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.     

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Enzymatic hydrolysis of cod (Gadus morhua) by-products: Optimization of yield and properties of lipid and protein fractions

The main products of hydrolysis of fish by-products are hydrolysed protein and oil. The aim of this work was to study the effect of initial heat inactivation of endogenous enzymes, addition of water prior to hydrolysis, use of different commercial enzymes and combination of enzymes on the yield and purity of the protein and oil fractions after enzymatic hydrolysis of cod by-products. This study was designed to examine how all these factors were effective for destroying protein–lipid complexes in order to obtain pure oil and protein fractions and reduce the insoluble fraction.

Initial heating of raw material changed both raw material properties and inactivated endogenous enzymes thereby influencing the following hydrolysis. High amount of lipids in raw material combined with initial heating caused formation of protein–lipid complexes which was found in all protein containing fractions. The main constituents of the lipids in the complexes were phospholipids and other polar lipids. Insoluble protein–lipid complexes formed lead to increased amount of sludge, reduced FPH yield and high amount of lipids in FPH. The highest amount of separated oil was obtained in the experiments after initial heating without added water. These treatments also reduce amount of emulsion, which is not a desirable product after hydrolysis. Initial heating caused denaturation of protein, which decreased their emulsifying properties.

Results showed that it is not possible to obtain all desirable quality indicators such as: maximum oil and FPH yield, minimum emulsion and sludge yield and the highest protein recovery in FPH with the lowest amount of lipids in FPH fraction by using only one hydrolysis process. Therefore, the aim and requirements for the final products should be prioritised and defined very clearly before the process is designed taking into account the composition of raw material. Hydrolysis of unheated raw material with Alcalase and addition of water was the best compromise taking into account the mentioned quality indicators.     

A Quality by Experimental Design Approach to Assess the Effect of Formulation and Process Variables on the Extrusion and Spheronization of Drug-Loaded Pellets Containing Polyplasdone<Superscript>®</Superscript> XL-10

Successful pellet production has been reported in literature with cross-linked poly(vinylpyrrolidone), Polyplasdone^® XL-10 and INF-10. In the present study, a quality by experimental design approach was used to assess several formulation and process parameter effects on the characteristics of Polyplasdone^® XL-10 pellets, including pellet size, shape, yield, usable yield, friability, and number of fines. The hypothesis is that design of experiments and appropriate data analysis allow optimization of the Polyplasdone product. High drug loading was achieved using caffeine, a moderately soluble drug to allow in vitro release studies. A five-factor, two-level, half-fractional factorial design (Resolution V) with center point batches allowed mathematical modeling of the influence of the factors and their two-factor interactions on five of the responses. The five factors were Polyplasdone^® level in the powder blend, volume of water in the wet massing step, wet mixing time, spheronizer speed, and spheronization time. Each factor and/or its two-factor interaction with another factor influenced pellet characteristics. The behavior of these materials under various processing conditions and component levels during extrusion-spheronization have been assessed, discussed, and explained based on the results. Numerical optimization with a desirability of 0.974 was possible because curvature and lack of fit were not significant with any of the model equations. The values predicted by the optimization described well the observed responses. The hypothesis was thus supported.     

The influence of morphology on geldanamycin production in submerged fermentations of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The diverse morphology of the filamentous organism Streptomyces hygroscopicus var. geldanus was characterised by image analysis under various environmental conditions. In the presence of surfactant compounds, a significant decrease in the mean pellet diameter was observed. Cell aggregation was also influenced by spore inoculum level, with high concentrations reducing pellet size. In addition, the dispersion of pellets was found to increase with the inclusion of glass beads to submerged shake-flask cultures. In all cases, production of the secondary metabolite geldanamycin was determined to be dependent on the morphological profile of the organism, with a concomitant increase of 88% in geldanamycin yield observed as the mean pellet diameter was reduced by 70%. Thus, to maximise the yield of geldanamycin, it is necessary to limit pellet formation in Streptomyces hygroscopicus var. geldanus to an appropriate size.     

Bacterial colonization of pellet softening reactors used during drinking water treatment

Pellet softening reactors are used in centralized and decentralized drinking water treatment plants for the removal of calcium (hardness) through chemically induced precipitation of calcite. This is accomplished in fluidized pellet reactors, where a strong base is added to the influent to increase the pH and facilitate the process of precipitation on an added seeding material. Here we describe for the first time the opportunistic bacterial colonization of the calcite pellets in a full-scale pellet softening reactor and the functional contribution of these colonizing bacteria to the overall drinking water treatment process. ATP analysis, advanced microscopy, and community fingerprinting with denaturing gradient gel electrophoretic (DGGE) analysis were used to characterize the biomass on the pellets, while assimilable organic carbon (AOC), dissolved organic carbon, and flow cytometric analysis were used to characterize the impact of the biological processes on drinking water quality. The data revealed pellet colonization at concentrations in excess of 500 ng of ATP/g of pellet and reactor biomass concentrations as high as 220 mg of ATP/m(3) of reactor, comprising a wide variety of different microorganisms. These organisms removed as much as 60% of AOC from the water during treatment, thus contributing toward the biological stabilization of the drinking water. Notably, only a small fraction (about 60,000 cells/ml) of the bacteria in the reactors was released into the effluent under normal conditions, while the majority of the bacteria colonizing the pellets were captured in the calcite structures of the pellets and were removed as a reusable product.     

