Availability of iron from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility,dialysability) and uptake and transport by Caco-2 cells

Iron solubility, dialysability and transport and uptake (retention + transport) by Caco-2 cells as indicators of iron availability have been estimated in the in vitro gastrointestinal digests of infant foods (adapted, follow-up and toddler milk-based formulas and fruit juices containing milk and cereals (FMC)). Low correlation coefficients (in all cases R-squared ? 37.1%) were obtained between iron solubility or dialysability versus transport or uptake efficiency – a fact emphasizing the importance of incorporating Caco-2 cell cultures to in vitro systems in order to adapt the conditions to those found in in vivo assays. The highest uptake efficiency corresponded to FMC (25.6–26.1%) and toddler formulas (32.1–41.9%), the samples with the highest ascorbic acid contents and ascorbic acid/iron molar ratios. In addition, the toddler formulas contained caseinphosphopeptides with the cluster sequence SpSpSpEE, representing the binding site for minerals. In adapted formulas, greater iron uptake efficiency was obtained for the formulation containing ferrous lactate (22.7%) versus ferrous sulfate (4.7%).     

Mineral retention in three-week-old piglets fed goat and cow milk infant formulas

Goat milk and cow milk are commonly used in infant formula preparations and, as such, understanding the nutritional characteristics of infant formulas made from these milks is important. In this study, a goat milk infant formula was compared with an adapted (whey-enhanced) cow milk infant formula with respect to mineral absorption and deposition using the 3-wk-old piglet as a model for the 3-mo-old infant. Equal numbers of piglets (n = 8) were fed either the goat milk formula or the cow milk formula. The mineral composition of the prepared goat milk formula was higher than that of the prepared cow milk formula for most minerals, including calcium (75.1 vs. 56.7 mg/100 mL) but excluding iron, which was higher in the prepared cow milk formula (0.92 vs. 0.74 mg/100 mL). The amounts of calcium, phosphorus, and manganese absorbed by the piglets were significantly higher for the goat milk formula, whereas the amounts of zinc, iron, and magnesium absorbed were significantly higher for the cow milk formula. Apparent mineral absorption, relative to intake, was statistically higher in the cow milk formula for calcium and phosphorus, although the actual differences were very small (less than 1.3%). For copper, zinc, iron, and magnesium there was no significant difference between treatments in apparent mineral absorption, whereas for manganese, absorption was higher for the goat milk infant formula. The absolute mineral deposition was higher in piglets fed the goat milk formula for calcium, phosphorus, and manganese, whereas iron deposition was higher in the piglets fed cow milk formula. For all other minerals tested, there were no significant differences between treatments. The goat milk infant formula provided a pattern of mineral retention in the 3-wk-old piglet very similar to that of the adapted cow milk infant formula. The minor differences observed between the 2 appeared to be due to the different mineral contents of the 2 formulas.     

Addition of milk or caseinophosphopeptides to fruit beverages to improve iron bioavailability?

A study has been made of the influence of caseinophosphopeptides (CPPs) added to a fruit beverage versus milk based fruit beverages upon iron retention, transport and uptake, using a combined simulated gastrointestinal digestion/Caco-2 cell system. Grape concentrate, orange concentrate, and apricot puree were used for sample formulation. Eight samples were assayed with/without added Fe sulphate (3 mg/100 ml fruit beverage) and/or added Zn sulphate (1.6 mg/100 ml fruit beverage), with/without skimmed milk (11% v/v). The addition of milk to fruit beverages exerted a positive effect on iron retention, transport and uptake versus fruit beverages, and this effect was greater than that of CPPs added to soluble fractions of fruit beverages. The addition of CPPs to soluble fractions of fruit beverages improved iron transport. Iron supplementation increased Fe retention, transport and uptake – the effect being more notable in samples with milk. Zinc supplementation did not affect Fe retention, transport or uptake.     

Iron-Fortified Infant Cereals

Infants are particularly susceptible to iron deficiency and related anemia due to their high growth rates and the low iron content of breast milk and most unfortified weaning foods. Cows' milk also is poor in iron, and certain forms of cows' milk cause blood and thus iron loss from the gastrointestinal tract. Iron-fortified cereal-based complementary foods – infant cereals – are recommended to supply the iron needs of older infants. Fortified infant cereals contain much more iron than other fortified cereal products – up to ten or fifteen times as much. Highly or slightly soluble iron salts have excellent bioavailability, but affect color and reduce chemical stability, so these iron salts are not commonly used to fortify infant cereals. Insoluble sources of iron, such as the iron phosphates, were used historically to fortify infant cereals, but these sources have very poor bioavailability. Infants depending on these cereals for iron suffered from high rates of iron deficiency and anemia.

