Preparation and Characterization of Emulsion Gels from Whole Faba Bean Flour.

Faba bean protein has good functionalities, but it is little used in the food industry. This study identified a challenge from unfavourable starch gelation when utilizing faba bean for producing protein-based emulsion gel foods, and developed processing methods to overcome that. Two types of protein-based emulsion gel foods, namely yogurt and tofu analogue products, were prepared. The processing methods in this study involved steps of thermal pre-treatment of the beans, dehulling, milling, adding plant oil, homogenization, prevention of starch gelation, and inducing protein gelation. Two methods for preventing starch gelation were studied, namely starch removal and hydrolysis. The gel texture, water-holding capacity, and structural properties of the gel products were evaluated. Both starch-gelation prevention methods produced yogurt and tofu analogue products having typical emulsion gel properties. Hydrolysis of starch was favourable for producing the yogurt analogue, because the hydrolysate compounds improved the gel strength and viscosity. Moreover, it utilized the whole flour, meaning all the nutrients from the cotyledon were used and no side-stream was created. In contrast, starch removal was slightly better than hydrolysis for producing the tofu analogue, because the hydrolysate lowered the gel strength and water-holding capacity of the products. It is both possible and ecologically sustainable to utilize whole faba bean flour for making emulsion gel products.

Proposal for C-Hordein as Reference Material in Gluten Quantification.

The concentration of residual barley prolamin (hordein) in gluten-free products is overestimated by the R5 ELISA method when calibrated against the wheat gliadin standard. The reason for this may be that the composition of the gliadin standard is different from the composition of hordeins. This study showed that the recognition of whole hordein by R5 antibody mainly came from C-hordein, which is more reactive than the other hordeins. The proportion of C-hordein in total hordein ranged from 16 to 33% of common Finnish barley cultivars used in this study and was always higher than that of ω-gliadin, the homologous protein class in the gliadin standard, which may account for the overestimation. Thus, a hordein standard is needed for barley prolamin quantification instead of the gliadin standard. When gluten-free oat flour was spiked with barley flour, the prolamin concentration was overestimated 1.8-2.5 times with the gliadin standard, whereas estimates in the correct range were obtained when the standard was 40% C-hordein mixed with an inert protein. A preparative-scale method was developed to isolate and purify C-hordein, and C-hordein is proposed as a reference material to calibrate barley prolamin quantification in R5-based assays.

Degradation of C-hordein by metal-catalysed oxidation.

C-hordein is a monomeric prolamin protein in barley. The unusual primary structure of C-hordein has highly repetitive sequences and forms a secondary structure of beta-turns. C-hordein structure is similar to that of collagen protein, whose degradation by metal-catalysed oxidation has been intensively studied. No information exists on the metal catalysed oxidation of C-hordein, however. In this study, copper-catalysed hydrogen peroxide induced oxidation of C-hordein caused substantial degradation and formed some insoluble compounds. The use of a gliadin standard in R5 ELISA determinations causes an overestimation of hordeins in a sample. A C-hordein standard was therefore directly used as a standard, thus allowing the C-hordein to be analysed as its oxidised prolamin product. After 48 h of oxidation, the prolamin concentration of oxidised C-hordein decreased to 20% of its original amount for competitive ELISA, and to 3% for sandwich ELISA methods. Carbonyl groups were formed during the oxidation. Backbone fragmentation and side-chain modification suggested structural changes of R5 epitopes in C-hordein. Oxidation is an alternative to enzymatic hydrolysis when degrading and modifying C-hordein.

In situ enrichment of folate by microorganisms in beta-glucan rich oat and barley matrices.

