Studies on Enzymatic Liquefaction and Saccharification of Starch

Liquefaction of corn starch is generally considered to be difficult to achieve. A double enzyme system, a combination of bacterial alpha-amylase and fungal glucoamylase, was tested for the production of dextrose from corn starch. The former liquefies starch and the latter hydrolyzes further into glucose

Particularly various conditions for liquefaction of corn starch were examined. As criteria of solubilization of starch molecule, turbidity of filtrate obtained while the solution was hot, iodine coloration value, filterability and turbidity of the saccharified solution after the addition of alcohol were measured.

Starch slurry containing bacterial alpha-amylase was poured continuously in fine stream into hot water with vigorous agitation. Liquefaction at 92°C was better than at 87°C for dissolving corn starch. It was indispensable to apply heat treatment under high pressure after liquefaction followed by the second addition of bacterial alpha-amylase at 90°C to render remaining tightly bound starch micelle to be broken down. Heat treatment was continued at 120°C or at higher temperature. Then, solution was cooled rapidly down to about 90°C. Immediately bacterial alpha-amylase was added. In case the second addition of bacterial alpha-amylase was carried out after cooling to 55°C or after holding for 10min at 80°C, no effect was observed by the second addition. These findings were confirmed in the experiments on commercial production scale.     

Enhancement of the Stability of Insoluble Calcium Particles Using a Phospholipid Coating

Calcium is one of the most important elements in the human body. Insoluble calcium particles are often used in calcium-fortified food products, such as calcium-fortified milk, dairy beverages or protein powders. However, their suspension may be unstable often leading to precipitation in such products. In this study, three different kinds of insoluble calcium particles, i.e. hydroxyapatite (HA), tricalcium phosphate (TCP) and calcium carbonate (CaCO₃) were coated with amphiphilic phospholipids using a solvent-exchange method. Suspension stability of these insoluble calcium particles was effectively improved with phospholipid coating, especially for HA and TCP, as more phospholipids were coated on the surface of these two calcium particles than CaCO₃. Phospholipid coating increased the electrostatic repulsions between particles, preventing the particles from aggregating and precipitating. In addition, the digestibility of phospholipid-coated insoluble calcium particles was tested in simulated gastric juice, and the dissolution time of these insoluble calcium particles was prolonged through phospholipid coating.     

Evaluation and Elimination of the Interference by Starch in the Biuret Determination of Wheat Protein

In the biuret determination of wheat protein, starch, color substances and lipids extractable with an alkaline solution such as biuret reagent were found to interfere with the biuret method. Comparative evaluation of their effects revealed that starch, a major component of wheat, has the most significant effect on the biuret method. In the presence of starch, the correlation between Kjeldahl protein and the optical density of biuret was poor.

It was found that several organic solvents effectively retarded solubilization of wheat starch in the biuret reaction mixture. By treatment of the wheat samples with these solvents prior to the biuret procedure, the effect of starch was completely eliminated and the optical density of biuret became closely correlated with Kjeldahl protein.     

Oxidative Degradation of Starch by Free Radical Systems

A new method for preparation of chemically modified starch by means of free radical systems was developed. The method is characterized is that, aqueous starch suspension is treated with hydrogen peroxide in the presence of free radical-producing synergists such as ascorbic acid, hydrazine, or hydroxylamine to give products of remarkably reduced viscosity which can be regulated arbitrarily by use of appropriate levels of the reagents.

Chemical and physical changes in the modified starch together with the reaction mechanism were also studied.     

A rapid laboratory test for determining death in some armored scale species (Coccoidea: Diaspididae)

A rapid laboratory test for determining death in the Florida red scale (Chrysomphalus aonidum (L.)) and the California red scale (Aonidiella aurantii mask.) based on enzymatic activity is described. This test is based on the ability of the live scale enzymes to react with iodine, yielding iodine products which in turn are unable to give the blue color reaction with starch. The reaction seems to be due to the decrease in the oxidation number of the iodine from I⁰ — I^-1 by the scale reductive enzymes. The longevity of the females after detachment from the host or after treatment with insecticides commonly used in the field, was determined by this test.

