Development of a three‐tier in vitro system,using Caco‐2 cells,to assess the effects of lactate on iron uptake and transport from rye bread following in vitro digestion

A three‐tier Caco‐2 cell system was developed to assess simultaneously iron dialysability, uptake and transport across the Caco‐2 monolayer from an in vitro digested food matrix. The effect of lactate (0–200 mmol L⁻¹) on iron absorption from rye bread subjected to simulated peptic (pH 5.5) and pancreatic digestion (pH 6.5) was investigated to model absorption pre and post the sphincter of Oddi. Lactate increased dialysability (11.8%, P < 0.05) in peptic digests whereas it reduced it in pancreatic digests (4.9%, P < 0.001). Iron uptake from the peptic digests was in the region of 39–76 pmol mg⁻¹ protein whereas it decreased from 281 to 51 pmol mg⁻¹ protein in pancreatic digests. Iron transport was calculated for the peptic digests from [¹⁴C]polyethylene glycol movement and only at 200 mmol L⁻¹ lactate was there any detectable transcellular transport (180 pmol mg⁻¹ protein, P < 0.05). Iron absorption was positively correlated to dialysable iron for both digests (R² = 0.48 and 0.41, respectively, P < 0.01) and the effect of lactate was therefore associated mainly with iron bioaccessibility. The three‐tier system showed the potential to obtain detailed insight into each step involved in iron transport across the monolayer from a food mixture. Copyright © 2006 Society of Chemical Industry     

Dephytinisation of Sangak and Barbari bread made from different extraction rate flours increases iron and zinc bioaccessibility in Caco‐2 cells

Bread is a staple food in many countries and an important source of iron and zinc. The bioavailability of these minerals is generally low because of the content of phytic acid. Traditional Iranian breads were prepared with flours of different extraction rates, Sangak at 93% and Barbari at 82%. Breads were dephytinised by addition of Aspergillus niger phytase during in vitro digestion. The effect upon iron and zinc bioaccessibility in the Caco‐2 cell model was investigated. The cellular uptake of iron and zinc was lower from Sangak, compared to Barbari, despite higher mineral content in Sangak. Dephytinisation of both breads increased iron uptake in the Caco‐2 cells (0.65 vs. 1.64 in Sangak and 0.77 vs. 1.97 ng mg^?1 protein in Barbari). Zinc uptake increased from 0.98 to 2.8 in Sangak and from 1.4 to 2.9 ng mg^?1 protein in Barbari. Thus, dephytinisation substantially improves iron and zinc bioaccessibility.     

The use of an enzymatic extraction procedure for the enhancement of highland barley (Hordeum vulgare L.) phenolic and antioxidant compounds

A multistep enzymatic extraction method was compared with a conventional chemical extraction process to evaluate the phenolic content and antioxidant activity of highland barley (Z2, Zangqing 25; CHQK, Changheiqingke). The main phenolic compound extracted was (+)‐catechin, followed by ferulic acid, p‐coumaric acid, caffeic acid and chlorogenic acid. The multi‐enzymatic digestion yielded a higher retrieval of (+)‐catechin compared to the conventional chemical extraction procedure (P < 0.05). Compounds obtained from the multi‐enzymatic digestion process exhibited significantly higher (P < 0.05) antioxidant activities determined by oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) when compared to chemical extracts. These results suggest that highland barley subjected to in vitro multi‐enzymatic digestion exhibits a higher phenolic content and antioxidant activity than the chemical extraction, and this multi‐enzymatic digestion coupled with the CAA assay may be a valuable tool to evaluate the antioxidant potential of wholegrains and fruits, as well as vegetables.     