The objective was to study folate production of yeast strains, bacteria isolated from oat bran, and selected lactic acid bacteria as well as one propionibacterium in oat and barley based models. Simultaneously, we aimed at sustaining the stability of viscosity, representing the physicochemical state of beta-glucan. Total folate contents were determined microbiologically and vitamers for selected samples by UHPLC. Folate in yeast cells comprised mainly 5-methyltetrahydrofolate and tetrahydrofolate. Folate production by microbes in YPD medium was different to that in cereal fermentations where vitamers included 5-methyltetrahydrofolate, 5,10-methenyltetrahydrofolate and formylated derivatives. Microbes producing significant amounts of folate without affecting viscosity were Saccharomyces cerevisiae ALKO743 and Candida milleri ABM4949 among yeasts and Pseudomonas sp. ON8 and Janthinobacterium sp. RB4 among bacteria. Net folate production was up to 120 ng/g after 24 h fermentation and could increase during 2-week storage. Glucose addition increased the proportion of 5-methyltetrahydrofolate. Streptococcus thermophilus ABM5097, Lactobacillus reuteri, and Propionibacterium sp. ABM5378 produced folate but in lower concentrations. Both endogenous and added microbes contribute to folate enhancement. Selection of microbes with folate producing capability and limited hydrolytic activity will enable the development of products rich in folate and beta-glucan.

Testing safety of germinated rye sourdough in a celiac disease model based on the adoptive transfer of prolamin-primed memory T cells into lymphopenic mice.

UNLABELLED: The current treatment for celiac disease is strict gluten-free diet. Technical processing may render gluten-containing foods safe for consumption by celiac patients, but so far in vivo safety testing can only be performed on patients. We modified a celiac disease mouse model to test antigenicity and inflammatory effects of germinated rye sourdough, a food product characterized by extensive prolamin hydrolysis. Lymphopenic Rag1-/- or nude mice were injected with splenic CD4+CD62L-CD44high-memory T cells from gliadin- or secalin-immunized wild-type donor mice. We found that: 1) Rag1-/- recipients challenged with wheat or rye gluten lost more body weight and developed more severe histological duodenitis than mice on gluten-free diet. This correlated with increased secretion of IFNγ, IL-2, and IL-17 by secalin-restimulated splenocytes. 2) In vitro gluten testing using competitive R5 ELISA demonstrated extensive degradation of the gluten R5 epitope in germinated rye sourdough. 3) However, in nude recipients challenged with germinated rye sourdough (vs. native rye sourdough), serum anti-secalin IgG/CD4+ T helper 1-associated IgG2c titers were only reduced, but not eliminated. In addition, there were no reductions in body weight loss, histological duodenitis, or T cell cytokine secretion in Rag1-/- recipients challenged accordingly. IN CONCLUSION: 1) prolamin-primed CD4+CD62L-CD44high-memory T cells induce gluten-sensitive enteropathy in Rag1-/- mice. 2) Hydrolysis of secalins in germinated rye sourdough remains incomplete. Secalin peptides retain B and T cell stimulatory capacity and remain harmful to the intestinal mucosa in this celiac disease model. 3) Current antibody-based prolamin detection methods may fail to detect antigenic gluten fragments in processed cereal food products.

Oxidative modification of a proline-rich gliadin peptide.

Prolamins are proline-rich proteins occurring in cereal grains. Prolamins of wheat, barley and rye, or gluten protein, can cause coeliac disease in individuals not tolerating gluten. Degrading harmful prolamins can reduce their toxicity. A model peptide sequenced in α-gliadin, 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), was chosen for our study. The metal-catalysed oxidation of 33-mer was studied, instead of enzymatic hydrolysis. Peptide 33-mer was treated in several oxidative systems. Iron-catalysed hydrogen peroxide-induced oxidation showed the greatest modification of 33-mer. Carbonyl groups and dityrosine cross-links were readily formed. At best, the immunological activity of 33-mer was reduced to 18% of its initial level after 24h of oxidation. Oxidative treatment can be further applied for the modification of cereal prolamin proteins, since it appears to be a potential alternative for reduction of coeliac immunological activities in gluten proteins.

Isolation and characterization of folate-producing bacteria from oat bran and rye flakes.

