Wheat Protein Research Articles

Gluten - a nutritional enemy or indispensable diet ingredient?

Introduction: Gluten is a mixture of wheat, rye and barley storage proteins. These corps have provided human beings worldwide with energy and nutrients for centuries. However, an increase in the occurrence of symptoms after the consumption of gluten has been observed recently. Although celiac disease is an indication of strict adherence to the gluten-free diet (GFD), the popularity of GFD is growing among healthy people. The risks of unsupported gluten elimination should be assessed before the introduction of GFD. 
 Purpose: The study aims to define GFD and indicate who should follow the diet, as well as present difficulties concerning strict adherence to GFD and possible consequences of eliminating gluten on one’s own. 
 Description of the state of knowledge: Regarding the mechanism, in which the symptoms after gluten consumption develop, the following diseases have been distinguished: celiac disease (CD), wheat allergy and non-celiac gluten sensitivity. Currently, the only indication of strict long-life adherence to GFD is CD. Such a restrictive approach may cause financial and social problems. Although the introduction of GFD improves the quality of life in CD patients, it does not lead to the immediate disappearance of the symptoms. Healthy people, who decide to eliminate gluten, should balance their diet carefully, as GFD might cause nutritional deficiencies and metabolic diseases. 
 Summary: Strict long-life adherence to GFD is indicated in CD. This treatment may be problematic, which often is not evident to people who eliminate gluten on their own. Unbalanced GFD can lead to nutritional deficiencies as well as metabolic diseases.

A Comparative Evaluation of the Structural and Biomechanical Properties of Food-Grade Biopolymers as Potential Hydrogel Building Blocks.

The aim of this study was to conduct a comparative assessment of the structural and biomechanical properties of eight selected food-grade biopolymers (pea protein, wheat protein, gellan gum, konjac gum, inulin, maltodextrin, psyllium, and tara gum) as potential hydrogel building blocks. The prepared samples were investigated in terms of the volumetric gelling index, microrheological parameters, physical stability, and color parameters. Pea protein, gellan gum, konjac gum, and psyllium samples had high VGI values (100%), low solid–liquid balance (SLB < 0.5), and high macroscopic viscosity index (MVI) values (53.50, 59.98, 81.58, and 45.62 nm−2, respectively) in comparison with the samples prepared using wheat protein, maltodextrin, and tara gum (SLB > 0.5, MVI: 13.58, 0.04, and 0.25 nm−2, respectively). Inulin had the highest elasticity index value (31.05 nm−2) and MVI value (590.17 nm−2). The instability index was the lowest in the case of pea protein, gellan gum, konjac gum, and inulin (below 0.02). The color parameters and whiteness index (WI) of each biopolymer differed significantly from one another. Based on the obtained results, pea protein, gellan gum, konjac gum, and psyllium hydrogels had similar structural and biomechanical properties, while inulin hydrogel had the most diverse properties. Wheat protein, maltodextrin, and tara gum did not form a gel structure.

Recovery of Grain Yield and Protein with Fertilizer Application Post Nitrogen Stress in Winter Wheat (Triticum aestivum L.)

Unfavorable weather conditions and lack of appropriate farm machinery often delay N application. This results in nitrogen (N) deficiency during the vegetative and early reproductive growth stages of winter wheat. The objective of this study was to evaluate the impact of N application timings (from tillering to flag leaf growth stages) on winter wheat grain yield and protein. The study was conducted across 12 site–years in Oklahoma, US. The treatments included a non-fertilized check, a pre-plant application of 100 kg N ha−1, and ten in-season application timings at 100 kg of N ha−1. The in-season treatment applications were initiated at the point when an N deficiency was visually identified by comparing the pre-plant treatment to the non-fertilized check. The treatments were applied in a progressive order every seven growing degree days (GDD &gt; 0 °C) until a cumulative GDD of 63 was reached after visual deficiency (DAVD). All in-season treatments increased grain yield and protein as compared to the non-fertilized check, showing that N was a yield-limiting factor. The nitrogen applications made post Feekes 8 decreased grain yield when compared to pre-plant applications. Across this data set, that timing corresponded to a range of 21 to 63DAVD. The results suggested that forgoing N application until the growth stage Feekes 7, even when the visual N deficiency was highly apparent before that stage, had no negative impact on the yield, and it even increased the yield as compared to the pre-plant application in some cases. The plant developmental stage at which the N application takes place is more critical than the level of N deficiency. Our results show that N fertilizer applications should be made posteriorly to the crop dormancy to maximize both yield and protein, and that plants can recover from N deficiency when applications are made until the late-vegetative phase (Feekes 7). This document shows that winter wheat producers have a much wider N application window than traditionally believed.

