Fermentation of pseudocereals quinoa, canihua, and amaranth to improve mineral accessibility through degradation of phytate.

BACKGROUND: Pseudocereals are nutrient-rich grains with high mineral content but also phytate content. Phytate is a mineral absorption inhibitor. The study's aim was to evaluate phytate degradation during spontaneous fermentation and during Lactobacillus plantarum 299v® fermentation of quinoa, canihua, and amaranth grains and flours. It also aimed to evaluate the accessibility of iron, zinc, and calcium and to estimate their bioavailability before and after the fermentation of flours with starter culture. Lactic acid, pH, phytate, and mineral content were analyzed during fermentation. RESULTS: Higher phytate degradation was found during the fermentation of flours (64-93%) than during that of grains (12-51%). Results suggest that phytate degradation was mainly due to endogenous phytase activity in different pseudocereals rather than the phytase produced by added microorganisms. The addition of Lactobacillus plantarum 299v® resulted in a higher level of lactic acid (76.8-82.4 g kg-1 DM) during fermentation, and a relatively quicker reduction in pH to 4 than in spontaneous fermentation. Mineral accessibility was increased (1.7-4.6-fold) and phytate : mineral molar ratios were reduced (1.5-4.2-fold) in agreement with phytate degradation (1.8-4.2-fold) in fermented flours. The reduced molar ratios were still above the threshold value for the improved estimated mineral bioavailability of mainly iron. CONCLUSION: Fermentation proved to be effective for degrading phytate in pseudocereal flours, but less so in grains. Fermentation with Lactobacillus plantarum 299v® improved mineral accessibility and estimated bioavailability in flours. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa.

BACKGROUND: Quinoa is a pseudocereal with relatively high content of proteins and minerals that also contains mineral inhibitors such as phytate. The aim of the present study was to evaluate lactic acid fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa. Various processes were evaluated, and quinoa grains were dry-roasted, milled, and fermented, either with or without the addition of wheat phytase or activated quinoa phytase (added as back-slop starter), for 10 hr. In other processes, raw quinoa flour was fermented for 10 hr or 4 hr and dry-roasted. Hedonic sensory evaluation was then performed to evaluate the acceptability of the fermented flours prepared as porridges. RESULTS: The combined dry roasting and fermentation processes significantly (p < .05) degraded phytate between 30% and 73% from initial content. The most effective process was fermentation of raw quinoa flour followed by dry roasting, which improved the estimated zinc and iron bioavailability. Particularly, estimated zinc bioavailability improved from low (Phy:Zn 25.4, Phy·Zn:Ca 295) to moderate (Phy:Zn 7.14, Phy·Zn:Ca 81.5). Phytate degradation was mainly attributed to the activation of endogenous phytase during fermentation. Dry roasting was effective in improving the sensory attributes of the fermented quinoa flour. Porridge made with raw quinoa flour fermented for 4 hr and dry-roasted was more favorable to overall acceptability than that which was fermented for 10 hr and dry-roasted. CONCLUSION: Fermentation of quinoa flour for 4 hr followed by dry roasting was successful in improving both nutritional and sensory attributes of the final product.

Bioactivity of Cod and Chicken Protein Hydrolysates before and after in vitro Gastrointestinal Digestion.

Bioactivity of cod (Gadus morhua) and chicken (Gallus domesticus) protein hydrolysates before and after in vitro gastrointestinal (GI) digestion was investigated using yeast Saccharomyces cerevisiae as a model organism. Both hydrolysates were exposed to in vitro GI digestion prior to cellular exposure to simulate digestion conditions in the human body and therefore investigate the role of modulations in the GI tract on the cell response. The effect of digested and undigested hydrolysates on intracellular oxidation, cellular metabolic energy and proteome level was investigated. No difference in the effect on intracellular oxidation activity was obtained between cod and chicken hydrolysates, while higher affect on intracellular oxidation was provided by digested hydrolysates, with relative values of intracellular oxidation of cod of (70.2±0.8) and chicken of (74.5±1.4) % than by undigested ones, where values of cod and chicken were (95.5±1.2) and (90.5±0.7) %, respectively. Neither species nor digestion had any effect on cellular metabolic energy. At proteome level, digested hydrolysates gave again significantly stronger responses than undigested counterparts; cod peptides here also gave somewhat stronger response than chicken peptides. The knowledge of the action of food protein hydrolysates and their digests within live cells, also at proteome level, is important for further validation of their activity in higher eukaryotes to develop new functional food ingredients, such as in this case chicken and cod muscle-derived peptides.

Herring and chicken/pork meals lead to differences in plasma levels of TCA intermediates and arginine metabolites in overweight and obese men and women.