The aim of this research was to identify endogenous bacteria in commercial oat bran and rye flake products in order to study their folate production capability while maintaining the soluble dietary fibre components in physiologically active, unhydrolyzed form. Fourty-two bacteria were isolated from three different oat bran products and 26 bacteria from one rye flake consumer product. The bacteria were tentatively identified by sequence analysis of the 16S rRNA genes. The identification results revealed up to 18 distinct bacterial species belonging to 13 genera in oat bran, and 11 species belonging to 10 genera in rye flakes. The most common bacterial genus in oat bran was Pantoea, followed by Acinetobacter, Bacillus, and Staphylococcus. Pantoea species dominated also in rye flakes. The extracellular enzymatic activities of the isolates were studied by substrate hydrolysis plate assays. Nearly 80% of the isolates hydrolyzed carboxymethylcellulose, whereas starch-degrading activities were surprisingly rare (10%). Beta-glucan was hydrolyzed by 19% of the isolates. Protease, lipase or xylanase activity was expressed by 24%, 29%, and 16%, respectively, of the isolates. Representatives of the genera Bacillus, Curtobacterium, Pedobacter, and Sanguibacter showed the highest diversity of enzymatic activities, whereas members of Janthinobacterium and Staphylococcus possessed no hydrolytic activities for the substrates studied. Production capability for total folates was analyzed from aerobic cell cultures at the stationary growth phase. The amount of folates was determined separately for the cell mass and the supernatant by microbiological assay. For comparison, folate production was also examined in a number of common lactic acid bacteria. The best producers in oat bran belonged to the genera Bacillus, Janthinobacterium, Pantoea, and Pseudomonas, and those in rye flakes to Chryseobacterium, Erwinia, Plantibacter, and Pseudomonas. Supernatant folate contents were high for Bacillus, Erwinia, Janthinobacterium, Pseudomonas, and Sanguibacter. Compared to the endogenous bacteria, lactic acid bacteria were poor folate producers. The results of this work provide the first insight into the potential role of endogenous microflora in modulating the nutrient levels of oat and rye based cereal products, and pave way to future innovations of nutritionally improved cereal foods.

Production of folate by bacteria isolated from oat bran.

Twenty bacteria isolated from three commercial oat bran products were tested for their folate production capability. The bacteria as well as some reference organisms were grown until early stationary phase on a rich medium (YPD), and the amount of total folate in the separated cell mass and the culture medium (supernatant) was determined by microbiological assay. Folate vitamer distribution was determined for eight bacteria including one isolated from rye flakes. For seven bacteria the effect of temperature and pH on folate production was studied in more detail. Relatively large amount of folate was both produced in the cell biomass (up to 20.8microg/g) and released to the culture medium (up to 0.38microg/g) by studied bacteria. The best producers were characterized as Bacillus subtilis ON4, Chryseobacterium sp. NR7, Janthinobacterium sp. RB4, Pantoea agglomerans ON2, and Pseudomonas sp ON8. The level of folate released in culture medium was the highest for B. subtilis ON5, Chryseobacterium sp. NR7, Curtobacterium sp. ON7, Enterococcus durans ON9, Janthinobacterium sp. RB4, Paenibacillus sp. ON10, Propionibacterium sp. RB9, and Staphylococcus kloosii RB7. Marked differences in the distribution of folate vitamers among the bacterial strains were revealed by the HPLC analysis. The main vitamers were tetrahydrofolate, 5,10-methenyltetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate. Increase in the folate content during bacterial growth was accompanied by proportional increase in the 5-methyltetrahydrofolate content and decrease of 5-formyltetrahydrofolate. 10-Formylfolic acid dominated in the culture media of four bacteria, and Janthinobacterium sp. RB4 was also found to excrete 5-methyltetrahydrofolate. Intracellular folate content was higher when the bacteria were grown at 28 degrees C than at 18 degrees C or 37 degrees C and also higher at pH 7 than at pH 5.5.

Functional characteristics of egg white proteins within wheat, rye, and germinated-rye sourdoughs.

Egg white (EW) proteins are functional proteins, which possess certain biological activities (antimicrobial, antigenic, and peptidase-inhibitory) that may influence the food processing or vice versa can be affected by processing. This study investigated the behavior of EW proteins within sourdough systems with respect to proteolysis and fermentation parameters, and the ability of EW to build foam structures with sourdoughs. Of the EW proteins, ovotransferrin was hydrolyzed in all sourdoughs (wheat, rye, and germinated-rye), whereas the breakdown of ovalbumin was specific for germinated-rye sourdoughs, with the cysteine endopeptidases being responsible for the hydrolysis. The presence of EW in sourdough fermentations had no influence on the prolamin hydrolysis or the growth of starter culture, indicating that the peptidase-inhibitory and antimicrobial properties of EW play no important role in sourdoughs. EW foams, however, appeared as potential structure builders in sourdough applications and could serve as alternative structural agents in the production of baked goods with low gluten content.

Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods.

The assortment and quality of bakery products designed for celiac patients may be improved by designing whole grain ingredients with low residual prolamin contents. The main objective of this study was to evaluate the extent of prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs (GRSDs). Size-exclusion chromatography analyses, the fate of fluorescent prolamin, and immunological analyses determined the extent of prolamin hydrolysis and pentosan solubilization. Hydrolysis of rye prolamins was extensive in GRSDs, and according to enzyme-linked immunosorbent assay analyses, more than 99.5% of the prolamins were hydrolyzed. Pentosan solubilization occurred in native-rye sourdoughs, whereas in GRSDs, pentosans were partially hydrolyzed to monosaccharides. Test baking showed that the use of GRSD improved the overall quality of oat bread and that an estimated daily gluten intake from 100 g of bread would be less than 10 mg. However, the clinical safety must be assured before making any recommendations for celiac patients to use such products.

Unkilned and large amounts of oats in the coeliac disease diet: a randomized, controlled study.

OBJECTIVE: There is evidence for the long-term safety of oats as part of a gluten-free diet in coeliac disease (CD). Oats is generally processed by kilning, which theoretically may change its antigenic properties and be the reason that it is tolerated by patients with CD. The aim of this study was to investigate the suitability of large amounts of unkilned oats, comparing its use with kilned oats in adult coeliac patients. MATERIAL AND METHODS: The study group included 13 men and 19 women with CD in remission. The goal of daily intake of oats was 100 g during one year. These patients using oats as part of their gluten-free diet were randomized to two treatment groups. One group used regular oats and the other unkilned oats. After 6 months the patients changed the treatment groups. Food intake, symptoms, histology of the small intestine and the levels of endomysial antibodies were noted. RESULTS: No marked changes were found in the duodenal biopsies, in the levels of endomysial antibodies or in the well-being of the patients. Compliance with the diet did not change during the follow-up. CONCLUSIONS: Large amounts of both unkilned and regular kilned oats are well tolerated by adult patients with CD. Oats is therefore not harmful, even in its unkilned form, which indicates that its antigenic nature is not changed by common industrial food processing in such a way that would prevent the provoking of CD.

Prolamin hydrolysis in wheat sourdoughs with differing proteolytic activities.

The prolamins of wheat, rye, and barley contain structures that are harmful to gluten-sensitive people, and an extensive degradation of these prolamins during food processing might eliminate this problem. Sourdough fermentation is a cereal food process during which some protein degradation occurs. In this study, the prolamin hydrolysis that occurred in a high-proteolytic-activity germinated-wheat sourdough (GWSD) was compared with that of wheat sourdough systems which contained moderate or no proteolytic activities. Virtually all of the wheat prolamins (gliadins and glutenins) were degraded during the GWSD fermentation. Quantification of its prolamin levels confirmed that extensive prolamin hydrolysis had occurred in the GWSD. This hydrolysis was attributed to the cysteine proteinase activities of the germinated wheat. The use of high-proteolytic sourdoughs in baking could make it possible to prepare new low-prolamin cereal-based products for use by gluten-sensitive people, who could then diversify their diets by including these whole-grain containing products into their every-day diets.

Effects of yeasts and bacteria on the levels of folates in rye sourdoughs.

