nutritional value. The unicellular red microalga Galdieria sulphuraria has a very high daily productivity and its cultivation under acidic conditions avoided any bacterial contamination. G. sulphuraria can be cultured under autotrophic and heterotrophic conditions: in this study a screening of 43 strains showed that in the latter case a duplication of biomass production was obtained. The proximate composition (protein, carbohydrates, fiber and lipids), the micronutrient content (carotenoids, phycobiliproteins, chlorophylls and vitamins) together with the antioxidant activity of the biomass produced by a selected strain of G. sulphuraria under both cultivation conditions were determined. Results showed that the material is rich in proteins (26-32%) and polysaccharides (63-69%) and poor in lipids. Under heterotrophic cultivation conditions, the lipid moiety mainly contained monounsaturated fatty acids. Among micronutrients, some B group vitamins are present, beta-carotene is the main carotenoid and phycobiliproteins are present under both cultivating conditions. G. sulphuraria proteins are strictly associated with polysaccharide components and therefore not digestible. In the second part of the work, an extraction protocol using Viscozyme L, a commercial enzymatic preparation containing a mixture of polysaccharidases, was developed which made G. sulphuraria proteins a good substrate for human gastrointestinal enzymes. All in all, the data suggested that G. sulphuraria biomass has a potential use as food ingredients both for protein-rich or insoluble dietary fibre-rich applications. The low concentration of lipids and the absence of green color make this microalgae source particularly useful for the addition to many food preparations.

Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes

Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17–41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products.