Bioactivities of Pseudocereal Fractionated Seed Proteins and Derived Peptides Relevant for Maintaining Human Well-Being.

Food proteins and peptides are able to exert a variety of well-known bioactivities, some of which are related to well-being and disease prevention in humans and animals. Currently, an active trend in research focuses on chronic inflammation and oxidative stress, delineating their major pathogenetic role in age-related diseases and in some forms of cancer. The present study aims to investigate the potential effects of pseudocereal proteins and their derived peptides on chronic inflammation and oxidative stress. After purification and attribution to protein classes according to classic Osborne's classification, the immune-modulating, antioxidant, and trypsin inhibitor activities of proteins from quinoa (Chenopodium quinoa Willd.), amaranth (Amaranthus retroflexus L.), and buckwheat (Fagopyrum esculentum Moench) seeds have been assessed in vitro. The peptides generated by simulated gastro-intestinal digestion of each fraction have been also investigated for the selected bioactivities. None of the proteins or peptides elicited inflammation in Caco-2 cells; furthermore, all protein fractions showed different degrees of protection of cells from IL-1ß-induced inflammation. Immune-modulating and antioxidant activities were, in general, higher for the albumin fraction. Overall, seed proteins can express these bioactivities mainly after hydrolysis. On the contrary, higher trypsin inhibitor activity was expressed by globulins in their intact form. These findings lay the foundations for the exploitation of these pseudocereal seeds as source of anti-inflammatory molecules.

Exploiting the potential of Sudanese sorghum landraces in biofortification: Physicochemical quality of the grain of sorghum (Sorghum bicolor L. Moench) landraces.

This study aimed to describe the phytonutrients and antioxidant activity, protein content, in vitro protein digestibility (IVPD), protein fraction, and bioavailability of Fe and Zn in the grains of five sorghum landraces grown in Sudan. The results showed significant differences in all quality tests among the landraces. The Tetron landrace showed the highest percentage of crude protein and IVPD among the landraces. Additionally, most of the landrace grains had high contents of Fe and Zn with a high rate of bioavailability. The Kolom 4055 and Wad akar exhibited significantly higher total phenolic contents, with antioxidant activity of 79.3% and 83.4%, respectively. The glutelin content was relatively higher compared to the other fractions, irrespective of sorghum landraces. The principal components cumulatively accounted for 89.3% of the total variation among the five sorghum landraces. It can be concluded that these landraces could be used in the improvement of new value-added crops using the by-products of sorghum grains.

Animal, Plant, Collagen and Blended Dietary Proteins: Effects on Musculoskeletal Outcomes.

Dietary protein is critical for the maintenance of musculoskeletal health, whereappropriate intake (i.e., source, dose, timing) can mitigate declines in muscle and bone mass and/orfunction. Animal-derived protein is a potent anabolic source due to rapid digestion and absorptionkinetics stimulating robust increases in muscle protein synthesis and promoting bone accretion andmaintenance. However, global concerns surrounding environmental sustainability has led to anincreasing interest in plant- and collagen-derived protein as alternative or adjunct dietary sources.This is despite the lower anabolic profile of plant and collagen protein due to the inferior essentialamino acid profile (e.g., lower leucine content) and subordinate digestibility (versus animal). Thisreview evaluates the efficacy of animal-, plant- and collagen-derived proteins in isolation, and asprotein blends, for augmenting muscle and bone metabolism and health in the context of ageing,exercise and energy restriction.

Harnessing Microbes for Sustainable Development: Food Fermentation as a Tool for Improving the Nutritional Quality of Alternative Protein Sources.

In order to support the multiple levels of sustainable development, the nutritional quality of plant-based protein sources needs to be improved by food technological means. Microbial fermentation is an ancient food technology, utilizing dynamic populations of microorganisms and possessing a high potential to modify chemical composition and cell structures of plants and thus to remove undesirable compounds and to increase bioavailability of nutrients. In addition, fermentation can be used to improve food safety. In this review, the effects of fermentation on the protein digestibility and micronutrient availability in plant-derived raw materials are surveyed. The main focus is on the most important legume, cereal, and pseudocereal species (Cicer arietinum, Phaseolus vulgaris, Vicia faba, Lupinus angustifolius, Pisum sativum, Glycine max; Avena sativa, Secale cereale, Triticum aestivum, Triticum durum, Sorghum bicolor; and Chenopodium quinoa, respectively) of the agrifood sector. Furthermore, the current knowledge regarding the in vivo health effects of fermented foods is examined, and the critical points of fermentation technology from the health and food safety point of view are discussed.