Fermentation of rye dough is often accompanied with an increase in folate content. In this study, three sourdough yeasts, Candida milleri CBS 8195, Saccharomyces cerevisiae TS 146, and Torulaspora delbrueckii TS 207; a control, baker's yeast S. cerevisiae ALKO 743; and four Lactobacillus spp., L. acidophilus TSB 262, L. brevis TSB 307, L. plantarum TSB 304, and L. sanfranciscensis TSB 299 originally isolated from rye sourdough were examined for their abilities to produce or consume folates. The microorganisms were grown in yeast extract-peptone-d-glucose medium as well as in small-scale fermentations that modelled the sourdough fermentation step used in rye baking. Total folate contents were determined using Lactobacillus rhamnosus (ATCC 7469) as the growth indicator organism. The microorganisms studied did not excrete folates into the media in significant amounts. Yeasts increased the folate contents of sterilised rye flour-water mixtures from 6.5 microg/100 g to between 15 and 23 microg/100 g after 19-h fermentation, whereas lactic acid bacteria decreased it to between 2.9 and 4.2 microg/100 g. Strains of Lactobacillus bulgaricus, L. casei, L. curvatus, L. fermentum, L. helveticus, Pediococcus spp., and Streptococcus thermophilus that were also tested gave folate contents after fermentation that varied between 2 and 10.4 microg/100 g. Although the four Lactobacillus spp. from sourdough consumed folates their effect on folate contents in co-cultivations was minimal. It was concluded that the increase of folate content during fermentation was mainly due to folate synthesis by yeasts. Fermentation of non-sterilised flour-water mixtures as such resulted in three-fold increases in the folate contents. Two folate producing bacteria were isolated from the non-sterilised flour and identified as Enterobacter cowanii and Pantoea agglomerans.

Bread fortified with cholecalciferol increases the serum 25-hydroxyvitamin D concentration in women as effectively as a cholecalciferol supplement.

Fortification of foods is a feasible way of preventing low vitamin D status. Bread could be a suitable vehicle for fortification because it is a common part of diets worldwide. The bioavailability of cholecalciferol from bread is not known. We studied cholecalciferol stability, the concentration of the added cholecalciferol, the dispersion of cholecalciferol in bread, and the bioavailability of cholecalciferol from fortified bread. Three batches of fortified low-fiber wheat and high-fiber rye breads were baked; from each batch, 3 samples of dough and bread were analyzed for their cholecalciferol content. In a single-blind bioavailability study, 41 healthy women, 25-45 y old, with mean serum 25-hydroxyvitamin D concentration 29 nmol/L (range 12-45 nmol/L), were randomly assigned to 4 study groups. Each group consumed fortified wheat bread, fortified rye bread, regular wheat bread (control), or regular wheat bread and a cholecalciferol supplement (vitamin D control) daily for 3 wk. The daily dose of vitamin D was 10 mug in all groups except the control group. The vitamin dispersed evenly in the breads and was stable. Both fortified breads increased serum 25-hydroxyvitamin D concentration as effectively as the cholecalciferol supplement. Supplementation or fortification did not affect serum intact parathyroid hormone concentration or urinary calcium excretion. In conclusion, fortified bread is a safe and feasible way to improve vitamin D nutrition.

Functionality of endogenous folates from rye and orange juice using human in vivo model.

BACKGROUND: Cereals contribute about a quarter of the daily folate intake from a typical diet in several European countries. However, studies on bioavailability of endogenous folates, in particular of cereal sources, are scarce. AIM OF THE STUDY: We aimed to study how well natural folates from rye (different rye breads and muesli made of malted rye) and orange juice function in improving folate status of human volunteers compared to a diet containing folic acid fortified wheat bread. METHODS: Healthy human volunteers aged 20-66 y took part in a four-week intervention trial in which bread, breakfast cereal and juice were provided. The study had a parallel design with two groups, 1) rye and orange juice group (33 volunteers) and 2) fortified wheat bread and apple juice group (31 volunteers). The test foods provided on average 184 microg and 188 microg folate per day in rye and wheat groups, respectively. Test foods were consumed as part of the subjects' normal diet. RESULTS: In both groups statistically significant increases in serum and red cell folates were observed after the intervention period. The serum folate increased 26 % and 31 %, and red cell folate levels increased 17 % and 15 % in rye and orange juice and wheat and apple juice groups, respectively. The effects did not differ between the rye and wheat groups. Increases in serum and red cell folate were more profound among subjects with low starting folate levels. Decrease in the plasma homocysteine concentrations was observed only in the highest tertile of both groups but not in the group means. CONCLUSIONS: Endogenous folates incorporated into a healthy diet, even in moderate amounts, is an efficient way to improve folate status among healthy adults. Folates from different rye products and orange juice showed good bioavailability that was similar to folic acid from fortified white bread.