Temporally coordinated expression of nuclear genes encoding chloroplast proteins in wheat promotes Puccinia striiformis f. sp. tritici infection

Targeting host processes that allow pathogens to thrive can be invaluable in resistance breeding. Here, we generated a deep-sequencing transcriptome time course for Puccinia striiformis f. sp. tritici (Pst) infection on wheat and compared datasets from three wheat varieties with different levels of susceptibility to two tested pathogen isolates. We sought genes specifically altered in a susceptible host as candidates that might support colonisation. Host responses differed between Pst-varietal pairs most prominently early during infection. Notably, however, nuclear genes encoding chloroplast-localised proteins (NGCPs) exhibited temporal coordination of expression profiles that differed at later time points in relation to Pst susceptibility. Disrupting one such NGCP, encoding the chloroplast-localised RNA binding protein TaCSP41a, led to lower Pst susceptibility. These analyses thus highlight NGCPs as prime targets for Pst manipulation during infection and point to TaCSP41a disruption as a potential source of Pst resistance for breeding programmes.

Grain yield, chlorophyll and protein contents of elite wheat genotypes under drought stress

BackgroundDrought stress at different growth stages significantly alters growth, yield, and quality traits of wheat. However, great variability exists among genotypes regarding their response to drought stress. Therefore, determining the impacts of drought stress on yield and quality traits would help to select the superior genotypes. MethodsThis study investigated the effects of drought stress on wheat grain yield, chlorophyll, and protein contents. Fourteen (14) recently developed elite bread wheat (Triticum aestivum L.) genotypes were used in this study for evaluation under irrigated (full irrigation) and drought conditions (half of normal irrigation). The data relating to growth, yield and protein contents were recorded. ResultsSignificant differences (P ≤ 0.01) were noted among genotypes for all recorded traits. Drought stress significantly reduced the days to 50 % heading, days to 50 % maturity, grain filling, plant height (cm), number of spikes per m2, chlorophyll index (SPAD), peduncle length (cm), number of grains spike-1, thousand grain weight (g) and grain yield (kg ha−1). However, protein contents were increased under drought stress. Correlation analysis showed significant positive association of grain yield with thousand grain weight, number of spikes per m2, spike length, chlorophyll index, grain filling period and number of grains spike-1 under both irrigated and drought stress conditions. The protein contents expressed positive and negative relationship with yield under drought stress and irrigated conditions, respectively. Bioplot analysis revealed that genotype ‘V-19618′ and ‘V-19600′ proved superior under drought conditions regarding grain yield and related traits, while genotype ‘V-19574′ proved better under both irrigated and drought conditions. ConclusionsThese identified genotypes, i.e., ‘V-19618′ and ‘V-19600′ can be utilized in future wheat breeding programs to induce desirable characters for producing drought tolerant wheat genotypes.

Digestibility of wheat alpha-amylase/trypsin inhibitors using a caricain digestive supplement.

Wheat is a major source of nutrition, though in susceptible people it can elicit inappropriate immune responses. Wheat allergy and non-celiac wheat sensitivity are caused by various wheat proteins, including alpha-amylase trypsin inhibitors (ATIs). These proteins, like the gluten proteins which can cause celiac disease, are incompletely digested in the stomach such that immunogenic epitopes reach the lower digestive system where they elicit the undesirable immune response. The only completely effective treatment for these immune reactions is to eliminate the food trigger from the diet, though inadvertent or accidental consumption can still cause debilitating symptoms in susceptible people. One approach used is to prevent the causal proteins from provoking an immune reaction by enhancing their digestion using digestive protease supplements that act in the stomach or intestine, cleaving them to prevent or quench the harmful immune response. In this study, a digestive supplement enriched in caricain, an enzyme naturally present in papaya latex originally designed to act against gluten proteins was assessed for its ability to digest wheat ATIs. The digestion efficiency was quantitatively measured using liquid chromatography-mass spectrometry, including examination of the cleavage sites and the peptide products. The peptide products were measured across a digestion time course under conditions that mimic gastric digestion in vivo, involving the use of pepsin uniquely or in combination with the supplement to test for additive effects. The detection of diverse cleavage sites in the caricain supplement-treated samples suggests the presence of several proteolytic enzymes that act synergistically. Caricain showed rapid action in vitro against known immunogenic ATIs, indicating its utility for digestion of wheat ATIs in the upper digestive tract.

