Actinidin reduces gluten-derived immunogenic peptides reaching the small intestine in an in vitro semi-dynamic gastrointestinal tract digestion model.

Actinidin, a cysteine protease in green kiwifruit (Actinidia deliciosa), has been identified as a potential enzyme to hydrolyse gluten within the lumen of the gastrointestinal tract (GIT). The present study aimed to further evaluate the effect of purified actinidin sourced from green kiwifruit on the digestion of gluten and the release of immunogenic peptides during GIT digestion using an in vitro semi-dynamic GIT digestion model. Purified gluten was digested for 180 min with or without actinidin and subsequently analysed for free amino groups (o-phthaldialdehyde) to determine the degree of hydrolysis (DH), gluten R5 epitopes (ELISA), and peptide profiles (mass spectrometry). Strong interactions were observed between treatment (GIT digestion with or without actinidin) and digestion time for the DH of gluten (P < 0.01), amount of free amino groups released into the small intestine (P < 0.01), and amount of gluten epitopes present in the small intestine (P < 0.001). The rate of increase of DH of gluten and the amount of R5 epitopes present in the small intestine during the first 30 min of GIT digestion with actinidin was 0.3%/min and 4.8 ng/g of gluten respectively, whereas it was 0.01%/min and 60.9 ng/g of gluten respectively without actinidin. These results were corroborated by untargeted peptidomics, with a 1.5-fold lower number of known immunogenic epitopes reaching the small intestine at 30 min of GIT digestion when actinidin was present compared to the control. Present results demonstrate that actinidin enhanced the rate of proteolysis of gluten and reduced the number of immunogenic gluten epitopes reaching the small intestine during simulated semi-dynamic GIT digestion.

Rapid proteolysis of gluten-derived immunogenic peptides in bread by actinidin in a combined in vivo and in vitro oro-gastrointestinal digestion model.

This study aimed to determine the ability of actinidin, a cysteine protease in green kiwifruit (Actinidia deliciosa), to hydrolyse wheat proteins and gluten-derived immunogenic peptides from a commonly consumed food matrix (bread) using a combined in vivo and in vitro oro-gastrointestinal tract (GIT) model. A chewed and spat composite bolus of bread was in vitro digested with or without purified actinidin using a human gastric simulator (HGS). Gastric digestion was conducted for 150 min with gastric emptying occurring at different time points. Emptied samples were immediately digested under simulated small intestinal conditions. Gastric and small intestinal aliquots were collected to quantify peptide profiles and nine marker immunogenic peptides (by untargeted and targeted mass spectrometry, respectively), R5 epitopes (by monoclonal antibody-based competition assay), and free amino groups released by digestion (by the o-phthaldialdehyde method). There was a significant effect (P < 0.05) of actinidin and digestion time on the hydrolysis of wheat proteins and the amount of gluten R5 epitopes of that material emptying the HGS. Actinidin accelerated 1.2-fold the gastric hydrolysis of wheat proteins during the first 20 min of digestion, which was reflected in a faster (5.5 µg min-1) reduction in the evolution of R5 epitopes. Actinidin accelerated (P < 0.05) the rate of disappearance of most of the immunogenic marker peptides. For example, in the first 20 min of small intestinal digestion, the 33-mer peptide decreased (P < 0.05) 2-fold faster (0.25 vs. 0.12 µg g-1 of bread per min) in the presence of actinidin than in the control. Untargeted peptidomics showed actinidin decreased the amounts of known immunogenic peptides in the simulated small intestinal digestion. These findings demonstrated that actinidin accelerates the hydrolysis of wheat proteins and known gluten immunogenic peptides in a commonly consumed food matrix (bread) in a combined in vivo and in vitro oro-GIT digestion model.

The kiwifruit enzyme actinidin enhances the hydrolysis of gluten proteins during simulated gastrointestinal digestion.

This study investigated the effect of actinidin, a cysteine protease in kiwifruit, on the hydrolysis of gluten proteins and digestion-resistant gluten peptides (synthetic 33-mer peptide and pentapeptide epitopes) under static simulated gastrointestinal conditions. Actinidin efficacy in hydrolysing gliadin was compared with that of other gluten-degrading enzymes. Actinidin hydrolysed usually resistant peptide bonds adjacent to proline residues in the 33-mer peptide. The gastric degree of hydrolysis of gluten proteins was influenced by an interaction between pH and actinidin concentration (P < 0.05), whereas the pentapeptide epitopes hydrolysis was influenced only by the actinidin concentration (P < 0.05). The rate of gastric degree of hydrolysis of gliadin was greater (P < 0.05) by actinidin (0.8%/min) when compared to papain, bromelain, and one commercial enzyme (on average 0.4%/min), while all exogenous enzymes were able to hydrolyse the pentapeptide epitopes effectively. Actinidin is able to hydrolyse gluten proteins under simulated gastric conditions.