The nature of mycobacterial acid-fastness.

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbol-fuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

The Nature of Mycobacterial Acid-Fastness

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbolfuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

Biosynthesis of glycoproteins by membranes of Acer pseudoplatanus. Incorporation of mannose and N-acetylglucosamine.

Membrane preparations from Acer pseudoplatanus suspension cultures were demonstrated to incorporate radioactivity from GDP-[U-14C]mannose and UDP-N-acetyl-[6-(3)H]glucosamine into high-molecular-weight polymers characterized as glycoprotein. From 20 to 25% of the 14C was incorporated as fucose with the remainder as mannose, whereas 90% of the 3H was incorporated as N-acetylglucosamine with the remainder as N-acetylgalactosamine. Pronase digestion yielded radioactive glycopeptides that were separated into four fractions by gel-permeation chromatography and paper electrophoresis. The isolated glycopeptides differed in molecular weight and isotopes incorporated, as well as in amino-acid and monosaccharide composition. The membrane preparation also incorporated radioactivity from the added nucleotides into chloroform/methanol (2:1, v/v)- and chloroform/methanol/water (10:10:3, by vol.)-soluble lipids, and into an insoluble pellet.     

Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is able to grow in toxic concentrations of n-butanol up to 1.5 % (volume in volume) as the sole carbon and energy source. Morphology changes in the cells growing on increasing concentrations of n-butanol were observed. The size of the bacteria decreased with increasing concentrations of n-butanol, also leading to an enhanced ratio between the surface and volume of the cells. This is in complete contradiction to the reaction of glucose-grown cells to which n-butanol had been added as a toxin. Similar differences were found in typical adaptive responses to toxic organic compounds, namely changes in fatty acid composition of membrane lipids and the activity of catalase. In both cases, reactions depending on the n-butanol concentrations could be observed when the toxin was added to glucose-grown cells, whereas no reaction was observable when the cells were growing in n-butanol as the sole carbon and energy source. This is another proof for the observation that there are certain differences between the adaptive strategies of cells when adapting to high concentrations of a growth substrate and those when adapting to a toxin added to growing cells.     

Comparison of Light and Electron Microscopic Determinations of the Number of Bacteria and Algae in Lake Water

Determinations of the number of microorganisms in lake water samples with the bright-field light microscope were performed using conventional counting chambers. Determinations with the fluorescence microscope were carried out after staining the organisms with acridine orange and filtering them onto Nuclepore filters. For transmission electron microscopy, a water sample was concentrated by centrifugation. The pellet was solidifed in agar, fixed, dehydrated, embedded in Epon, and cut into thin sections. The number and area of organism profiles per unit area of the sections were determined. The number of organisms per unit volume of the pellet was then calculated using stereological formulae. The corresponding number in the lake water was obtained from the ratio of volume of solidified pellet/volume of water sample. Control experiments with pure cultures of bacteria and algae showed good agreement between light and electron microscopic counts. This was also true for most lake water samples, but the electron microscopic preparations from some samples contained small vibrio-like bodies and ill-defined structures that made a precise comparison more difficult. Bacteria and small blue-green and green algae could not always be differentiated with the light microscope, but this was easily done by electron microscopy. Our results show that transmission electron microscopy can be used for checking light microscopic counts of microorganisms in lake water.     

Optimization and monitoring of water soluble substrate for synthesis of mannosylerythritol lipids by Pseudozyma antarctica (ATCC 32657)

Biosurfactants are amphipathic molecules, consisting of hydrophobic and hydrophilic domains, which can partition at the interfaces between different fluid phases, such as oil/water or water/air interfaces. Mannosylerithritol lipids (MELs) are a type of glycolipid. The yeast Candida (Pseudozyma) antarctica (ATCC 32657) secretes an extracellular MEL, with biosurfactant properties, when grown on a water soluble and insoluble substrate, such as glycerine or oils. Value added MEL was biologically synthesized, by using two different types of honey as natural and newer water soluble carbon source. Those types of honey as a carbon source were optimized at different concentration of total carbohydrate present in the honey. Further optimized substrate concentration of honey was monitored for 15 days, for studies of MEL yield and cell growth concentration per day on shake flask batch culture. Surfactant properties, such as surface tension, interfacial tension, foaming, emulsification and wetting of MEL, were observed. Produced MEL was confirmed by thin layer chromatography (TLC), Fourier Transform Infrared (FTIR) spectra, and ¹H Nuclear Magnetic Resonance (NMR) techniques.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