Differences in bread protein digestibility traced to wheat cultivar traits

Wheat grain storage proteins, i.e. the gluten proteins, and alpha-amylase trypsin inhibitors are partially resistant to gastrointestinal digestion. They figure among the wheat components that are potential triggers of non-celiac wheat sensitivity. To explore the digestibility of bread wheat proteins in relation to heritable plant traits, old and modern wheat varieties grown in two environments were genotyped, phenotyped, and processed into breads, which were partially digested in vitro in a system resembling human digestion. The extent of proteolysis measured after 2 h of digestion was variable. Digestion of proteins present in bread made from old wheat cultivars registered before 1960 and modern cultivars registered after 1960 was similar. No major plant trait was correlated to partial protein digestibility of bread from cultivars with high grain protein content. The protein digestibility of bread from high yield cultivars was influenced by protein composition, as well as the genetic distance per chromosome from a reference variety. Improving high yield cultivars by breeding for highly digestible proteins might help to mitigate adverse reactions to a range of wheat proteins in susceptible individuals.

Spatially varying wheat protein premiums

Spatially varying wheat protein premiums

Exogenous melatonin improved photosynthetic efficiency of photosystem II by reversible phosphorylation of thylakoid proteins in wheat under osmotic stress.

It has been well demonstrated that melatonin plays an important protective role in photosynthesis of plants under various environmental stresses, while the detailed mechanisms by which melatonin protects photosystem II (PSII) under environmental stress are still unclear. In the study, the effects of melatonin on photosynthetic efficiency, energy dissipation, PSII protein composition, and reversible phosphorylation of thylakoid proteins were investigated in wheat plants under osmotic stress. The results showed that osmotic stress significantly reduced pigment content, photochemical efficiency of PSII, oxygen-evolving activity, and dissipation of excess excitation energy, while 25 μM melatonin applications greatly alleviated their decline under osmotic stress. Western blot data of PSII proteins revealed that melatonin upregulated the levels of D1, Lhcb5, Lhcb6, PsbQ, and PsbS proteins in wheat exposed to osmotic stress. In addition, thylakoid membrane proteins were strongly phosphorylated in wheat under osmotic stress with or without melatonin. Furthermore, the results from PSII protein dephosphorylation showed that exogenous melatonin promoted the dephosphorylation of LCHII, CP43, and D1 under osmotic stress. Therefore, our findings suggest that melatonin can provide an effective protection for the photosynthetic apparatus by the regulation of PSII proteins and the reversible phosphorylation of thylakoid proteins under drought stress.

Study of Noodle Quality Based on Protein Properties of Three Wheat Varieties

Waxy wheat offers unique benefits in food processing, including improving the smoothness and performance of the product. However, waxy wheat is not yet commercially available. The protein characteristics, including the protein content, subunit distribution, secondary structure, chemical interactions, and microstructure of the gluten, were explored to realize the full potential of waxy wheat. The results showed that the noodles prepared from waxy wheat had a gentle and glutinous texture compared with GY2018 and YM13. Partial-waxy and waxy wheat had a lower gluten index and glutenin macropolymer (GMP) content than GY2018, indicating a reduced gluten strength. Confocal laser scanning microscopy (CLSM) images showed that the starch granules were not securely attached to the partial-waxy and waxy wheat protein matrix. In addition, the waxy protein chains appeared more elongated and they weakened the protein network. In particular, HMW-GS subunit 2 + 12 may be the essential cause of the weak dough from SKN1. Compared with GY2018 and YM13, SKN1 had the highest number of free sulfhydryl groups. Rather than ionic bonds, hydrophobic interactions increased the gluten network in GY2018, YM13, and SKN1. The weak molecular forces in the gluten will result in a soft noodle texture.