The Rate at Which Digested Protein Enters the Small Intestine Modulates the Rate of Amino Acid Digestibility throughout the Small Intestine of Growing Pigs.

Background: Actinidin, a cysteine protease in kiwifruit (KF), increases both the gastric digestion and gastric-emptying rate of beef muscle protein. Objective: This study aimed to determine the relation between the rate of digested nitrogen entering the small intestine (SI; a function of the extent of gastric digestion and gastric-emptying rate) and the disappearance of amino acids (AAs) in different parts of the SI at set times postfeeding. Methods: Male 9-wk-old pigs (n = 90; mean ± SD body weight: 28 ± 2.9 kg) were fed a diet containing 14% beef for 3 d. The beef-based diet was supplemented with green KF pulp (containing actinidin), gold KF pulp supplemented with actinidin, or gold KF pulp alone (no actinidin). The KF or actinidin amounts corresponded to the intake of 2 KFs/human meal. On day 3, pigs were killed at 0.5, 1, 3, 5, and 7 h postprandially. Stomach chyme was analyzed to determine the rate of digested nitrogen entering the SI. Apparent AA digestibility at set times was determined in the proximal, medial, and distal SI. Polynomial and correlation analyses were conducted. Results: The rate of digested nitrogen entering the SI was higher (P < 0.001) with actinidin (e.g., >44% at 5 h postprandially). Actinidin also increased the apparent AA digestibility at the proximal and medial SI (P ≤ 0.05) at set times (e.g., 42% and 15% greater for arginine, respectively), but not in general for the distal SI (P > 0.05). At the proximal SI, apparent AA digestibility was correlated more strongly with the digested nitrogen entering the SI (r = 0.73, P < 0.001; n = 57) than with gastric emptying (r = 0.64, P < 0.001) or gastric protein digestion (r = 0.57, P < 0.001). Similar trends were observed for the medial SI. Conclusion: The rate of digested nitrogen entering the SI is an accurate predictor of the rate of AA digestibility and the location of AA absorption in the pig SI.

Dietary actinidin from kiwifruit (Actinidia deliciosa cv. Hayward) increases gastric digestion and the gastric emptying rate of several dietary proteins in growing rats.

Dietary actinidin influences the extent to which some dietary proteins are digested in the stomach, and it is hypothesized that the latter modulation will in turn affect their gastric emptying rate (GE). In this study, the effect of dietary actinidin on GE and gastric digestion of 6 dietary protein sources was determined in growing rats. Each dietary protein source [beef muscle, gelatin, gluten, soy protein isolate (SPI), whey protein isolate, and zein] was included in 2 semisynthetic diets as the sole nitrogen source. For each protein source, 1 of the 2 diets contained actinidin [76.5 U/g dry matter (DM)] in the form of ground freeze-dried green kiwifruit (Actinidia deliciosa cv. Hayward), whereas the other diet contained freeze-dried gold kiwifruit (Actinidia chinensis cv. Hort16A), which is devoid of actinidin (3.4 U/g DM). For both diets, dietary kiwifruit represented 20% of the diet on a DM basis. The real-time GE was determined in rats gavaged with a single dose of the diets using magnetic resonance spectroscopy over 150 min (n = 8 per diet). Gastric protein digestion was determined based on the free amino groups in the stomach chyme collected from rats fed the diets (n = 8 per diet) that were later killed. GE differed across the protein sources [e.g., the half gastric emptying time (T(½)) ranged from 157 min for gluten to 266 min for zein] (P < 0.05). Dietary actinidin increased the gastric digestion of beef muscle (0.6-fold), gluten (3.2-fold), and SPI (0.6-fold) and increased the GE of the diets containing beef muscle (43% T(½)) and zein (23% T(½); P < 0.05). There was an inverse correlation between gastric protein digestion and DM retained in the stomach (r = -0.67; P < 0.05). In conclusion, dietary actinidin increased gastric protein digestion and accelerated the GE for several dietary protein sources. GE may be influenced by gastric protein digestion, and dietary actinidin can be used to modulate GE and protein digestion in the stomach of some dietary protein sources but not others.

Actinidin from kiwifruit (Actinidia deliciosa cv. Hayward) increases the digestion and rate of gastric emptying of meat proteins in the growing pig.