Elemental iron of small particle size, particularly electrolytic iron, currently is the generally accepted vehicle for infant cereal fortification. Iron-fortified cereal made with electrolytic iron reduces iron deficiency and related anemia in several settings but unfortunately is not fully protective in all. Ascorbic acid is a known enhancer of iron bioavailability but ascorbic acid is heat-labile and ascorbic activity declines rapidly during storage. Nonetheless, adding ascorbic acid during processing appears to improve the availability of electrolytic iron and thus the reliability of iron-fortified infant cereal as a means of preventing iron deficiency in older infants.     

Effect of phytate reduction of sorghum,through genetic modification,on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay,Caco-2 cell uptake assay,and suckling rat pup absorption model

Improved iron and zinc availability from sorghum, a commonly consumed staple, will benefit many malnourished communities in rural Africa burdened with high prevalence of iron and zinc deficiency. This research compared the effect of genetic phytate reduction in sorghum on iron and zinc bioaccessibility and uptake measured by in vitro dialysability and Caco-2 cell uptake assays to that of iron and zinc absorption measured by a suckling rat pup model. The phytate reduction (80–86%) in these sorghums significantly increased zinc availability. The Caco-2 cell method, but not the dialysability assay, proved useful in estimating zinc absorption. The measured increase in iron availability differed between the methods, possibly due to the effect of varying mineral (Ca, Fe, Zn, P) contents of the sorghums. This effect was most prominent in the iron uptake results. More research is needed to determine the effect of naturally occurring variations in mineral contents of sorghum on the iron uptake by Caco-2 cells.     

A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula

In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results.     

The activity of single gliadin components in a foetal chick intestine assay for coeliac disease

 The in vitro methods proposed for estimating calcium bioavailability can be useful in studying the effect of dietetic factors. The aim of our work was to study the possible influence of different infant formula components – calcium, phosphorus, lactose, citric acid, ascorbic acid and proteins – on calcium bioavailability, considering dialysability to be an estimate of bioavailability. The 1981 method of Miller et al. with slight modifications (concerted action n^o 10 – FLAIR project) was applied to 18 commercial infant formulas of six different types: adapted, follow-up, preterm, hydrolysates, lactose-free and soy-based. Significant positive linear correlations between the amount of calcium dialysed and the calcium and phosphorus contents were found. However, there were no statistically significant correlations between the amount of calcium dialysed and the lactose, ascorbic acid or citric acid contents, In the case of proteins an exponential relationship was found between the amount of calcium dialysed and the protein content. Received: 21 April 1998 / Revised version: 5 October 1998     

Concentrations of cadmium and lead in different types of milk

 Concentrations of Pb and Cd were determined in samples of human, raw and pasteurized cow's and goat's milk and powdered infant formula. The following mean Cd concentrations (and ranges) were recorded: in human milk, 2.70 μg/l (0.6–11.3, n=55); in raw cow's milk, 4.88 μg/l (0.7–23.1, n=47); in pasteurized cow's milk, 4.30 μg/l (3.4–5.9, n=6); in goat's milk, 7.81 μg/l (1.0–18.4, n=38); and in powdered, infant formula, 3.81 μg/l (3.4–4.1, n=5). The concentrations (and ranges) of Pb were: in human milk, 8.34 μg/l (0.1–32.3, n=55); in raw cow's milk, 14.82 μg/l (1.3–39.1, n=28); in pasteurized cow's milk, 10.25 μg/l (6.9–19.6, n=6); in goat's milk, 11.86 μg/l (0.4–38.5, n=36); and in powdered, infant formula, 8.30 μg/l (5.1–10.6, n=5). Our data were within the normal ranges for each kind of milk. The Cd and Pb concentrations in goat's milk were significantly higher than the concentrations observed in the other milks, whereas human milk and powdered infant formula presented the lowest Cd and Pb concentrations. A considerable decrease in the concentration of Cd with the stage of lactation was observed. The concentrations of Cd and Pb in human, cow's and goat's milk also varied according to the time of year. The concentrations of Pb and Cd in the different milks did not present any risk to human health (infants or adults). Received: 26 May 1998     

Ferritin synthesis by Caco-2 cells as an indicator of iron bioavailability: Application to milk-based infant formulas

The bioavailability of iron from milk-based infant formulas was estimated by an in vitro system including enzymatic digestion, iron uptake by Caco-2 cells and ferritin determination via an enzymoimmunoassay (ELISA). Positive correlations (p < 0.01) were found between the Fe(II) added to Caco-2 cells and ferritin synthesis and between the amount of dialyzed iron added to the cell culture and ferritin synthesis. The comparison of the bioavailability of iron from different milk-based formulas showed that adapted formulas having the same composition but differing in the iron salts added yielded similar ferritin levels. The same happened with follow-up formulas differing only in the presence or absence of bifidobacterium added. However, significant differences in the amount of ferritin synthesized were recorded between the two analyzed toddler formulas. Such differences could be attributed firstly to the ascorbic acid content and perhaps also to the manufacturing process involved, because one formula was in liquid form while the other was powdered.     