Effects of decolorization on aggregation behavior of highland barley proteins: Comparison with wheat proteins

To explore effects of decoloration on highland barley proteins (HBPs) aggregation, physicochemicalproperties, structuralcharacteristics, and functional characteristics of decolorized highland barley proteins (DHBPs) were investigated. In order to compare the aggregation of HBPs, wheat proteins (WPs) were selected as a control group. The solubility, turbidity, particle size and surfacehydrophobicity of HBPs increased obviously (P < 0.05) after decoloration, but still lower than WPs. Electrophoretic analysis showed that the bands amount of HBPs and DHBPs were lower than WPs, while decolorization had no effect on the bands amount of HBPs. Moreover, chemical bonds contents of DHBPs were higher than HBPs, but significant lower (P < 0.05) than WPs. The Fourier-transform infrared spectroscope showed that secondary structures of HBPs were more flexible after decoloration. Besides, foaming property, emulsifying property and water holding capacity of DHBPs increased significantly (P < 0.05). To conclude, the aggregation behavior and functional properties of HBPs were improved by the decolorization.

Inactivation of a wheat protein kinase gene confers broad-spectrum resistance to rust fungi

Wheat crops are frequently devastated by pandemic stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). Here, we identify and characterize a wheat receptor-like cytoplasmic kinase gene, TaPsIPK1, that confers susceptibility to this pathogen. PsSpg1, a secreted fungal effector vital for Pst virulence, can bind TaPsIPK1, enhance its kinase activity, and promote its nuclear localization, where it phosphorylates the transcription factor TaCBF1d for gene regulation. The phosphorylation of TaCBF1d switches its transcriptional activity on the downstream genes. CRISPR-Cas9 inactivation of TaPsIPK1 in wheat confers broad-spectrum resistance against Pst without impacting important agronomic traits in two years of field tests. The disruption of TaPsIPK1 leads to immune priming without constitutive activation of defense responses. Taken together, TaPsIPK1 is a susceptibility gene known to be targeted by rust effectors, and it has great potential for developing durable resistance against rust by genetic modifications.

TaEXPB5 functions as a gene related to pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm

The thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) is completely male sterile under low temperature (< 18 ℃) during Zadoks growth stages 45–52, whereas its fertility can be restored under hot temperature (≥ 20 ℃). The K-TCMS line may facilitate hybrid breeding and hybrid wheat production. Therefore, to elucidate the molecular mechanisms of its male sterility/fertility conversion, we conducted the association analysis of proteins and transcript expression to screen fertility related genes using RNA-seq, iTRAQ, and PRM-based assay. A gene encoding expansin protein in wheat, TaEXPB5, was isolated in K-TCMS line KTM3315A, which upregulated expression in the fertility anthers. Subcellular localization analysis suggested that TaEXPB5 protein localized to nucleus and cell wall. The silencing of TaEXPB5 displayed pollen abortion and the declination of fertility. Further, cytological investigation indicated that the silencing of TaEXPB5 induced the early degradation of tapetum and abnormal development of pollen wall. These results implied that TaEXPB5 may be essential for anther or pollen development and male fertility of KTM3315A. These findings provide a novel insight into molecular mechanism of fertility conversion for thermo-sensitive cytoplasmic male-sterility wheat, and contribute to the molecular breeding of hybrid wheat in the future.

Rationale for field-specific on-farm precision experimentation

Uncertainties in farming necessitate detailed knowledge of the production efficiencies to maintain sustainability. To accomplish ecologically based agriculture, with the goal of intensification by maximizing production and profit as well as minimizing environmental impact, we hypothesized that a site-specific knowledge base can be efficiently achieved through modern precision agriculture (PA) technologies at the field scale. The two goals of this study were to quantify the spatiotemporal variation of crop responses and the variables driving crop production, crop quality, and field-scale farmer net-return. We conducted on-farm experimentation (OFE) on several fields for three years where we varied nitrogen fertilizer rate as a management input, to induce changes in crop response. Using a Monte Carlo approach, we assessed the probability that crop responses varied across fields and between years. To determine the drivers of crop production, quality, and net-return, we performed sensitivity analyses to assess the impact of variation in the environment with the most influence on crop responses and farmer profits. Our analysis provided evidence that the degree of the response of winter wheat yield and protein content to variable nitrogen fertilizer rates are not homogenous across time and space. Elevation as a covariate to nitrogen fertilizer rate was the primary influence on predicted yields and protein across most fields, yet not among all fields and across years in fields. The drivers of net-return varied among fields and across years primarily between yield and protein. However, in some cases the most influential factor was the base price received, controlled by the grain elevators that growers sell to, indicating that in some fields and years, farmer’s net-returns are dictated by variables outside of a farmer’s control or ability to manage. These results provide basic evidence justifying the use of OFE for farm management and suggest that management needs to be specific to each field and point in time, with recommendations being made specifically for a field based on information gathered from that field. On-farm experimentation will enable farmers to identify these drivers and understand how their inputs influence yield and protein within fields. Using information provided by OFE with decision support systems can enable farmers to make informed management decisions that maximize their profits and increase the efficiency of chemical inputs, such as nitrogen fertilizer.