Influence of nutritional and antinutritional components on dough rheology and in vitro protein & starch digestibility of minor millets.

The nutritional and antinutritional components of minor millets were correlated with mixolab dough mixing behavior and in vitro protein and starch digestibility. Total arabinoxylan (r = -0.53, p < 0.05) and dietary fiber (r = -0.66, p < 0.05) content significantly (p < 0.05) increased protein weakening. Peak viscosity negatively correlated with phenolic (r = -0.55, p < 0.05) content. The dietary fiber and phenolics suppressed retrogradation. Protein digestibility negatively correlated with tannin (r = -0.70, p < 0.05), phytic acid (r = -0.69, p < 0.05), phenolics (r = -0.79, p < 0.05), flavonoids (r = -0.72, p < 0.05) and total dietary fiber content (r = -0.84, p < 0.05). A positive correlation of resistant starch (RS) with total dietary fiber (r = 0.85, p < 0.05), phenolics (r = 0.89, p < 0.05), flavonoids (r = 0.83, p < 0.05), phytic acid (r = 0.43, p < 0.05) and tannin content (r = 0.79, p < 0.05) was observed. Millets predicted lower glycemic index than wheat and it was found to be negatively associated with the RS (r = -0.96, p < 0.05) and total dietary fiber content (r = -0.89, p < 0.05) and positively correlated (r = 0.98, p < 0.05) with rapidly digestible starch. The millets may be diversified for personalized nutrition and development of functional food.

Non-starch constituents influence the in vitro digestibility of naked oat (Avena nuda L.) starch.

The present study investigated the effects of proteins, lipids and ß-glucan in naked oat flour (NOF) on the in vitro digestibility of starch. The content of rapidly digested starch (RDS) increased, and the content of resistant starch (RS) decreased in NOF after removing the non-starch constituents. The estimated glycemic index (eGI) of starch in NOF increased after the removal of the non-starch constituents, with a decreasing order of naked oat starch (NOS) > de-ß-glucan flour > de-proteins flour > de-lipids flour > NOF. NOS was found to have an A-type crystalline pattern, but the removal of proteins or ß-glucan rendered NOS a V-type crystalline pattern. The relative crystallinity decreased after removing non-starch constituents. The in vitro digestibility was positively correlated with the short-range molecular order and negatively correlated with the relative crystallinity. These results clearly illustrate the effects of non-starch constituents on the low digestibility of naked oat.

Bioadhesive Food Protein Nanoparticles as Pediatric Oral Drug Delivery System.

The goal of this study was to develop bioadhesive food protein nanoparticles using zein (Z), a hydrophobic corn protein, as the core and whey protein (WP) as the shell for oral pediatric drug delivery applications. Lopinavir (LPV), an antiretroviral drug, and fenretinide, an investigational anticancer agent, were used as model drugs in the study. The particle size of ZWP nanoparticles was in the range of 200-250 nm, and the drug encapsulation efficiency was >70%. The nanoparticles showed sustained drug release in simulated gastrointestinal fluids. ZWP nanoparticles enhanced the permeability of LPV and fenretinide across Caco-2 cell monolayers. In both ex vivo and in vivo studies, ZWP nanoparticles were found to be strongly bioadhesive. ZWP nanoparticles enhanced the oral bioavailability of LPV and fenretinide by 4 and 7-fold, respectively. ZWP nanoparticles also significantly increased the half-life of both drugs. The nanoparticles did not show any immunogenicity in mice. Overall, the study demonstrates the feasibility of developing safe and effective food protein-based nanoparticles for pediatric oral drug delivery.

The effect of cell wall encapsulation on macronutrients digestion: A case study in kidney beans.