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Résumé Un test basé sur l'activité enzymatique pour déterminer rapidement la mort chez le pou de Floride (Chrysomphalus aonidum L.) et la cochenille rouge de Californie (Aonidiella auranlii Mask.) est décrit. Ce test repose sur la capacité des enzymes de la cochenille vivante de réagir avec l'iode en donnant des produits iodés ne pouvant plus colorer l'amidon. Cette réaction semble due à la diminution du degré d'oxidation de l'iode de I⁰ —I^-1 par l'entremise des enzymes réducteurs de la cochenille.La longévité des femelles, détachées de leur hôte ou après certains traitements insecticides usuels, a été déterminée au moyen de ce test.

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Contribution from The Volcani Institute of Agricultural Research, Bet Dagan, Israel. 1969 Series; No. 1581-E.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Auxin and gibberellin effects on cell growth and starch during abscission in cotton

An increase in starch content of cells in the abscission zone of the cotton explant appeared correlated with an increase in number of cells. A large increase in the number of cells in the abscission zone, concomitant with an increase in starch content, followed treatment with gibberellin as compared to auxin. In the final stages of abscission starch was hydrolyzed in the cells of the separation layer. Some starch remained after the petiole abscised.

A positive phloroglucinol-hydrochloric acid reaction in the cells of the petiole distal to the line of separation indicated the presence, not of lignin, but of soluble sugars and uronic acids. This reaction was especially intense following gibberellic acid treatment.

It was concluded that gibberellin in accelerating abscission leads to (1) an increase in cell number and starch content in the abscission zone, (2) the hydrolysis of starch in the separation layer just before abscission, and (3) the breakdown of polysaccharides and the release of soluble sugars and uronic acids. Auxin, an abscission retardant, either delays or prevents these events.

     

The use of C14-labelled substrate in histochemical demonstration of different forms of phosphorylase

Active and total phosphorylase activity, using labelled C¹⁴-glucose-1-phosphate as the substrate, is demonstrated by histoautoradiographic method. This method can demonstrate the polysaccharide synthesizedin vitro by phosphorylase without intervention from the unlabelled pre-existing glycogen. C¹⁴-glucose can not replace C¹⁴-glucose-1-phosphate as substrate. The distribution of phosphorylase in tissue sections, except in cases of very low activity, is similar to that obtained by customary dilute Lugol's iodine staining method. The relative difference of intensity between active and total phosphorylase, as revealed by iodine staining, is also reflected by histoautoradiographic method. Histoautoradiographic method has several advantages over the iodine staining method. This method is more sensitive for demonstration of very low phosphorylase activity which may escape detection by iodine staining. Branching enzyme activity, especially when it favors synthesis of glycogen type of polysaccharide instead of amylopectin type, can be better detected by this method. Active phosphorylase substrate medium can be used to demonstrate this activity in plant tissues, where the presence of pre-existing starch often prohibits the use of iodine staining method. Stripping film method for autoradiography is recommended for the study of this enzyme activity.     

Charakterisierung der Stärkehydrolysate nach Einwirkung einer thermostabilen α-Amylase

The action of thermostable α-amylase produced by Bacillus licheniformis 44MB82 strain on soluble and insoluble starch, amylose and amylopectin at temperatures 30°C and 90°C was studied. The hydrolysis of soluble starch proceeded rapidly for 10 to 15 minutes after which the maltodextrins thus formed were further dissociated. In the course of 60-minutes enzyme treatment mainly glucose, maltose and maltosugars (from G₃ to G₆) as low molecular weight products were found and the formation of maltcse and maltotriose was increased by the longer treatment. The hydrolysis of insoluble starch and amylopectin proceeded in the same way while the amylose was hydrolysed slowly.     

Induced Disruption of Sieve Element Plastids in Heracleum mantegazzianum L.

Sieve element plastids of Heracleum mantegazzianum L. were disruptedin fresh tissue by treatment with iodine. Their breakdown andthe consequent release of starch grains into the sieve elementlumen was recorded by cin?micrography. We discuss how plastidsburst to release the particles so frequently seen in sieve tubesunder the light microscope.     

Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.

Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.

The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.

     

Block-Surface Staining for Differentiation of Starch and Cell Walls in Wheat Endosperm

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starch granule distribution in cells of wheat [Triticum aestivum L.] caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 μm³ to 22,000 μm³ with approximately 90% of the granules measuring less than 752 μm³ (ca. 11 μm in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells.     