Gluten-free raw materials in the technology of instant pasta

In this work, the possibility of producing instant pasta products from unconventional raw materials was considered. These raw materials do not contain wheat gliadin or proteins with similar properties (secalin, hordein, avenin). Rice, buckwheat and soybean flours were selected as raw materials for the study. In the course of the work, laboratory studies were carried out to identify structure formers and plasticizers, as well as their optimal dosages and ratios, capable of imitating the properties of wheat gluten in pasta prepared according to a traditional recipe. The choice of food additives was based on the results of the analysis of their influence on the physicochemical properties of semi-finished and finished products, as well as on the technological parameters of production. On the basis of the conducted studies of nutritional value, physical and chemical properties and organoleptic characteristics of the samples of instant pasta from non-traditional raw materials were studied. A conclusion was made about the prospects of using non-traditional raw materials in the production of this type of products. 14 recipes were developed based on mixtures of rice, buckwheat and soy flour. Instant pasta made from gluten-free raw materials with the use of structuring agents is characterized by the best values for the proportion of dry substances passing into the cooking water during brewing and a higher protein content, as well as a lower fat content in the finished product in comparison with instant pasta made from wheat flour. The organoleptic analysis of the prepared samples of pasta showed the possibility of creating a product that surpasses the traditional one in terms of not only physical and chemical properties and nutritional value, but also the best taste, texture, smell and colour.

How Does the Environment Affect Wheat Yield and Protein Content Response to Drought? A Meta-Analysis.

Wheat (Triticum aestivum L.) is one of the most significant cereal crops grown in the semi-arid and temperate regions of the world, but few studies comprehensively explore how the environment affects wheat yield and protein content response to drought by means of meta-analysis. Therefore, we collected data about grain yield (GY), grain protein yield (GPY), grain protein content (GPC), and grain nitrogen content (GNC), and conducted a meta-analysis on 48 previously published data sets that originate from 15 countries. Our results showed that drought significantly decreased GY and GPY by 57.32 and 46.04%, but significantly increased GPC and GNC by 9.38 and 9.27%, respectively. The responses of wheat GY and GNC to drought were mainly related to the drought type, while the GPY was mainly related to the precipitation. The yield reduction due to continuous drought stress (CD, 83.60%) was significantly greater than that of terminal drought stress (TD, 26.43%). The relationship between the precipitation and GPY increased in accordance with linear functions, and this negative drought effect was completely eliminated when the precipitation was more than 513 mm. Sandy soils and high nitrogen application level significantly mitigated the negative effects of drought, but was not the main factor affecting the drought response of wheat. Compared with spring wheat, the drought resistance effect of winter wheat was more obvious. Evaluation of these models can improve our quantitative understanding of drought on wheat yield and food security, minimizing the negative impact of drought on crop production.

Brewing by-product valorisation: trub debittered for nutritional and quality improvement of pasta

Trub, a brewing by-product, can be used as alternative ingredient for foods nutritional enrichment after its bitter compounds extraction. Study presents the optimisation of bitter compounds extraction from trub by Box-Behnken design, and use of debittered trub (DT) as new ingredient to enrich pasta. Bitterness extraction process was evaluated at different pH levels, time and extraction steps, and physical-chemical properties of DT (obtained under optimal conditions) were evaluated. Pasta was enriched with DT (5%, 10% and 15%) and its physical-chemical and quality properties were evaluated. Protein structure and chemical composition of trub were altered after process, also modifying its technological properties. Pasta with 10% DT increased in 33.51% protein content. Interaction of DT and wheat proteins resulted in a more compact structure, and DT water absorption capacity provided pasta texture changes. DT use improved pasta nutritional and quality properties, enabling trub valorisation and its use as vegetable proteins alternative source.

