Surface Layer Properties of Dough Liquor Components: Are They Key Parameters in Gas Retention in Bread Dough?

Gas cell stability during bread making is controlled by both surface and bulk properties. This paper is focused on studying the surface properties of the water-soluble phase of the dough, the dough liquor (with and without lipids), as well as the composition of the air/water interface. Using infrared reflection measurements, we showed that in lipid-poor liquor, proteins are the dominant species present at the air/water interface. With complete liquor (including the lipids), a mixed interface of protein and lipids is obtained. However, the presence of lipids in the surface layer did not significantly affect the surface pressure. We also added enzymes to the flour to evaluate in what way the surface-active properties of the liquor components can be affected. These results were compared to the effect of adding a surfactant [diacetyl tartaric esters of mono- and diglycerides (DATEM)]. Biobake 10804, a xylanase that increased the arabinoxylan content of the dough liquor, decreased the surface pressure and increased the dilational modulus in lipid-poor liquor. This effect was not observed with the liquor including the lipids. Lipopan 50 BG, a 1,3-specific lipase, increased the surface pressure of the liquor that included the lipids. Lipopan F BG, which converts polar lipids to their lyso form, strongly increased the surface pressure not only in the lipid-containing liquor but also in the lipid-poor liquor. DATEM, as expected, increased the surface pressure while strongly decreasing the dilational modulus. Results of these studies were used to help explain changes in loaf volume observed in a series of baking tests, using the same enzymes and additives. This led to the conclusion that the effect of surface-active components alone cannot account for the larger loaf volumes observed. Clearly, both the effect of bulk and interfacial rheological properties should be considered together when explaining gas cell stability.Presented at the 2005 AACC annual Meeting, Orlando, FL, September 11–14.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

The protein synthesis inhibitors from wheat, barley, and rye have identical antigenic determinants

We have purified the protein synthesis inhibitors from wheat (tritin) and investigated their antigenic relationship to inhibitors from other grains and more distantly related plants. Three forms of tritin (tritin 1, 2, and 3) indistinguishable with regards to their molecular weight and enzymatic properties were found. Antiserum against the most abundant tritin, tritin 2, cross reacted with tritin 1, tritin 3, and with the translation inhibitors from barley and rye, but not with other plant-derived translational inhibitors. The results indicate that only closely related plants contain antigenically similar protein synthesis inhibitors.     

Proteomic analysis of amphiphilic proteins of hexaploid wheat kernels

Wheat proteins and specially gluten proteins have been well studied and are closely associated with baking products. Amphiphilic proteins (proteins that are soluble using nonionic detergent Triton X-114 ) also play an important role in wheat quality. Some of them, like puroindolines, are lipid binding proteins, and are strongly linked to dough foaming properties and to fine crumb texture. However many amphiphilic proteins are still unknown and both their physiological and technological functions remain to be analysed. In order to explore these proteins, proteomic analysis was carried out using 81 F9 lines, progeny obtained from an interspecific cross "W7984"x"Opata", and already used to built a map of more than 2000 molecular markers (International Triticeae Mapping Initiative, ITMImap). Two-dimensional electrophoresis (immobilized pH gradient (pH 6-11)x sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed on amphiphilic proteins with three to five replicates for each line. Silver stained gels were analysed using Melanie 3 software. Genetic determinism was carried out on 170 spots segregating between the two parental hexaplo?d wheats. Many of these spots were mapped on different chromosomes of the ITMImap. Spots of interest were identified using matrix-assisted laser desorption/ionization-time of flight and some of them were partly sequenced using electrospray ionization-tandem mass spectrometry. This proteomic approach provided some very useful information about some proteic components linked to bread wheat quality and particularly to kernel hardness.     

Secretion of alpha-amylase and multiple forms of glucoamylase by the yeast Trichosporon pullulans

Trichosporon pullulans IGC 3488 produced extracellular alpha-amylase and glucoamylase activities when grown in batches in a medium containing corn steep liquor and soluble starch or corn starch. alpha-Amylase, unlike glucoamylase activity, was secreted biphasically. For both amylases the maximum concentration was found in stationary phase cultures. The amylolytic enzymes, previously concentrated by ammonium sulfate precipitation, were separated into a glucoamylase fraction and an alpha-amylase fraction by Ultrogel AcA 54 gel filtration. Pullulanase activity was located in the glucoamylase fraction, whereas cyclodextrinase activity was restricted to the alpha-amylase fraction. Isoamylase and alpha-glucosidase were not detected. Electrophoretic analysis showed that alpha-amylase activity was due to a single protein. Glucoamylase, however, occurred in multiple forms. The four glucoamylases and the alpha-amylase were glycoproteins.     

Identification of thioredoxin h-reducible disulphides in proteomes by differential labelling of cysteines: insight into recognition and regulation of proteins in barley seeds by thioredoxin h

Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.     

Proteins influencing foam formation in wine and beer: the role of yeast

This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa--characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins.     