High temperature storage of infant formula milk powder for prediction of storage stability at ambient conditions

Hexanal formation and pentanal formation were found to be early signs of quality deterioration of both a high lipid infant formula milk powder and a high protein infant formula milk powder with similar lactose and water content; these aldehydes were sensitive predictors for shelf-life at ambient conditions using short term storage at temperatures up to 55 °C. Higher temperature loads such as 70 °C were found to induce rapid lactose crystallisation entailing a rising water activity, higher levels of free radicals, rapid browning and formation of Strecker aldehydes from protein degradation, of which all were found to be unsuitable for shelf-life prediction. Advanced glycation end products were not detected during storage at 25 °C for up to 18 weeks and the rate of oxygen consumption for the reconstituted milk drink was not affected by storage of the infant formulas at 25 °C; neither can be recommended for shelf-life prediction.     

Survival and growth of Enterobacter sakazakii in infant cereal as affected by composition, reconstitution liquid, and storage temperature

Invasive infections caused by Enterobacter sakazakii have occurred predominantly in low-birth-weight neonates and infants younger than 2 months of age. However, infections have also occurred in healthy infants up to 8 months of age and in immunocompromised children up to 4 years of age. The ability of E. sakazakii to survive and grow in infant cereals as affected by composition of the cereal, composition of the reconstitution liquid, and temperature is unknown. A study was done to determine the survival and growth characteristics of E. sakazakii initially at populations of 0.005 and 0.52 CFU/ml of infant rice cereal, oatmeal cereal, or rice with mixed fruit cereal reconstituted with water, milk, or apple juice. Reconstituted cereals were stored at 4, 12, 21, and 30 degrees C, and populations were monitored for up to 72 h. Growth did not occur in reconstituted cereals stored at 4 degrees C or in cereals reconstituted with apple juice and stored at 12 degrees C. Populations (> or =1 CFU/ml) were detected in cereals reconstituted with water or milk and stored at 12, 21, and 30 degres C for 24, 8, and 4 h, respectively. The composition of infant cereals did not markedly affect the survival or growth of E. sakazakii in reconstituted cereals. Populations of E. sakazakii in reconstituted cereal decreased with increases in populations of mesophilic aerobic microflora up to 8 to 9 log CFU/ml, which was concurrent with decreases in pH. E. sakazakii, initially at 2.62 log CFU/ml of rice cereal reconstituted with apple juice (pH 4.32), survived at 40C for at least 14 days. The pathogen grew at 21 and 30 degrees C within 2 days and then decreased to undetectable levels (<1 CFU/10 ml) in cereal stored at 21 degrees C for 5 days or 30'C for 4 days. Initially, at 7.32 log CFU/ml, E. sakazakii was detected in rice cereal stored at 4 degrees C for 50 days. It is recommended that reconstituted infant cereals stored at 21 or 30 degrees C be discarded within 4 h after preparation or stored at -40C, temperatures at which E. sakazakii will not grow.     

Nano Zinc Oxide-Loaded Calcium Alginate Films with Potential Antibacterial Properties

In this work, zinc oxide nanoparticles-loaded calcium alginate films were investigated for their moisture uptake behavior at different temperatures. The equilibrium uptake data was interpreted quantitatively by GAB isotherm models. The monolayer moisture contents were 0.301 ± 0.003, 0.0214 ± 0.092, and 0.171 ± 0.102 at 20, 30, and 37°C, respectively. The water vapor transmission rate was found to be 0.816 ± 0.143, 1.42 ± 0.045, and 1.632 ± 0.064 g s⁻¹ m⁻² respectively. For the moisture content range of 0.2 to 0.6, the net ∆H and ∆S values were found to be 22.73 to 11.14 KJ/mol and 0.064 to 0.034 KJ/mol/K, respectively. The moisture uptake of films increased with water activity but showed negative temperature dependence. The enthalpy of sorption (∆H) and differential entropy (∆S) were determined at different moisture content values, ranging from 0.2 to 0.6 g/g db. The two parameters showed a higher degree of correlation. The equilibrium moisture content data was used to evaluate harmonic mean temperature T _hm. Finally, the biocidal action of films was tested against model bacteria Escherichia coli.     