Cotyledon cells in kidney beans naturally encapsulate starch and proteins limiting the access of digestive enzymes to their substrates. In this study, we investigated the effect of cell wall on bean protein digestibility and its relationship with starch digestion. Results showed that proteins contained in the cytoplasmic matrix influence the rate at which starch is digested in-vitro. Confocal laser scanning microscopy revealed that storage proteins in the cytoplasm act as a second encapsulation system preventing starch digestion. This microstructural organization only affected starch since no changes in protein digestion rate or extent were observed due to the presence of starch granules. Fourier transform infrared spectroscopy revealed that cellular entrapment limited protein denaturation induced by thermal treatments. High concentrations of a fraction resistant to digestion were found in proteins that were heated when entrapped within intact cotyledon cells, compared to those thermally treated as bean flour.

Impact of cooking temperature on the quality of quick cooking brown rice.

Three cooking temperatures (72, 80, and 88 °C) were applied to two rice genotypes (Puitá Inta CL and INOV CL) for preparing quick cooking brown rice. Samples were analyzed for cooking time, color, scanning electron microscopy (SEM), damaged grains, amylose, protein content and extractability, differential scanning calorimetry (DSC), X-ray diffraction (XRD), sensory properties, and in vitro digestion. Cooking time was reduced from 23.0-23.6 to 5.5-6.9 min when the highest temperature was applied, depending on genotype. The greatest grain deformation was observed for treatments from Puitá Inta CL. XRD showed greater ability of brown rice from Puitá Inta CL to gelatinize at 88 °C. Appearance, texture, and flavor of quick cooking brown rice prepared at 88 °C was inferior to its brown rice counterparts. Starch digestibility decreased by around 20-22% in 88 °C-prepared-quick cooking brown rice. Lower digestibility values were determined for 88 °C-treated-INOV CL, and were associated with grain integrity.

Protein-quality evaluation of complementary foods in Indian children.

BACKGROUND: The types of food in complementary feeding of infants and young children are important for growth and development. Food protein quality, as measured by the Digestible Indispensable Amino Acid Score (DIAAS), requires the determination of true ileal digestibility of indispensable amino acids (IAAs) in children. OBJECTIVES: First, the aim of this study was to measure the true ileal IAA digestibility of 4 (rice, finger millet, mung bean, and hen egg) commonly consumed complementary foods in children aged <2 y using the dual-isotope tracer method. Second, we calculated the DIAAS of complementary feeding diets and their relation to stunting in a representative Indian rural population. DESIGN: Rice, finger millet, and mung bean were intrinsically labeled with deuterium oxide (2H2O), whereas egg was labeled through oral dosing of hens with a uniformly 2H-labeled amino acid mixture. True ileal IAA digestibility was determined by the dual-isotope tracer technique. The DIAAS of complementary food protein was calculated in children aged 1-3 y from a nationally representative survey to evaluate its relation with stunting. RESULTS: True ileal IAA digestibility was lowest in mung bean (65.2% ± 7.1%), followed by finger millet (68.4 %± 5.3%) and rice (78.5% ± 3.5%), and was highest for egg (87.4% ± 4.0%). There was a significant inverse correlation of complementary food DIAAS with stunting in survey data (r = -0.66, P = 0.044). The addition of egg or milk to nationally representative complementary diets theoretically improved the DIAAS from 80 to 100. CONCLUSIONS: The true ileal IAA digestibility of 4 foods commonly consumed in complementary diets showed that the DIAAS was associated with stunting and reinforces the importance of including animal source food (ASF) in diets to improve growth. This trial was registered at http://ctri.nic.in/clinicaltrials/login.php as CTRI/2017/02/007921.

Extraction and characterization of starch granule-associated proteins from rice that affect in vitro starch digestibility.

Starch granule-associated proteins (SGAPs) including granule-surface proteins and granule-channel proteins in waxy, low- and high-amylose rice starch were extracted and identified. The in vitro digestibility of starch was investigated before and after the extraction of granule-channel proteins or total SGAPs. The results showed that 10 types of major differentially expressed proteins (DEPs) including 14-3-3-like protein and ribosomal protein were found among starches. In addition, the lack of only granule-channel proteins or total SGAPs led to significant and different changes in the levels of rapidly digestible starch, slowly digestible starch and resistant starch. Possible mechanisms are related to the accessibility of amylase into starch granules and structural properties of SGAPs. This study provides more information about DEPs in rice starch with different amylose content and supports further study on the relationship between SGAPs and in vitro starch digestibility.

Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum.

Kafirins are the major storage proteins in sorghum (Sorghum bicolor) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the k1C family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the k1C genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple k1C family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all k1C genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.