Behavior and viability of tobacco protoplasts in response to electrofusion parameters

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.

Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

     

Hydrogen peroxide and lignification

The production of hydrogen peroxide in plant tissue is demonstrated quickly with a simple histochemical test. The test solution, 50 mM potassium iodide in a 4% (w/v) potato starch suspension, is applied to the cut surface of the tissue to be tested. Hydrogen peroxide oxidizes iodide ions to iodine; the iodine is complexed by the starch to form a blue-purple color. This test detects hydrogen peroxide production in cells undergoing lignification, i.e. tracheary elements and phloem fibers, and in some epidermal cells. In addition there is a rapid production of hydrogen peroxide in crushed cells. The test is negative under (i) anaerobic conditions and (ii) in the presence of catalase.     

Simultaneous determination of T-, B- and mixed rosette-forming lymphocytes in human peripheral blood]

Preliminary treatment of erythrocyte suspension with trypan blue solution did not affect their capacity to participate in the reaction of spontaneous rosette formation, but permitted to differentiate them in a mixture with other erythrocytes after staining with methyl-green-pyronine on a fixed slide. By using a mixture of such marked erythrocytes sensitized with antibodies and a complement, the presence in the peripheral blood of healthy people of a small lymphocyte subpopulation connecting these two types of indicator particles was demonstrated.     

Expression of a wild-type GBSS gene introduced into an amylose-free potato mutant by Agrobacterium tumefaciens and the inheritance of the inserts at the microsporic level

Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Transformation of a diploid amylose-free (amf) potato mutant with the gene encoding GBSS leads to the restoration of amylose synthesis. Transformants were obtained which had wild-type levels of both GBSS activity and amylose content. It proved to be difficult to increase the amylose content above that of the wild-type potato by the introduction of additional copies of the wild-type GBSS gene. Staining of starch with iodine was suitable for investigating the degree of expression of the inserted GBSS gene in transgenic amf plants. Of the 19 investigated transformants, four had only red-staining starch in tubers indicating that no complementation of the amf mutation had occured. Fifteen complemented transformants had only blue-staining starch in tubers or tubers of different staining categories (blue, mixed and red), caused either by full or partial expression of the inserted gene. Complementation was also found in the microspores. The segregation of blue- and red-staining microspores was used to analyse the inheritance of the introduced GBSS genes. A comparison of the results from microspore staining and Southern hybridisation indicated that, in three tetraploid transgenics, the gene was probably inserted before (duplex), and in all others after, chromosome doubling (simplex). The partial complementation was not due to methylation of the HPAII/MSPI site in the promoter region. Partially complemented plants had low levels of mRNA as was found when the GBSS expression levels were inhibited by anti-sense technology.     

Efficacy of Fe3O4/Starch Nanoparticles on Sporosarcina pasteurii Performance in MICP Process

The microbial induced calcite precipitation (MICP) has been explored using well-known urease producer bacterium Sporosarcina pasteurii for many applications including soil stabilization. Urease enzyme hydrolyzes urea and in the presence of calcium chloride causes calcium carbonate precipitation between sand particles increasing sand stiffness and strength. In this study, the liquefied soil samples from Anzali coast were positioned inside injection columns by standard positioning technique. The columns were treated by injecting S. pasteurii suspension and cementation solution (CaCl₂ and urea). The effect of different conditions consisting of number of injections, injection intervals, flow rate, and ratio of injection solution on unconfined compression strength (USC) of sands formed inside the columns were evaluated. The results indicated that soil strength was increased when ratio of reactant solutions and injection time were elevated. Moreover, the maximum Ca-precipitation in MICP reaction in liquid medium was obtained while Fe₃O₄/starch concentration and time of addition of nanoparticle to culture medium were 10.8?mg/L and 1.4?h, respectively. The USC results showed that the columns injected by bacterial suspension treated by Fe₃O₄/starch under optimized conditions improved the soil strength up to 1200?kPa in comparison to the control column as 220?kPa.     

Causal basis of gravity stimulus nullification by clinostat rotation

Using appropriate clinostat rotation methods, it has been shown that increases in root growth and geotropic curvature of oat coleoptiles are related to and explained (within the limits of auxin economy) by increased rates of plant respiration imparted by nullification of the gravitational stimulus. Increased respiration with decreased gravitational stimulus, in turn, is explained by increased uniformity in intracellular distribution of metabolically active graviprecipitable particles.