Permeability of the Cell Envelope and Osmotic Behavior in Saccharomyces cerevisiae

Bakers' yeast (Saccharomyces cerevisiae) was equilibrated with distilled water and then packed into standardized pellets by centrifugation. The fractional space (S value) that was accessible to passive permeation was probed with a variety of mono- and divalent salts, mono- and disaccharides, polyols, substrates and products of beta-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC 3.1.3.2), and a cross-linked polymer of sucrose (Ficoll 400). A simple but very reproducible method was developed to measure pellet volume. At the limit of zero osmolality for bathing medium, the interstitial space was 0.223 ml/ml of pellet, and the aqueous volume of cell envelopes was 0.117 ml/ml of pellet. Thus the cell envelope for this yeast, under these conditions, was approximately 15% of the total cell volume. At a finite osmolality, the space in a yeast pellet that was accessible to salt was accounted for by the sum of initial interstitial space, the volume of the cell envelopes, and the volume of water abstracted from the cells by osmosis. Plots of S value versus osmolality were linear for uncharged probes and curvilinear for all salts. When Ficoll and potassium thiocyanate were presented to the yeast in admixture, the S values for the salt increased continuously over the range of osmolality studied. However, the S values for Ficoll 400 (which did not penetrate the cell wall) were lower by an amount equilivalent to the cell envelopes; they increased in parallel with the S curve for salt up to 1.15 osmol/kg and then plateaued. The results support the concept of incipient plasmolysis at 1.15 osmol/kg, and the separation of protoplasm from the cell wall is indicated with more concentrated solutions. Such cells were still viable if slowly diluted in distilled water, but they were injured by the shock of rapid dilution. However, shocking the cells did not release beta-fructofuranosidase into the medium. The complete accessibility of salts toward killed cells was demonstrated with yeast that had been pretreated with heat, organic solvents, or glutaraldehyde.     

Metallic Zinc Reduction of Disulfide Bonds between Cysteine Residues in Peptides and Proteins

The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mercapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within 1 h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).     

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.     

Large-scale isolation of a native deoxyribonucleohistone complex from baker''s yeast.

A method for large scale isolation of a native deoxyribonucleohistone complex from yeast is described. Crude chromatin, obtained after disrupting yeast cells at low ionic strength, contains a large amount of lipids, partially due to contaminating membranes. Most of them are removed by a Triton X-100 treatment, followed by step-gradient centrifugation. About 90% of the pellet may be solubilized by mild procedures, the composition of the soluble material being: histone/DNA = 1.0;nonhistone proteins/DNA = 0.55; RNA/DNA = 0.18. Histones can be obtained with high purity. Micrococcal nuclease digests DNA to yield a series of oligomeric fragments, with an average repeat length of about 160 base pairs. Circular dichroism spectra show that (theta) 270 is reduced by about 30% when compared to pure DNA and that chromosomal proteins are not denatured. These results indicate that the components of the complex conserve the native state.     

Surface effects in preparation of cell-size liposomes

Effects of surface type and area were shown to be important in the yield of cell-size liposomes, but not in determining their size. The liposomes were prepared by dissolving lipids in a chloroform-methanol solution and then evaporating the solvent under nitrogen in the presence of glass beads. After evaporation of the solvent, which was rapid due to the increased surface area, the dried lipids were then swollen in water at high temperatures (higher than the phase transition of the lipids), which led to formation of giant liposomes. The number of liposomes prepared in the presence of pyrex glass beads, which increase more than 100-times the surface area of lipid-glass contact, is more than 5-times larger than in the control experiments without glass beads. The yield of liposomes in the presence of another type of glass bead was almost the same as in the control experiments. These effects may be due to long- and short-range intermolecular interactions in the glass/water/lipid system.     

Involvement of Norepinephrine in the Production of a Flower-Inducing Substance in the Water Extract of Lemna

A flower-inducing substance (FIS) is produced by incubationof the pellet of centrifuged ho-mogenates of Lemna paucicostata441 (P441) with commercial enzyme preparations such as catalase,cellulase, lipase and proteinase K. The active component inthese preparations was identified as tyrosine. The tyrosinemetabolites, dopa, dopamine, norepinephrine (NE) and epinephrine,were also effective for the production of FIS, and NE the mosteffective. Addition of only 1 µg NE to the pellet obtainedfrom 100 mg fresh weight of P441 produced FIS without incubation.NE added to the pellet heated after resuspension also producedFIS but that added to the pellet resuspended in boiling waterimmediately after separation did not. A heat-stable substance(s)produced by a heat-unstable reaction in the pellet may reactwith NE to produce FIS or interact with NE to induce flowering.About 400 ng per g fresh weight of NE was detected in the waterextract of P441 plants, but only about 0.5 ng in the hot-acidicwater extract of the plants, which suggests that most of theNE was produced after homogenization. (Received October 17, 1990; Accepted December 28, 1990)