SCOPE: What effect does replacing chicken or pork with herring as the main dietary source of protein have on the human plasma metabolome? METHOD AND RESULTS: A randomised crossover trial with 15 healthy obese men and women (age 24-70 years). Subjects were randomly assigned to four weeks of herring diet or a reference diet of chicken and lean pork, five meals per week, followed by a washout and the other intervention arm. Fasting blood serum metabolites were analysed at 0, 2 and 4 weeks for eleven subjects with available samples, using GC-MS based metabolomics. The herring diet decreased plasma citrate, fumarate, isocitrate, glycolate, oxalate, agmatine and methyhistidine and increased asparagine, ornithine, glutamine and the hexosamine glucosamine. Modelling found that the tricarboxylic acid cycle, glyoxylate, and arginine metabolism were affected by the intervention. The effect on arginine metabolism was supported by an increase in blood nitric oxide in males on the herring diet. CONCLUSION: The results suggest that eating herring instead of chicken and lean pork leads to important metabolic effects, particularly on energy and amino acid metabolism. Our findings support the hypothesis that there are metabolic effects of herring intake unrelated to the long chain n-3 polyunsaturated fatty acid content.

Postprandial lipid and insulin responses among healthy, overweight men to mixed meals served with baked herring, pickled herring or baked, minced beef.

PURPOSE: The aim was to compare postprandial lipid, insulin and vitamin D responses after consumption of three otherwise identical meals served either with baked herring, pickled herring or with baked, minced beef. METHODS: Seventeen healthy, overweight men (mean age 58 years, BMI 26.4-29.5 kg/m(2)) consumed standardized lunches together with baked herring, pickled herring or baked, minced beef on three occasions in a crossover design. Blood samples were taken just before and up to 7 h after the meal. The postprandial response was measured as serum concentrations of triglycerides (TG), total cholesterol and lipoproteins (LDL, HDL and VLDL), insulin, 25-OH vitamin D and plasma fatty acid composition. RESULTS: There was no difference in postprandial lipid responses between the two herring meals, whereas a slower TG clearance was observed after the baked, minced beef meal. The 150 g servings of baked and pickled herring provided 3.3 and 2.8 g of long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), respectively, which was reflected in a substantial postprandial increase in plasma LC n-3 PUFA levels. The pickled herring contained 22% sugar and consequently gave a higher insulin response compared with the other two meals. CONCLUSIONS: Both pickled and baked herring are good sources of LC n-3 PUFA in the diet, but the presence of sugar in pickled herring should be taken into consideration, especially if large amounts are consumed. The faster postprandial TG clearance after a meal with baked herring compared with baked beef supports previous studies on the beneficial effects of herring on cardiovascular health.

Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies.

The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 µM Fe). Caco-2 cells were exposed to 0, 80 and 120 µM Fe and responded with increased hepcidin production at 120 µM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered.

Degradation of phytate by Pichia kudriavzevii TY13 and Hanseniaspora guilliermondii TY14 in Tanzanian togwa.

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3µmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement.

Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential.

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP(6) degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP(6) degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P(5) or D/L-Ins(1,2,3,4,6)P(5); D/L-Ins(1,2,3,4)P(4); to finally Ins(1,2,3)P(3) and D/L-Ins(1,2,4)P(3)/D/L-Ins(1,3,4)P(3). This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.

Hemoglobin-mediated lipid oxidation and compositional characteristics of washed fish mince model systems made from cod (Gadus morhua), herring (Clupea harengus), and salmon (Salmo salar) muscle.

The use of washed cod light muscle minces in mechanistic studies of hemoglobin (Hb)-mediated fish lipid oxidation has largely increased in the past 5 years. Although cod light muscle has a low level of intrinsic lipid oxidation catalysts, a prerequisite for a good oxidation model system, we believe it cannot fully mimic the oxidation kinetics taking place in other fish species being more susceptible to lipid oxidation. The aim of this study was to systematically investigate whether washed mince model systems useful in Hb-mediated oxidation studies could be prepared also from herring (Clupea harengus) and salmon (Salmo salar) light muscles. The kinetics of oxidation in the washed models was measured during ice storage (+/-Hb), and the results were related to compositional differences. Minces from cod, herring, and salmon light muscles were washed 3 times with 3 volumes of water and buffer. A 20 microM portion of Hb and 200 ppm streptomycin was then added, followed by adjustment of pH and moisture to 6.3 and 86%, respectively. Samples with or without Hb were then stored on ice, and oxidation was followed as peroxide value (PV), rancid odor, redness (a*) loss and yellowness (b*). Prior to storage, all minces and models were also analyzed for total lipids, fatty acids, alpha-tocopherol, proteins, Hb, Fe, Cu, and Zn. Hb-mediated lipid oxidation appeared within 2 days on ice in all models. Small differences in the oxidation rates ranked the models as herring > cod > salmon. These differences were ascribed to more preformed peroxides and trace elements in the herring model, and more antioxidants in the salmon model. Controls, without Hb, stayed stable in all cases except herring, where a very slight oxidation appeared, especially if the herring raw material had been prefrozen. In conclusion, fattier fish like dark muscle species and salmonoids are useful for making washed mince model systems and would be a better choice than cod if there is an interest in the oxidation kinetics of such species.