Oat seedlings grown with nullification of the gravitational stimulus show a more uniform distribution of graviprecipitable protoplasmic inclusions. Respiration involves reactions between particles of sufficient mass to be precipitated in 1 × g unidirectional force field and particles of such size that they are distributed throughout the protoplasm due to thermal energy. When plants are grown without nullification of the gravitational stimulus, graviprecipitable protoplasmic inclusions tend to accumulate in the lower portions of the cells. Respiration rates are consequently limited due to a relatively low concentration of the smaller particles in the region of high concentration of the larger particles. Distribution of the larger particles throughout the protoplasm negates the reaction rate limiting effects of these intracellular concentration gradients.

It has been shown using starch particles to index graviprecipitation of intracellular particles that enhancement of respiration by nullification of the gravitational stimulus may be induced, maintained, discontinued, and reinduced by treatments which concomitantly result in the more uniform distribution of graviprecipitable protoplasmic inclusions, the maintenance of this uniform distribution, the discontinuation of this uniform distribution and its reinduction.

The degree of uniformity of intracellular particle distribution in response to treatments only partially nullifying the gravitational stimulus is shown wholly consonant with growth responses of roots and coleoptiles when the growth is measured as the expression of increased respiration in the absence of auxin limitation.

The findings collectively indicate that gravity sensing by plants is broadly based on the physicochemical relationship between particle distribution and the expression of respiratory metabolism as growth.

     

Fractionation of a commercial cellulase preparation from Penicillium funiculosum and its use in the purification of a water soluble α-glucan from milled barley

A commercial enzyme preparation from a selected strain of Penicillium funiculosum has been partially purified using a single stage chromatofocusing fractionation to produce an amylase-free mixture of hydrolytic enzymes. This mixture has been used to remove the non-starch polysaccharides from aqueous extracts of laboratory milled barley. The structure of the resulting purified α-glucan fraction has been examined by gel filtration before and after enzymic debranching and by iodine staining. The mild damage caused to the starch within the barley kernel releases a low molecular weight amylopectin molecule with no detectable amylose in the product. In this respect the product is different from that produced after severe, direct grinding of the purified barley starch where some amylose appears in the water soluble extract. Although the small amount of α-glucan is not of any quantitative industrial significance in itself, it does reflect the extent and type of physical damage which is taking place in the starch granule. The model proposed to explain these results - a starch granule with a solid amylose/amylopectin core but with a number of patches of protruding amylopectin clusters - may have important implications in an industrial context.     

The Use Of Lead Tetraacetate,Benzidine, O-Dianisidine and A “Film Test” In Investigating The Periodic-Acid-Schiff Technic

In order to obtain a better understanding of the “periodic-acid-Schiff” reaction, also known as the “periodic-acid fuchsin-sulfurous-acid” reaction, three types of investigations were carried out

1) The Schiff reagent was replaced by other aldehyde reagents: benzidine or o-dianisidine. There was no significant change in the histological distribution and intensity of the reactions occurring after periodic acid oxidation.

2) Periodic acid was replaced by another oxidizing agent: lead tetraacetate (dissolved in acetic acid). There was no significant change in the histological distribution of the reactions with the Schiff reagent, but some change in their intensity. It was concluded that 1,2-glycols and a-amino alcohols play the main role in the reactions with both oxidants. The presence of α-hydroxy acids in some types of mucous cells is suggested by the results with lead tetraacetate.

Incidently, glycogen and starch are not sufficiently oxidized by lead tetraacetate (in acetic acid) at room temperature to give positive reactions with the Schiff reagent, while cellulose and other periodic-acid-Schiff reactive substances are.

3) The staining of films of presumed reactive substances with the periodic-acid-Schiff technic C O the intense reactivity of many polysaccharides, mucopolysaccharides and mucoproteins, but not of ordinary proteins. (Hyaluronic and chondroitin sulfuric acid are, however, not reactive in vitro).

In conclusion, the periodic-acid-Schiff technic consists of an oxidation of 1,2-glycols and a-amino alcohols to produce aldehyde groups, which are then stained by the Schiff reagent. The “film test” reveals that these radicals are present in certain polysaccharides, mucopolysaccharides and mucoproteins.