Does the addition of caseinophosphopeptides or milk improve zinc in vitro bioavailability in fruit beverages?

The influence of caseinophosphopeptides (CPPs) added to fruit beverage versus milk based fruit beverage upon zinc retention, transport, and uptake, as well as the influence of Fe supplementation, were studied using a combined simulated gastrointestinal digestion/Caco-2 cell system.Zinc retention, transport, and uptake of milk based fruit beverage was 4- to 5-fold greater than that of fruit beverages with or without CPPs – no statistically significant differences being observed in relation to the presence or absence of CPPs. Possibly, a slow release of CPPs throughout the digestive tract, as can be expected to take place during the digestion of casein, has a more beneficial effect than the addition of preformed CPPs upon Zn availability.A significantly negative Fe × Zn interaction in relation to Zn retention and uptake was observed in fruit beverages. This should be taken into account in the formulation of such beverages, where the supplementing of both of these mineral elements is common practice.     

Lactic Fermentation of Non-Tannin and High-Tannin Cereals: Effects on In Vitro Estimation of Iron Availability and Phytate Hydrolysis

The effect on iron availability estimated in vitro and phytate hydrolysis was investigated in non-tannin and high-tannin cereals, lactic fermented as flour/water slurries or gruels. A natural starter culture initiated fermentation and addition of germinated flour and phytase in the fermentation process was tested. Lactic fermentation of nontannin cereals with added flour germinated sorghum seeds or wheat phytase increased iron solubility from about 4% up to 9 and 50%, respectively. Soaking flour in water before adding starter culture had a similar effect. The increase in soluble iron was strongly related to enzymatic deeradation of phytate (p<0.001). The reduction of inositol hexa- and pentaphosphates was about 50% with added germinated flour. Reduction was > 90% after soaking the flour prior to fermentation and almost complete with 50 mg phytase added. High-tannin cereals showed a minor increase in soluble iron after fermentation, ascribed to the inhibitory effect of tannins (both on iron solubility and on enzymatic hydrolysis of phytate). Lactic-fermented cereal foods have a potential in developing countries to improve iron nutrition.     

Physicochemical characteristics of weaning food formulated from different blends of cereal and soybean

 The physicochemical characteristics of porridges of adult and infant weaning food prepared from fermented blends of cereals and soybean are reported in this study. The pH of the formulated flour blends was less than 5.0 within 12 h of fermentation with F15B and F410B, attaining a pH of 3.4. Titratable acidity, which was not detected in unfermented samples, increased as the fermentation progressed. An increase in the gross energy content was noted with F25B, yielding a value of 439.0 kcal/100 g. All the fermented flour blends reconstituted well in boiling water and the water-holding capacity also increased. There was a slight difference in the bulk densities of the loose and packed flour. The index of gelatinization of unfermented formulated flour ranged from 82 (B. U.) in F15A to 334 (B. U.) in F310A. However, there was an increase in the values of the index of gelatinization of the blends at the end of the fermentation. All the blends had relatively low viscosities except F310B. Based on the parameters considered in this study, F15B is recommended as a potential infant and adult food. Received: 10 June 1996 / Revised version: 17 September 1997     

Ascorbate enhances iron uptake into intestinal cells through formation of a FeCl3–ascorbate complex

It has been well documented that ascorbate enhances iron uptake, with a proposed mechanism based on reduction to the more absorbable ferrous form. We have performed a study on the effects of ascorbate on ferric iron uptake in the human epithelial Caco-2 cell-line. Ascorbate increased uptake in a concentration-dependent manner with a significant difference between iron uptake and reduction. Uptake kinetics are characteristic of a non-essential activator and the formation of an Fe³⁺–ascorbate complex. This investigation provides evidence that ascorbate enhances the apical uptake of ferric iron into Caco-2 cells through the formation of a Fe³⁺–ascorbate complex.     

Casein phosphopeptides released by simulated gastrointestinal digestion of infant formulas and their potential role in mineral binding

Adapted and follow-up milk-based infant formulas were subjected to gastrointestinal digestion simulating physiological conditions. The naturally occurring casein phosphopeptides (CPPs) generated were fractionated by anion exchange high-performance liquid chromatography and sequenced by tandem mass spectrometry. In both infant formula digests, a total of 19 CCPs from bovine casein were identified, of which 7 corresponded to α_s1-casein and 12 to α_s2-casein. Most CPPs had the cluster sequence SpSpSpEE, representing the binding sites for minerals. The distribution of calcium, iron and zinc content in CPP fractions eluted from the anion exchange column was also studied. The results obtained suggest that calcium could be preferably bound to CPPs with the cluster sequence SpSpSpEE, whereas iron and zinc could be bound to CPPs containing the phosphorylated cluster and phosphoserine residues.     