The Effect of Using Wheat Protein as a Protein Source on Some Blood Parameters in Juvenile Sea Bass (Dicentrarchus labrax) Feeds

In this study, wheat protein was substituted for fish meal at different rates (25%, 50%, 75%, 100%) in sea bass (Dicentrarchus labrax) fish feed and the effects on some blood parameters were investigated. The initial weight of 450 sea bass fish used in the study was calculated as 2.33 ± 0.2 g. Sea bass fry, 30 in each aquarium, were fed with experimental feeds for 60 days in triplicates. At the end of the experiment, hematological and biochemical blood parameters were analyzed. As a result, it was found that the use of wheat protein at a high rate had statistically significant effects on blood parameters.

An Adjuvant-Free Mouse Model Using Skin Sensitization Without Tape-Stripping Followed by Oral Elicitation of Anaphylaxis: A Novel Pre-Clinical Tool for Testing Intrinsic Wheat Allergenicity.

Wheat is a major food allergen per the regulatory bodies of various nations. Hypersensitivity reactions to wheat have been steadily increasing for reasons that are not completely understood. Wheat-allergy models typically use adjuvants to induce sensitization to wheat proteins followed by an intraperitoneal challenge to elicit anaphylaxis. Although these models are very useful, they lack the ability to reveal the intrinsic allergenicity potential of wheat. To improve the mouse model of wheat allergy, we tested the hypothesis that repeated skin application of salt-soluble protein extract (SSPE) from durum wheat will clinically sensitize the mice to oral anaphylaxis to SSPE. Balb/c mice were bred and maintained on a plant-protein-free diet and used in the experiments. Adult female mice were exposed to SSPE once a week for 9 weeks via a solution on intact skin. Sensitization was measured by SSPE-specific IgE (sIgE) antibody and total IgE (tIgE) levels. Oral anaphylaxis was quantified by hypothermic shock response (HSR), and mucosal mast cell response (MMCR) was quantified by measuring MMCP-1 after oral challenge. Using single mouse data, correlation analyses were performed to determine the relationship among the allergenicity readouts. Spleen cytokines were quantified using a protein microarray method. Our results show that (i) repeated skin exposures to SSPE elicited robust increases in the sIgE and tIgE levels; (ii) skin exposure to SSPE was sufficient to sensitize mice for oral anaphylaxis and MMCR; (iii) both HSR and MMCR showed a strong correlation with each other, as well as with sIgE, and a modest correlation with tIgE levels; (iv) selected Th2/Th17/Th1 cytokines were elevated in skin-sensitized mice; and (v) oral allergen-challenged mice showed selective elevation of IL-6 and a panel of chemokines compared to saline-challenged mice. Together, we report the development and characterization of a novel adjuvant-free wheat-allergy mouse model that uses skin sensitization without tape-stripping followed by oral elicitation of anaphylaxis. Furthermore, validation of quantifiable wheat allergenicity readouts makes this model particularly suitable as a pre-clinical testing tool to assess the intrinsic sensitization/oral-anaphylaxis elicitation potential of novel wheat proteins (e.g., processed wheat) and to develop hypo/non-allergenic wheat products.

Characterization and Cellular Uptake of Peptides Derived from In Vitro Digestion of Meat Analogues Produced by a Sustainable Extrusion Process.

Whether proteins in meat analogues (MAs) have the ability to provide equivalent nutrition as those in animal meat remains unknown. Herein, a MA was produced by high-moisture extrusion using soy and wheat proteins. The physicochemical properties, in vitro digestion, and cellular uptake of the released peptides were systematically compared between the MA and the chicken breast (CB). The MA showed a higher hardness but a lower degree of texturization than the CB. After simulated digestion, soluble peptides in the MA had a higher molecular weight and higher hydrophobicity. No observable cytotoxicity or inflammatory response to Caco-2 cells was found for both MA and CB digests. The former exhibited less permeability of peptides across Caco-2 cells. Liquid chromatography with tandem mass spectrometry found that the identified peptides in MA and CB digests contained 7-30 and 7-20 amino acid residues, respectively, and they became shorter after cellular transportation. The amino acid composition showed fewer essential and non-essential amino acids in the MA permeate than in the CB permeate.