Does the pelleting process affect the nutritive value of a pre‐starter diet for suckling piglets? Ex vivo studies on mineral absorption

BACKGROUND: The effects of pelleting on the extent of the Maillard reaction (MR) and on calcium, magnesium and zinc solubility and absorption were analysed in a conventional pre‐starter diet for suckling piglets. Development was tested measuring colour, absorbance (280/420 nm), fluorescence, residual free lysine, furosine, hydroxymethylfurfural (HMF) and furfural contents before and after pelleting. Fluorescence, absorbance and mineral solubility were also measured after in vitro digestion of diets. The effects on mineral absorption were tested using Caco‐2 cells. RESULTS: MR indexes confirmed the development of the reaction during the pelleting of this particular diet compared with the meal diet. The CIE‐Lab colour parameters showed a decrease in luminosity (L*) and progress of the colour to the red zone (a*) in the pelleted diet. A 36% decrease in free lysine content was observed. Significant correlations were observed between fluorescence intensity and furosine levels, HMF and furfural. The pelleting process did not modify calcium and magnesium solubility after in vitro digestion, but soluble zinc increased. The efficiency of calcium and zinc transport across Caco‐2 cell monolayers was greater in the pelleted diet. CONCLUSIONS: Evidence of MR development is shown, resulting in various nutritional consequences. Optimisation of pelleting could result in a better formulation of diets for feedstuffs. Copyright © 2010 Society of Chemical Industry     

Effect of in vitro‐simulated gastrointestinal digestion on the stability and antioxidant activity of blueberry polyphenols and their cellular antioxidant activity towards HepG2 cells

In this study we investigated the influence of in vitro‐simulated gastrointestinal digestion on the bioaccessibility and antioxidant activity of polyphenols from blueberries (Vaccinium spp.). Total phenolic, anthocyanin, and flavonoid content was determined, and extract and digesta compositions were analysed by HPLC‐ESI‐MS/MS. The phenolic compounds were relatively stable under a gastric environment, whereas polyphenols and anthocyanins were unstable under an intestinal environment. The bioaccessibility of polyphenol, anthocyanin, and flavonoid was greatly decreased after the intestinal digestion, and the recoveries were only 13.93%, 1.95%, and 15.68% (the IN sample), respectively. Polyphenolic profile alteration occurred during in vitro‐simulated gastrointestinal digestion. Changes of phenolic compound antioxidant activity during digestion correlated with polyphenol, flavonoid, and caffeic acid concentrations. Digested extract cellular antioxidant activity was lower than non‐digested extract activity (P < 0.05). Polyphenol dose–response correlations with cellular antioxidant activity were observed. These results indicated that in vitro‐simulated gastrointestinal digestion significantly impact polyphenols and their antioxidant activity.     

Fermented coconut jelly as a probiotic vehicle,physicochemical and microbiology characterisation during an in vitro digestion

There is little information on the survival of probiotics in plant-based foods after simulated gastric and intestinal conditions, likewise the microstructure arrangement in the no-dairy fermented food. This work aimed to study if the agar–agar in a fermented coconut jelly confers protection to probiotics, phenolic and antioxidant compounds during in vitro digestion. Samples containing higher agar–agar amounts tend to retain (P < 0.05) antioxidant and phenolic compounds in their network better, even after the in vitro digestion. Also, a compact and homogeneous microstructure was observed by the Confocal Laser Scanning Microscopy. The texture profile analysis shows that 1% of agar samples presented the maximum hardness (P < 0.05) due to more bonding points and intermolecular interactions. Finally, the survival of probiotics remained above the recommended values (10⁶–10⁷ CFU g⁻¹) after the in vitro digestion of a product with probiotic potential.     

Cooking does not impair the impact of pulsed electric field on the protein digestion of venison (Cervus elaphus) during in vitro gastrointestinal digestion

The present study was conducted to elucidate whether cooking impairs the positive effect of pulsed electric field (PEF) on the digestibility of venison during in vitro gastrointestinal protein digestion. Previous studies have used fresh uncooked meat to demonstrate the effect of PEF on protein digestibility during gastrointestinal digestion neglecting the effect that cooking could induce during meat preparation process. PEF-treated samples (T₁, 10 kV, 90 Hz, 20 µs) were cooked (core temperature of 75 °C) and subjected to in vitro simulated gastrointestinal protein digestion along with non-treated controls. A 3% increase of in vitro protein digestibility was found in cooked PEF-treated venison (P < 0.05). A positive (P < 0.05) impact of PEF processing was observed on overall protein digestion as measured by soluble protein (%) and SDS-PAGE. PEF did not change (P > 0.05) the release of minerals from cooked venison during digestion. Cooking had no negative influence on the mechanism through which PEF operates in improving the protein digestibility of venison.     