Foam separation of microbial cells

Batch foam separation has been employed to separate Saccharomyces carlsbergensis cells from their broth without the use of any external surface-active agent. A model has been developed to predict the foamate cell concentration as well as the variation of cell concentration in the bulk liquid in the foam column as a function of time. The model assumes a linear equilibrium relation between the cell concentrations at the interface and the bulk. The foam has interface as well as interstitial liquid. The interface is assumed to be in equilibrium with the interstitial liquid, which in turn is assumed to have the same concentration as the bulk. The interfacial area is calculated by assuming the foam bubbles to be pentagonal dodecahedral in shape. The model has been found to explain the results of foam separation of cells quite well, particularly with respect to the effect of bubble size and aeration rate.     

Inhibitors of animal cell-free protein synthesis from grains

We described earlier the purification and properties of a protein (tritin) from wheat that enzymatically inhibits translation in cell-free systems from animals but not plants. In this report, we have examined 11 additional grains (Family Gramineae) and three other seeds for the presence of tritin-like proteins. In addition to wheat species, barley, oats, rye, triticale and corn were found to be sources of inhibitor; no inhibitor could be detected in rice, millet, sesame, alfalfa, mung bean or common bean seeds. The inhibitors from barley and rye were purified and found to differ from tritin with respect to heat inactivation, although they are similar to tritin with respect to molecular weight, behavior during purification and specific activity. The inhibitor from corn was purified and found to differ from tritin with respect to heat inactivation and molecular weight, although it is similar to tritin in behavior during purification and specific activity. These inhibitors constitute 2–17% of the total extractable protein in these grain s. Thus, wheat, barley, rye and corn can serve as convenient sources of a family of closely related inhibitors of protein synthesis which, when conjugated with lectins, antibodies, or hormones, could prove useful as chimeric toxins.     

Insights into protective effects of medium additives on animal cells under fluid stresses: the hydrophobic interactions

Animal cells in suspension culture can suffer severe mechanical damage from bursting gas bubbles or other hydrodynamic force sources. Certain chemical additives in the culture media, particularly some surface-active chemicals, can effectively protect animal cells against such damage. Previously we proposed that the protective effect is associated with the adsorption of the additives in the cell membrane through hydrophobic binding of the surface-active molecules to the membrane. Adsorption of the additives to the cell membrane may lead to decreased hydrophobicity of the cell surface, thus eliminating cell adhesion to bubbles and reducing cell damage from bursting bubbles. In this study, we measured the hydrophobicity of two insect cell lines based on cell adhesion to hydrocarbon phase and its influence by surface-active chemicals, Pluronic F68, a methylcellulose and a polyethylene glycol. The experimental results showed strong support for the aforecited cell protection mechanism.     

An alpha-amylase inhibitor gene from Phaseolus coccineus encodes a protein with potential for control of coffee berry borer (Hypothenemus hampei)

Plant alpha-amylase inhibitors are proteins found in several plants, and play a key role in natural defenses. In this study, a gene encoding an alpha-amylase inhibitor, named alphaAI-Pc1, was isolated from cotyledons of Phaseolus coccineus. This inhibitor has an enhanced primary structure to P. vulgaris alpha-amylase inhibitors (alpha-AI1 and alpha-AI2). The alphaAI-Pc1 gene, constructed with the PHA-L phytohemaglutinin promoter, was introduced into tobacco plants, with its expression in regenerated (T0) and progeny (T1) transformant plants monitored by PCR amplification, enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis, respectively. Seed protein extracts from selected transformants reacted positively with a polyclonal antibody raised against alphaAI-1, while no reaction was observed with untransformed tobacco plants. Immunological assays showed that the alphaAI-Pc1 gene product represented up to 0.05% of total soluble proteins in T0 plants seeds. Furthermore, recombinant alphaAI-Pc1 expressed in tobacco plants was able to inhibit 65% of digestive H. hampei alpha-amylases. The data herein suggest that the protein encoded by the alphaAI-Pc1 gene has potential to be introduced into coffee plants in order to increase their resistance to the coffee berry borer.     

A mechanism for internal reflux in foam fractionation

Multiple equilibrium stages can be engendered in foam fractionation, a process used for the enrichment of streams of proteins, by returning some of the foamate stream to the top of the column as external reflux liquor. However, it was recognised, 40 years ago that reflux could be autogenously created through the coalescence of bubbles in fractionation columns. By invoking the hydrodynamic theory of rising foam, we suggest a mechanism for the creation of internal reflux in foam fractionation. This method can give internal reflux rate as a function of bubble size. However, since the bubble size profile in a rising foam cannot be estimated, we cannot yet estimate how internal reflux varies with position in the column.     

The characterisation and mapping of a family of LMW-gliadin genes: effects on dough properties and bread volume

Analysis of a cDNA library from wheat cv Wyuna endosperm, indicated a significant size and sequence variation among seed-endosperm protein genes. In this study, a family of low-molecular-weight seed protein genes are analysed that are related to the gliadins and the low-molecular-weight glutenin subunits. Sequence analysis and comparison of these proteins showed that they are closely related to a 17-kDa protein from barley, epsilon hordein, which plays a role in beer foam stability in the brewing industry. Mapping of these genes in wheat shows that they are located on group 7 and 4 chromosomes, as opposed to a group 1 and 6 location for the glutenins and gliadins. It is possible that this family of proteins forms a new class of seed-endosperm proteins important in defining the quality characteristics of wheat flour. Therefore, a representative gene from this family was expressed in Escherichia coli and the purified protein was supplemented into a base wheat flour. Rheological analysis showed that the protein effected dough strength and resistance break down during mixing of the dough, and provided a 20% increase in loaf height after baking.     

Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.     

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.     

The effect of carbon dioxide,aeration rate and sodium chloride on the secretion of proteins from growing bakers' yeast (Saccharomyces cerevisiae)

Since yeast may be an important microorganism for industrial use when its genes are modified by recombinant DNA techniques to overproduce certain proteins, (particularly those which are glycosylated), it is desirable to study how environmental variables affect its protein secretion ability. It is also of interest to understand how proteins such as proteases, lipases and amylases are excreted in solid matrices to develop a basis for learning more about solid fermentations. With these two applications in mind, the total protein excreted by both aerated and non-aerated Saccharomyces cerevisiae growing in a liquid batch culture (with varying levels of CO₂ and NaCl) was tracked. Using a modified Bradford method (Coomassie Blue dye-binding assay) for the concentration of total proteins in the extracellular fermentation broth, it has been determined that by 24 h of the run, excreted proteins rose to levels of about 10% of the total cell protein (500 μg ml^?1 protein from about 10 g of yeast, containing about 5 g total protein). No cell lysis was observed during the 24 h run. The highest protein levels at the top of the fermentor seemed to be those achieved in response to CO₂ alone. Additions of NaCl did not seem to enhance the secreted protein level. Large inconsistencies in replicating anaerobic runs for protein concentration appeared to be explained by noting that rising CO₂ bubbles may cause ‘foam fractionation’ of the proteins in the broth.     

Proteomic analysis of tilapia Oreochromis niloticus Streptococcus agalactiae strains with different genotypes and serotypes

Nine tilapia Oreochromis niloticus group B streptococcus (GBS) strains differing in serotype and genotype were selected and paired. Two‐dimensional difference gel electrophoresis (2D DIGE) and matrix‐assisted laser‐desorption ionization time‐of‐flight‐mass spectrometry (MALDI‐TOF‐MS) were used to analyse the protein profiles of the strain pairs. Forty‐three proteins corresponding to 66 spots were identified, of which 35 proteins were found in the seven selected strain pairs that represented pairs differing in genotype and serotype. Among the 35 proteins, numbers of differentially expressed proteins in strains of different serotypes were greater than found in strains of different genotypes, suggesting that serotype plays a more essential role than genotype in the differential protein expression among GBS strains. No distinct pattern was found with respect to genotype and the protein expression profile of GBS strains. Several proteins were identified as surface‐associated cytoplasmic proteins that possessed the typical immunity‐eliciting characteristics of surface proteins. The identified proteins were found to be involved in 16 biological processes and seven Kyoto encyclopaedia of genes and genomes (KEGG) pathways. The data, for the first time, identified differentially expressed proteins in O. niloticus GBS strains of different serotypes, which play a major role in immunogenicity of O. niloticus GBS than does genotype, offering further information for design of a vaccine against O. niloticus GBS.     

Ontogeny of alpha-amylase circadian rhythms in rat parotid gland

The content of alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) and total soluble proteins of parotid glands (from rats exposed to a photoperiod of 14 hr light: 10 hr dark), have been determined every 2 or 3 hr over 24 hr periods in 15, 25 and 90-day-old rats. In 35-, 45- and 72-day-old rats, determinations were performed only at 0100 and 1400 hr. The alpha-amylase and total soluble protein contents from 90-day-old rats show a circadian variation, with a maximum value at 2200 hr and a minimum at 1400 hr. Parotids from 15- and 25-day-old rats also show a circadian rhythm. The minimum value is recorded at 0100 hr and the maximum at 1400 hr. At day 35 and after, there is an inversion of the amylase rhythm. In immature rats, it appears that alpha-amylase and soluble protein are under the influence of another synchronizer, whose timing is independent of that imposed by mastication of solid food.     

Silicone antifoam performance enhancement by nonionic surfactants in potato medium

The ability of a silicone antifoam to retard foaming in a liquor prepared from potatoes is enhanced by the addition of ethoxylated nonionic surfactants. The enhancement is non-linear for surfactant concentration, with all 12 surfactants tested possessing a concentration at which foam heights strongly diminish, referred to as the surfactant critical antifoaming concentration (SCAFC). SCAFCs vary between surfactants, with lower values indicating better mass efficiency of antifoaming enhancement. SCAFCs decrease with degree of ethoxylation and decrease with the hydrophilic–lipophilic balance for ethoxylated nonionic surfactants. Surfactant addition produces a mixed water-surface layer containing surfactant and surface-active components in the potato medium. Surface tension reduction does not correlate well with antifoam performance enhancement. A model is proposed where surfactant adsorption promotes desorption of surface-active potato medium components from the water surface. At the SCAFC, desorption is not complete, yet the rate of bubble rupture is sufficiently enhanced to provide excellent foam control. Electronic Publication