The present study aimed to investigate the effect of dietary actinidin on the kinetics of gastric digestion of beef muscle proteins and on the rate of stomach emptying in growing pigs. For this purpose, 120 pigs (mean body weight 28 (sd 2·9) kg) were fed beef muscle protein-based diets containing either actinidin (fresh green kiwifruit pulp or gold kiwifruit pulp supplemented with purified actinidin) or no actinidin (fresh gold kiwifruit pulp or green kiwifruit pulp with inactivated actinidin). Additionally, fifteen pigs were fed with a protein-free diet to determine the endogenous protein flow. Pigs were euthanised at exactly 0·5, 1, 3, 5 and 7 h postprandially (n 6 per time point for each kiwifruit diet and n 3 for protein-free diet). Stomach chyme was collected for measuring gastric retention, actinidin activity, individual beef muscle protein digestion based on SDS-PAGE and the degree of hydrolysis based on the appearance of free amino groups. The stomach emptying of DM and N was faster when actinidin was present in the diet (P< 0·05): the half gastric emptying time of DM was 137 v. 172 min ( ± 7·4 min pooled standard error) for the diets with and without actinidin, respectively. The presence of dietary actinidin in the stomach chyme increased the digestion of beef muscle protein (P< 0·05) and, more specifically, those proteins with a high molecular weight (>34 kDa; P< 0·05). In conclusion, dietary actinidin fed in the form of fresh green kiwifruit increased the rate of gastric emptying and the digestion of several beef muscle proteins.

Actinidin enhances protein digestion in the small intestine as assessed using an in vitro digestion model.

This paper describes an in vitro study that tests the proposition that actinidin from green kiwifruit influences the digestion of proteins in the small intestine. Different food proteins, from sources including soy, meat, milk, and cereals, were incubated in the presence or absence of green kiwifruit extract (containing actinidin) using a two-stage in vitro digestion system consisting of an incubation with pepsin at stomach pH (simulating gastric digestion) and then with added pancreatin at small intestinal pH, simulating upper tract digestion in humans. The digests from the small intestinal stage (following the gastric digestion phase) were subjected to gel electrophoresis (SDS-PAGE) to assess loss of intact protein and development of large peptides during the in vitro simulated digestion. Kiwifruit extract influenced the digestion patterns of all of the proteins to various extents. For some proteins, actinidin had little impact on digestion. However, for other proteins, the presence of kiwifruit extract resulted in a substantially greater loss of intact protein and different peptide patterns from those seen after digestion with pepsin and pancreatin alone. In particular, enhanced digestion of whey protein isolate, zein, gluten, and gliadin was observed. In addition, reverse-phase HPLC (RP-HPLC) analysis showed that a 2.5 h incubation of sodium caseinate with kiwifruit extract alone resulted in approximately 45% loss of intact protein.

Actinidin enhances gastric protein digestion as assessed using an in vitro gastric digestion model.

Consumption of kiwifruit has long been claimed anecdotally to assist in gastric digestion. This has generally been assumed to be due to the presence of the proteolytic enzyme actinidin; however, there is little published evidence supporting this assumption. This paper reports the findings of an in vitro study that examined the effect of kiwifruit proteases (actinidin) on the digestion of a range of common food proteins under simulated gastric conditions. An extract from green kiwifruit containing actinidin was prepared. Several protein sources derived from soy, meat, milk, and cereals were incubated in the presence or absence of the kiwifruit extract using an in vitro digestion system consisting of incubation with pepsin at pH 1.9, simulating gastric digestion in humans. The digests were subjected to gel electrophoresis (SDS-PAGE). For some protein sources, simulated digestion in the presence of kiwifruit extract resulted in a substantially greater loss of intact protein and different peptide patterns from those seen after digestion with pepsin alone. As an example, the addition of actinidin extract enhanced the digestion of alpha-, beta-, and kappa-caseins in sodium caseinate by 37, 33, and 48%, respectively. Under simulated gastric conditions, kiwifruit extract containing actinidin enhanced the digestion of some, but not all, food proteins over and above that found with pepsin alone.

Milk and dairy products in the 21st century. Prepared for the 50th anniversary of the journal of agricultural and food chemistry.

Dairying into the 21st century will largely continue with the trends seen in the past few decades, although there is always the possibility of an unlikely but disruptive event. The politics of globalization will potentially be important in freeing up global trade in dairy products. Production on the farm will become increasingly efficient, resulting in continuing price benefits to the consumer. At the same time, increasing attention will be paid by the consumer, producer, and manufacturer to safety and quality issues. Environmental concerns will increase in importance, and the issue of methane production may be important for the industry over the next two decades. It is unlikely that genetically modified milk will be introduced soon, even if public acceptance ceases to be an issue; however, the use of genetic markers for accelerated genetic improvement of cows will have rapidly increasing importance. Despite increasing pressure from nonmilk alternatives, milk and dairy will still be the best sources of nutrition for the young and for traditional dairy products. Consumer concerns will be of overriding importance for the industry, and the safety of dairy foods must become absolute. Recent advances in the chemical, physical, and information sciences and technologies will be utilized to gain greater understanding of the increasingly complex food systems and to support the consumer objectives.