The inhibitory effect of albumin extracts from white beans (Phaseolus vulgaris L.) on in vitro iron and zinc dialysability: role of phytic acid

The role of phytic acid in determining iron and zinc dialysability in albumin extracts from raw, cooked and cooked/dephytinized white beans, has been investigated. Albumin extract from raw beans was characterized by high iron (19mg/100g), zinc (6.9mg/100g) and phytic acid (11.5μmol g⁻¹) contents and a low mineral dialysablity (Fe 0.48%, Zn 2.5%). Cooking did not influence the mineral dialysability from beans but significantly increased the dialysability of iron (1%, p < 0.001) and zinc (5.3%, p < 0.001) from the albumin extract. Slight modifications in the composition of inositol phosphates after cooking, both in beans and in albumin extract, were observed. The improvement in iron and zinc dialysability from cooked/dephytinized samples was strictly dependent on the residual IP(6 + 5) (inositol hexa + pentaphosphates) content. Compared to cooked beans, in cooked/dephytinized bean a reduction of IP(6 + 5) of 49% led to an increase of the iron and zinc dialysability (29% p < 0.05 and 42%, p < 0.001, respectively). Albumin extract from this sample showed a reduction of 58% in iron and of 45% in zinc content, an almost complete reduction in IP(6 + 5) content (0.6 μmol g⁻¹) and a strong increase in the iron and zinc dialysability. The albumin digests showed peptides of similar MW profiles but of different amino acid compositions. In particular, in peptides which derived from digestion of albumin extracted from cooked/dephytinized beans, a strong increase in cysteine content was found, indicating that, after the disruption of phytate-mineral-protein complexes cysteine-rich fragments were released. The study indicates that phytic acid is responsible for the low iron and zinc dialysability from the albumin bean fraction and indicates the significance of the amino acid composition of the protein digestion products for the enhancement of mineral dialysability.     

Simulated gastrointestinal digestion and in vitro colonic fermentation of date (Phoenix dactylifera L.) seed polyphenols

Date seeds are a by‐product of date fruit industry and a rich source of polyphenols. In this study, in vitro bioaccessibility and colonic fermentation of major polyphenols from date seed powder (DSP) and DSP‐fortified yoghurt (DSPY) were investigated using HPLC. Catechin, epicatechin and procyanidin A2, B1 and B2 were stable during simulated gastric and sequential intestinal digestion. Bioaccessibility was significantly (P < 0.05) higher for all compounds from DSPY compared with DSP. After in vitro colonic fermentation of insoluble digestion materials, most of the target compounds were metabolised by faecal bacteria to ferulic acid, 3‐hydroxyphenylacetic acid, 3‐phenylpropionic acid and 3‐(4‐hydroxyphenyl) propionic acid. DSPY contained significantly (P < 0.05) higher level of free polyphenols as indicated by higher bioaccessibility; however, the stability of the polyphenols and their fermentation products from DSPY were similar to that of DSP alone. These data would be useful in product developments incorporating DSP as a source of polyphenols in food products.     