Associations between urine excretion of isoflavonoids and cognition in postmenopausal women in the Women's Isoflavone Soy Health clinical trial.

OBJECTIVES: To determine effect of change in urine excretion of isoflavonoids on cognitive change. DESIGN: Post hoc analysis of isoflavonoid exposure (mean 2.7 years) during the randomized, placebo-controlled, double-blind Women's Isoflavone Soy Health trial. SETTING: General community. PARTICIPANTS: Healthy postmenopausal women (N = 350). INTERVENTION: Twenty-five grams of isoflavone-rich soy protein (91 mg of aglycone weight isoflavones: 52 mg genistein, 36 mg daidzein, 3 mg glycitein) or milk protein-matched placebo provided daily. MEASUREMENTS: Overnight urine excretion, fasting plasma levels of isoflavonoids, and cognitive function measured at baseline and endpoint. RESULTS: Three hundred women (age: mean 61, range 45-92) completed both cognitive assessments and did not use hormone replacement therapy during the trial. Mean on-trial change from baseline in urine excretion of isoflavonoids was not significantly associated with change in a composite score of global cognition (P = .39). Secondary analyses indicated that change in urine excretion of isoflavonoids was inversely associated with change in a factor score representing general intelligence (P = .02) but not with factor scores representing verbal or visual episodic memory. Mean differences in this general intelligence factor score between women in the lowest and highest quartiles of isoflavonoid change were equivalent to an approximate 4.4-year age-associated decline. Analyses based on plasma isoflavonoid levels yielded similar but attenuated results. CONCLUSION: In healthy postmenopausal women, long-term changes in isoflavonoids are not associated with global cognition, supporting clinical trial results, although greater isoflavonoid exposure from dietary supplements is associated with decrements in general intelligence but not memory; this finding requires confirmation in future studies.

Intraluminal immunoreactive caseinomacropeptide after milk protein ingestion in humans.

Caseinomacropeptide (CMP)is a 64 amino acids peptide which is the first product released after kappa-casein hydrolysis. The present work investigates the kinetics delivery of CMP in human jejunal lumen during the digestion of intrinsically [15N]-labelled casein, whey protein, yoghurt and pea flour meal. Effluents were collected through a nasointestinal tube and analysed for the enrichment in [15N] to evaluate the dietary nitrogen fraction. Detection and quantification of CMP was performed by an inhibition Elisa procedure. No trace of CMP was detected in the ileum of volunteers after the ingestion of the casein meal. The results showed that CMP appears in the jejunal effluents within the first 20 min after meal ingestion at a level varying from meal to meal. During digestion of whey protein, CMP appeared rapidly as a single peak and in high amounts, whereas it is discharged slowly in moderate proportions with the casein meal. These results demonstrate that CMP is emptied from the stomach in significant amounts during milk products digestion and support the hypothesis that food-born peptides could exert a physiological function. Moreover, in the present study a relation could be assumed between the amount of CMP in the meal and the stimulation of luminal endogenous nitrogen secretion. However, the specific physiological activity of CMP in humans, particularly on the digestion process, requires further studies.

[15N]-labeled pea flour protein nitrogen exhibits good ileal digestibility and postprandial retention in humans.

The aim of the present study was to evaluate postprandial absorption of pea protein as well as exogenous nitrogen retention in humans. For this purpose, after fasting overnight, seven healthy adults (4 males and 3 females) ingested [15N]-labeled pea protein (195 mmol N). Ileal effluents were collected for 8 h at 30-min intervals using a nasointestinal intubation technique. Urine and plasma samples were collected for 24 h. The [15N]-enrichment was determined in the intestinal samples, in the plasma amino acids and urea as well as in the urinary urea and ammonia fractions. The true gastroileal absorption of pea protein was 89.4 +/- 1.1%. This absorption was correlated with a significant increase (P < 0.05) in [15N]-enrichment in the plasma amino acids and in the nitrogen incorporated into the body urea pool for 1 h following pea ingestion. The enrichment remained significantly higher than the basal values in these pools 24 h after pea ingestion. The recovery of total urinary exogenous nitrogen after 22 h was 31.1 +/- 9.3 mmol N. Moreover, the kinetics of [15N]-labeled pea amino acids deamination reached a plateau of 39 mmol. Under these conditions, pea nitrogen retention represented 78% of the absorbed dietary nitrogen in healthy humans. The present results demonstrate the good true nitrogen digestibility and retention of pea protein in humans.