Absorption of zinc and retention of calcium: dose-dependent inhibition by phytate.

The dose-dependent inhibitory effect of sodium phytate (myo-inositol-hexaphosphate) on absorption of zinc and retention of calcium was studied in man. No systematic study of this dose-response effect has been reported to this time. Forty subjects were served meals containing white wheat rolls without/with additions of phytate. Ten subjects were given test meals containing one or two of the studied levels of phytate and in addition all subjects were served meals to which no phytate was added. The zinc content was 3.1 mg (47 micromol) and the calcium content 266 mg (6.6 mmol). The rolls were labelled extrinsically with radioisotopes, 65Zn and 47Ca, and whole-body retention of both minerals was measured. Totally 105 meals were served, 36 meals in which no phytate was added and 9-10 meals on each level of phytate. The zinc absorption in meals to which either 0, 25, 50, 75, 100, 140, 175 or 250 mg of phytate-P (0, 134, 269, 403, 538, 753, 941 or 1344 micromol phytate) had been added was 22%, 16%, 14%, 11%, 7%, 7%, 7% and 6%, respectively (mean values). The addition of 50 mg phytate-P or more significantly decreased zinc absorption (p=0.01) as compared to absorption from the test meals with no added phytate. The calcium retention at day 7 in the same meals was 31%, 28%, 27%, 26%, 22%, 19%, 14% and 11% (mean values). The addition of 100 mg phytate-P or more significantly decreased calcium retention (p=0.03) compared to the test meals with no added phytate. It was concluded that the inhibitory effect of phytate on the absorption of zinc and the retention of calcium was dose dependent.

Extrinsic labelling of zinc and calcium in bread.

This study investigated the effect of different means of extrinsic administration of 65Zn and 47Ca in white wheat flour bread on the measured absorption. Eight healthy subjects were served 80g of labelled bread as a standardized breakfast after an overnight fast on three occasions. Extrinsic labelling of the meals with 65Zn and 47Ca was done in three ways: (a) by adding the isotopes to the bread 16h before it was served, (b) by adding the isotopes shortly before serving or (c) by adding the isotopes to the water used in dough making. Zinc and calcium chloride corresponding to 3.2mg (49 micromol) zinc and 275mg (6.9mmol) calcium in one portion were added to the dough. Whole-body retention was measured by whole-body counting. The fractional absorption of zinc was (a) 0.243 +/- 0.122, (b) 0.217 +/- 0.101 and (c) 0.178 +/- 0.063 (mean +/- SD), and the fractional absorption of calcium (expressed as calcium retention on day 7) was (a) 0.351 +/- 0.108, (b) 0.357 +/- 0.131 and (c) 0.334 +/- 0.117 (mean+SD). No significant difference (p > 0.05) was seen between the different ways for either zinc nor calcium.

Simultaneous and sensitive analysis of Cu, Ni, Zn, Co, Mn, and Fe in food and biological samples by ion chromatography.

A sensitive and simple method for the simultaneous determination of nutritionally important minerals in food samples is in great demand. Ion chromatography coupled with UV-vis detection is shown to be an appropriate technique for this objective. The method is based on the formation of mineral complexes by pyridine-2,6-dicarboxylic acid in the mobile phase. The complexes are then postcolumn derivatized with 4-(2-pyridylazo)resorcinol (PAR), resulting in mineral-PAR complexes that are detected by UV-vis absorption at 500 nm. This facilitates the simultaneous separation and quantification of minerals in one chromatographic run. Within 16 min, Cu, Ni, Zn, Co, Mn, and Fe are analyzed. When a 50 microL injection volume is used, the average detection limit is 5 ppb in the injection liquid. The detection limit makes it a superior alternative to AAS and, in several applications, also an alternative to ICP-MS techniques. Different sample treatments were evaluated. The concentration of acid in the treated sample varied with the sample treatment, which may cause a limitation for the injection volume. A crucial prerequisite to achieve the reported detection limits and to obtain reliable results is to completely exclude all contamination from instruments and materials.