Influence of different types of protein on in vitro availability of intrinsic and extrinsic iron and zinc

The influence of beef, pork and soy proteins on intrinsic and extrinsic iron and zinc in vitro availability was investigated, by measuring mineral dialysability. Intrinsic iron dialysability (percentage of iron dialysed) was similar in the animal proteins and higher than that from soy protein isolate (P<0·001). Intrinsic zinc dialysability from pork protein was statistically (P<0·001) higher than that from other proteins studied. Beef and soy proteins do not differ in their percentages of zinc availability. The effect on the extrinsic iron availability was different among the three proteins studied, but not on the extrinsic zinc availability. The composition of amino acids and/or the small peptides produced during the digestion process of the protein can modify the iron, and probably the zinc, availability. The inorganic zinc addition (10 μg ml⁻¹) reduced the non-haem and extrinsic iron availability, while it did not affect the haem iron availability. At the concentrations used in this study (10 μg ml⁻¹) a decrease of zinc dialysability caused by the inorganic iron addition was not observed. ©1997 SCI     

Optimisation of experimental conditions for binding of divalent iron to bioactive casein phosphopeptides

Casein phosphopeptides (CPPs) are mineral‐binding bioactive peptides derived from enzymatic digestion of casein. They have been shown to increase iron bioavailability due to increasing iron solubility at the alkaline pH of the intestine. Thus, they are potentially applicable as an advantageous preparation for iron fortification of food. This study aimed to characterise a commercially available CPP and determines optimum experimental conditions for binding it with ferrous iron. The effect of three variables – pH (5–7.5), mass ratio of iron to CPP (MR) (0.04–0.16) and holding time (40–80 min) – on the CPP's iron content was investigated using the central composite design (CCD) in response surface methodology (RSM), and a quadratic model was developed. The optimisation in this survey was based on the maximum response. Among all the regression's terms, the main and quadratic effects of pH represented the most significant influences on the iron content (P‐value <0.05). The obtained optimum conditions were as follows: pH 6.5, MR 0.14 and holding time 72 min, resulting in binding 68.66 mg iron per gram of the CPP. The quantity of iron bound to the CPP depended considerably on the experimental conditions and proved the importance of optimisation.     

Antioxidant Peptide Fractions Isolated from Wheat Germ Protein with Subcritical Water Extraction and Its Transport Across Caco‐2 Cells

Wheat germ protein (WGP) was extracted with subcritical water and then hydrolyzed with Alcalase 2.4 L to obtain antioxidant hydrolysates. Wheat germ peptides (WG‐P, Mw < 1 kDa) were purified by using Sephadex G‐15 column chromatography. The results showed that WG‐P‐4 possessed the strongest DPPH radical scavenging activity in comparison with other peptides fractions. In addition, free amino acids and LC‐MS/MS analysis showed that Gly‐Pro‐Phe, Gly‐Pro‐Glu, and Phe‐Gly‐Glu were the major peptides of WG‐P‐4. Interestingly, the WG‐P‐4 fractions had good absorption characteristic. Moreover, the ratio of P_app both sides of apical compartment (AP) and basolateral compartment (BL) were between 0.5 and 1.0 on Caco‐2 cell model, which indicated that transmembrane transportation was mainly passive transport. Therefore, WG‐P could exert an effective antioxidant action by across the intestinal epithelium.     

The antioxidant activity and transcellular pathway of Asp‐Leu‐Glu‐Glu in a Caco‑2 cell monolayer

Asp‐Leu‐Glu‐Glu (DLEE) is one of the antioxidant peptides purified from Chinese dry‐cured Xuanwei ham in our previous study. In the current work, the stability in a simulated digestion system, the transportation pathway and the antioxidant ability of DLEE were further investigated in a Caco‐2 cell monolayer. In the simulated gastrointestinal digestion system, no oligopeptides were generated. In the transport trial, the inhibitors cytochalasin D increased the transport of DLEE across the Caco‐2 cell monolayer, with P_app values of 3.22 × 10^?6 cm s^?1. A decreased expression occludin was observed with the DLEE incubation in the cell monolayer, and the antioxidant activity showed to be increased gradually in basolateral side. This study indicates that the absorption of DLEE could mainly occur via paracellular transport and provides information about its antioxidant activity after being absorbed across a cell monolayer.     