Effects of polyphenol oxidation on in vitro iron availability in faba bean (Vicia faba L.) flour

Tannic acid (TA) and green tea extract (GTE) were added to faba bean flour, before and after incubation with polyphenol oxidase (mushroom tyrosinase), and the effect on in vitro iron availability was investigated. The inhibitory effect of TA and GTE was dose dependent, and the in vitro iron availability decreased from 64.5% in the pure faba bean flour to 9.8% (P < 0.05) with the addition of 1 mg of TA 1 g^?1 to faba bean flour. The addition of 5 mg of GTE resulted in low in vitro iron availability as after the addition of 10 mg of TA (11.6% against 10.3% for 1 mg TA). Incubation of the polyphenols with tyrosinase before addition to the faba bean flour significantly increased the in vitro iron availability. The increase in iron availability was significant (P < 0.05) when amounts of 0.2, 0.5 or 1 mg g^?1 TA or GTE added to faba bean flour, respectively, were oxidised, even with the lowest amount of tyrosinase (150 u). Oxidation of 1 mg TA or GTE with 150 u tyrosinase increased the in vitro iron availability from 10.3% to 15.5% and from 19.2% to 26.1%, respectively. At the 300‐u level, the addition of higher amounts of enzyme (from 600 to 900 u) did not have any effect. The results from the study therefore suggest that the oxidation of polyphenols may be a promising way to increase the availability of iron in polyphenol‐containing legume foods.     

Determination of Elements Profile of Some Wild Edible Plants

Fruits and leafy vegetables are believed to occupy a modest place as a source of trace elements due to their high water content. Most of nutrient requirements can be met by increasing the consumption of fruits and vegetables to 5–13 servings/day. In addition to meeting nutrient intake levels, greater consumption of fruits and vegetables is associated with reduced risk of cardiovascular disease, stroke, and cancers of the mouth, pharynx, esophagus, lungs, stomach, and colon. Therefore, vegetable and fruit consumption prevents sickness in population. Results in this research showed that Portulaca oleracia Linn. contains high value of macro-elements such as sodium (7.17 mg/g), potassium (14.71 mg/g), and calcium (18.71 mg/g), and high ash value in comparison with other plants. Also, Eulophia ochreata Lindl. has maximum micro-elements such as iron (5.04 mg/g) and zinc (3.83 mg/g) in comparison with other plants in this study. Therefore, we can conclude that P. oleracia Linn. has high nutritional values from the viewpoint of macro-elements such as sodium, potassium, and calcium; and because E. ochreata Lindl. has micro-elements in maximum amounts such as iron and zinc in comparison with other edible plants, it has high nutritional value from the viewpoint of the above trace (micro) elements. Momordica dioicia Roxb. or Cordia myxa Roxb have the lowest nutritional values because they have ash minimum values. M. dioicia Roxb. contains minimum values of sodium and calcium, but C. myxa Roxb. has zinc minimum value. Alocacia indica Sch., Asparagus officinalis DC., Chlorophytum comosum Linn., C. myxa Roxb., E. ochreata Lindl. have medium nutritional values.     

BEHAVIOR OF LISTERIA MONOCYTOGENES IN RECONSTITUTED INFANT CEREALS

Listeria monocytogenes may contaminate and grow in reconstituted infant cereals refrigerated or temperature abused before consumption. To assess this risk, a 5-strain composite of the pathogen was inoculated in rice, oatmeal, and wheat-rice-oatmeal infant cereals, hydrated (1/6, w/v) with apple juice, pasteurized milk (2% fat) or water, and stored at 4, 15, or 25C for 0, 8 and 24 h to simulate potential advance home preparation and abuse. L. monocytogenes survived with minimal (<0.6 log cfu per mL) or no growth in all cereal preparations at 4C, but it increased in cereals hydrated with water or milk by 2.4–3.3 and 5.3–6.4 log cfu per mL in 24 h at 15 and 25C, respectively. Reconstitution of cereals with water or milk supported growth (P < 0.05) even at 8 h of abuse. In cereals hydrated with apple juice and stored at 15 or 25C, populations of L. monocytogenes were maintained below 3.0 log cfu per mL at 24 h. These results indicate that refrigeration and/or reconstitution with an acid fruit juice may inhibit growth of L. monocytogenes in infant cereal meal preparations. However, in order to ensure prevention of growth, such meals should preferably be consumed soon after preparation or held at 4C for less than 8 h.