Absorption and excretion of the soy isoflavone genistein in rats.

Rodent models have been used to study the anticarcinogenic properties of the soy isoflavones, particularly genistein, but there is little information regarding the pharmacokinetics of the absorption and excretion of genistein. In this study, rats were given a single oral dose of genistein (20 mg/kg body weight) or an equivalent dose of its glycone forms, as an isoflavone-rich soy extract. Concentrations of genistein were measured in plasma, urine and feces at intervals up to 48 h after dosing. Plasma genistein concentration at 2 h after dosing was 11.0 +/- 2.3 mumol/L in genistein-treated rats compared with 4.93 +/- 0.22 mumol/L (P = 0.025) in soy extract-treated rats, but there were no significant differences at 8 h and later times. The mean urinary excretion rate during the first 2 h after dosing was more than 10 times higher in the genistein group compared with the soy extract group (0.27 +/- 0.08 mumol/h and 0.020 +/- 0.011 mumol/h, respectively, P = 0.017) but the percentage of dose recovered in urine over 48 h was not different between groups (19.9 +/- 2.4% genistein treated; 17.5 +/- 1.1% soy extract treated). There were no significant differences between groups in the recovery of genistein in feces (21.9 +/- 2.8% and 21.1 +/- 2.5% of dose, respectively). Only 6.1 +/- 0.9% of the daidzein from the soy extract was recovered in the feces. The results suggest that the extent of absorption of genistein is similar for the glycone and aglycone forms. Although higher initial plasma concentrations may be achieved with the aglycone, similar long-term concentrations exist for both forms of isoflavone.

Bioavailability of soybean isoflavones depends upon gut microflora in women.

Soybean isoflavones have been proposed to be anticarcinogenic, but their effective doses have not been established. To study their bioavailability, seven women consumed 3.4, 6.9, or 10.3 mumol isoflavones/kg body wt in soymilk in each of three meals of a liquid diet on one of three feeding days that were separated by 2-wk washout periods. Subjects were randomly assigned to doses in a cross-over design. Plasma, urine and fecal isoflavones were measured by reverse phase HPLC. In two subjects, fecal isoflavone recovery was 10-20 times that in the other five subjects. Average 48-h urinary recoveries of ingested daidzein and genistein were 16 +/- 4 and 10 +/- 4%, respectively, at all three doses among the five subjects excreting only small amounts of isoflavones in feces, whereas urinary recoveries of daidzein and genistein in the two subjects who excreted large amounts of fecal isoflavones were 32 +/- 5 and 37 +/- 6%, respectively. Urinary isoflavone excretion was nearly zero in all subjects at 48 h after dosing. Average plasma concentration of genistein at 24 h after the breakfast isoflavone dose in subjects excreting large amounts of fecal isoflavones was significantly greater by 2.5-fold than in subjects who excreted small amounts of fecal isoflavones (P < 0.05). In vitro anaerobic incubation of isoflavones with human feces showed that intestinal half-life of daidzein and genistein may be as little as 7.5 and 3.3 h, respectively. These data suggest that human isoflavone bioavailability depends upon the relative ability of gut microflora to degrade these compounds.

Soy protein and casein in cholesterol-enriched diets: effects on plasma lipoproteins in normolipidemic subjects.

The effect of dietary plant protein vs animal protein on plasma lipoproteins was investigated in a crossover study. Eleven normal subjects consumed formula diets containing 20% of calories as soy protein or casein, 27% as fat, and 53% as carbohydrate: the average cholesterol intake was 500 mg/d. After an initial decrease in plasma cholesterol and low-density-lipoprotein cholesterol (LDL-C) on both diets, the concentration of LDL-C stabilized during the soy-protein diet at a 16% lower level than on the casein diet (p less than 0.02) whereas the concentration of high-density-lipoprotein cholesterol (HDL-C) was 16% higher (p less than 0.01). In a similar earlier study with a cholesterol intake of less than 100 mg/d, casein and soy protein had indistinguishable effects on lipoprotein levels. Our two studies together suggest that the level of cholesterol intake may determine whether plant and animal dietary proteins have similar or different effects on plasma LDL and HDL concentrations.