Iron and calcium availability from digestion of infant cereals by Caco-2 cells

An adequate mineral intake during infancy is needed for normal growth and development. This study investigated the effect of dephytinization of four infant cereals and the use of water and follow-on formula as the liquid of reconstitution on the intestinal cell uptake of iron and calcium from infant cereals using a model that combines a simulated gastrointestinal digestion adapted to the gastrointestinal conditions of infants younger than 6 months and the Caco-2 cell line. Iron and calcium uptake by Caco-2 cells from most infant cereals was significantly (p < 0.05) improved when a phytase was added. When infant cereals were reconstituted with water, dephytinization increased iron (3.2–19.5 vs. 3–10%) and calcium (0.66–2.3 vs. 0.35–0.59%) availability compared to the same infant cereals reconstituted with a follow-on formula. We can conclude that dephytinization of infant cereals and water addition improved iron and calcium availability, depending on the infant cereal used.     

Spoilage of refrigerated (4 °C) Litopenaeus vannamei: cooperation between Shewanella species and contribution of cyclo‐(L‐Pro‐L‐Leu)‐dependent quorum sensing

The spoilage potential of Shewanella putrefaciens and S. baltica isolated from spoiled refrigerated Litopenaeus vannamei was evaluated by in vitro assays for trimethylamine oxide reduction, extracellular hydrolytic enzymes and biofilm formation, and in vivo inoculation into surface‐sterilised shrimp followed by microbial, biochemical and sensory analyses during storage for 5 days at 4 °C. S. baltica displayed higher spoilage potential than S. putrefaciens both in vitro and in vivo. Shrimp co‐inoculated with them had one‐day shorter shelf life than those mono‐inoculated, based on the results of bacterial density, volatile base nitrogen, trimethylamine, volatile organic compounds and sensory analysis, which strongly suggests cooperation of Shewanella species in shrimp spoilage. Exogenous cyclo‐(L‐Pro‐L‐Leu) boosted bacterial growth, extracellular protease and collagenase activities, and biofilm formation of S. putrefaciens and S. baltica at least before they entered the stationary phase, indicating that cyclo‐(L‐Pro‐L‐Leu)‐dependent quorum sensing, a recently suggested communication mechanism between them, contributes to the cooperation.     

Bioavailability of iron and zinc from multiple micronutrient fortified beverage premixes in Caco-2 cell model

Abstract: Iron and zinc deficiencies are the most prevalent nutrient deficiencies worldwide. They often coexist as the dietary factors, especially phytate, which impairs iron absorption also affects zinc absorption. Therefore, suitable strategies are required to control multiple micronutrient deficiencies in populations that subsist on high‐phytate foods such as the whole wheat flour based Indian bread (chapatti). The objective of the study, therefore, was to test the bioavailability of iron and zinc in 2 multiple micronutrient beverage premixes in the absence and presence of chapatti. The premix‐1 contained iron, zinc, and vitamin A while premix‐2 contained all micronutrients in premix‐1, plus folic acid and ascorbic acid. Ferritin induction and ⁶⁵Zn uptake were assessed using coupled in vitro digestion/Caco‐2 cell line model as the surrogate markers of iron and zinc bioavailability, respectively. The results show that iron bioavailability from premixes‐1 and 2 was similar in the absence of chapatti. However, premix‐2 showed significantly higher iron bioavailability compared to premix‐1 in the presence of chapatti. In contrast, the zinc uptake was similar from both premixes‐1 and 2 in the absence or presence of chapatti. These results suggest that both the premixes provide bioavailable minerals, but premix‐2 appears to be promising in the presence of foods that have high phytate.     

Effect of production environment, genotype and process on the mineral content of native bitter potato cultivars converted into white chuño

BACKGROUND: Variables and interaction effects affecting the mineral concentration of Andean bitter potatoes converted into so‐called white chuño are unknown. We report on the effect of three contrasting production environments (E) on the dry matter (DM), zinc, iron, calcium, potassium, magnesium, phosphorus and sodium concentration of four potato native bitter genotypes (G) processed (P) into two different ‘types’ of white chuño. RESULTS: The DM content and iron, calcium, magnesium and sodium concentration of white chuño are significantly dependent on E, G, P, and E × G × P interaction (predominantly at P < 0.01). In particular, the DM content and calcium concentration are influenced by all variables and possible interaction effects. The zinc and potassium concentration are not significantly dependent on E × G, G × P or E × G × P interaction effects, while the phosphorus concentration is not significantly affected by the G × P or E × G × P interaction effect. Zinc, phosphorus and magnesium concentrations decrease in the ranges of 48.3–81.5%, 61.2–73.0% and 62.0–89.7% respectively. The decrease in potassium is particularly severe, with 122‐ to 330‐fold losses. Iron and calcium increase by 11.2–45.6% and 74.5–714.9% respectively. CONCLUSION: E, G, P, and various interaction effects influence the mineral concentration of traditionally processed tubers. We speculate that mineral losses are caused by leaching, while increases of iron and calcium are a likely result of absorption. Copyright © 2012 Society of Chemical Industry     

Preparation and optimisation of liposome‐in‐alginate beads containing oyster hydrolysate for sustained release

Oyster (Crassostrea gigas) hydrolysate shows antihypertensive effect in our previous study. Oral administration of oyster hydrolysate can loss bioactive peptides due to enzymatic degradation in vivo. To maximise its bioavailability, liposome‐in‐alginate (LA) beads were used to encapsulate the oyster hydrolysates to protect from degradation and obtain sustained release. The preparation conditions of the LA beads were optimised by response surface method using a model peptide of tyrosylalanine (YA). Their characterisation, swelling and release properties were investigated. The optimised conditions for the concentration of calcium chloride, sodium alginate and the amount of ethanol‐dissolved lecithin (EDL) were 0.5 m , 3% and 95.4 mg, respectively. The encapsulation efficiencies of YA and the oyster hydrolysate in the optimised condition were 74.9% and 84.3%, respectively. The release time of the oyster hydrolysate in the simulated gastrointestinal fluid was up to 16 h. The LA beads can be recommended to encapsulate oyster hydrolysate for bioavailability improvement.     

Effects of different prebiotic stabilisers and types of molasses on physicochemical,sensory, colour and mineral characteristics of probiotic set yoghurt

The physicochemical, sensory, properties and mineral profile of probiotic set yoghurts prepared with different prebiotic stabilisers (pectin and guar) at 0.5% (w/v) and different molasses (grape, mulberry and carob) at 10% (v/v) were investigated. Guar‐supplemented yoghurts had the lowest titratable acidity values (P < 0.01) also the highest water‐holding capacity values (P < 0.01). However, guar had a negative effect on gel firmness and sensory properties of yoghurts. The effect of different stabilisers and molasses on colour L* and a*values was statistically significant (P < 0.01). Panelists gave the highest flavour scores to the pectin‐added yoghurts during storage period (P < 0.01).     

Optimisation of antioxidant hydrolysate production from sweet potato protein and effect of in vitro gastrointestinal digestion

In this study, response surface methodology (RSM) was applied to optimise antioxidant hydrolysate production from sweet potato protein (SPP). The effects of enzyme‐to‐substrate ratio, pH value and reaction temperature on ·OH radical scavenging activity and Fe²⁺‐chelating activity of antioxidant hydrolysates from SPP were investigated. The maximum ·OH radical scavenging activity (40.10%) and Fe²⁺‐chelating activity (74.37%) of SPP hydrolysates (SPPH) were obtained at an enzyme‐to‐substrate ratio of 4%, pH value of 7.8 and reaction temperature of 57 °C, which was in agreement with the predicted value (40.11% and 74.83%, respectively) estimated by RSM. The simulated in vitro gastrointestinal digestion (GID) dramatically modified molecular weights distribution, increased peptide (<3 kDa) concentration and highly retained antioxidant activity of SPPH, indicating potentially utilisation of SPPH as a functional supplement in food system.