Large Granule Fraction Research Articles

Intrinsic and extrinsic factors drive differences in the gelatinisation behaviour of barley and malt starch

We studied the impact of malting on barley starch gelatinisation properties and whether observed differences are due to changes in extrinsic or intrinsic factors. We isolated the total starch and large and small starch granules fractions from barley and malt samples and subjected them to DSC. The peak gelatinisation temperature for malt starch was, on average, 1.2 °C higher than for barley starch. The malting process and endosperm breakdown products were each responsible for half of this difference. The presence of water-extractable, non-starch components (sugars, minerals, protein and starch hydrolysis products,…) increased the intrinsic starch gelatinisation temperatures by 2.2–4.7 °C for barley and 3.6–5.3 °C for malt. The small starch granule fractions from barley had a 3.1 °C higher peak gelatinisation temperature than large granule fractions. No effect of malting was observed here. These findings indicate that matrix effects and starch granule size must be considered when addressing starch conversion during brewing.

Assessment of the intracellular distribution of copper in liver specimens from cats.

The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 μg/g dry weight, eight had copper concentrations between 100 and 180 μg/g, 14 had copper concentrations between 181 and 700 μg/g, and two had copper concentrations >700 μg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.

Partitioning of nutrient removal contribution between granules and flocs in a hybrid granular activated sludge system

Sludge granulation in continuous-flow systems is an emerging technology to intensify existing activated sludge infrastructure for nutrient removal. In these systems, the nutrient removal contributions and partitioning of microbial functions between granules and flocs can offer insights into process implementations. To this end, a reactor system that simulates the continuous-flow environment using an equal amount of initial granule and floc biomass was investigated. The two operational strategies for maintaining granule growth in the continuous-flow system were (a) the higher solids retention time (SRT) for the granules versus flocs, as well as (b) selective feeding of carbon to the granules. The SRT of the large granule fractions (>425 µm, LG) and floc/small granule fractions (<425 µm, FSG) were controlled at 20 and 2.7–6.0 days, respectively. Long term operation of the hybrid granule/floc system achieved high PO43- and NH4+ removal efficiencies. Higher polyphosphate-accumulating organisms (PAO) activity was observed in the FSG than LG, while ammonia-oxidizing bacteria (AOB) activities were similar in the two biomass fractions. Nitrite shunt was observed in the FSG, possibly due to out-competition by the high NOB activity in LG. More importantly, washing out the FSG caused a reduction in LG's AOB and PAO activity, indicating a possible dependency of LG on FSG for maintaining its nutrient removal capacity. Our findings highlighted the partitioning and potential competition/cooperation of key microbial functional groups between LG and FSG, facilitating nutrient removal in a hybrid granular activated sludge system, as well as implications for practical application of the treatment platform.

A new method to isolate and separate small and large starch granules from barley and malt

Large lenticular and small spherical starch granules are present in small grains in varying quantities and have different characteristics, affecting how the cereals perform during processing. Separation and isolation of these granules for characterisation is challenging, especially for germinating or malted cereals, because the latter can contain up to a 1000-fold higher α-amylase activity. In this work, a new but simple method is described to isolate starch from barley and malt and separate it in small and large starch granules while avoiding α-amylase activity. A protease treatment at pH 8.0 in the presence of 0.1 M EDTA is used to degrade the protein matrix around the starch granules in an aqueous cereal suspension. After sieving over a 38 μm sieve, highly pure (90.2–95.1 dm%) starch is obtained. Next, sequential sieving over 10 μm and 5 μm sieves results in isolated large and small starch granule fractions with a respective starch purity of 89.8%–93.8% and 76.8%–86.2% and a volume-based granule-type purity higher than 92.9%. Residual EDTA concentration and α-amylase activity in the isolated starch fractions are reduced to a minimum, preventing their interference during the further determination of the starch properties.

Subcellular distribution of hepatic copper in beef cattle receiving high copper supplementation

Previous studies of intensively reared cattle in NW Spain have reported significantly higher copper (Cu) accumulation in the liver in Holstein-Friesian (HF) animals than in Galician Blonde (GB) or GBxHF crosses when receiving a diet supplemented at the maximum Cu concentrations allowed in the EU legislation (35mg/kg). The present study aimed to evaluate whether this difference is due to the pattern of subcellular accumulation of Cu in the liver. For this purpose, liver samples from 10 GB, 9 HF and 10 GBxHF young bulls were analysed to determine the content of metallothionein (MT) and Cu and zinc (Zn) (in the liver (Cu-liver and Zn-liver) and bound to metallothionein (Cu-MT and Zn-MT)). The Cu distribution within the main subcellular compartments (nuclei, large granule, microsomes and cytosol) was also determined. Even though HF animals showed significantly higher (P<0.05) Cu concentrations in the liver (161±10mg/kg wet weight) compared with GB (132±8mg/kg), no breed-related differences were observed for any of the parameters considered in this study. Overall, the pattern of hepatic subcellular accumulation was similar to that previously described in cattle: (i) MT concentrations were lower than in other animal species but strongly related to hepatic Zn; (ii) a low proportion of Cu (6.61%) was bound to MT but this was strongly and negatively related to the Cu:Zn ratio in the liver cell; and (iii) the highest proportion of Cu (57.3%) was found in the large granule (lysosome containing) fraction. All these results indicate a low capacity of cattle to excrete Cu by the bile resulting in a high Cu accumulation in the liver cell.

Effect of granule size on physicochemical, morphological, thermal and pasting properties of native and 2-octenyl-1-ylsuccinylated potato starch prepared by dry heating under different pH conditions

Potato starch was fractionated into small granule fraction (SGF) and large granule fraction (LGF) and both fractions were then modified by octenyl succinylation at pH 4, 6 and 8 followed by dry heating at 130 °C for 2 h. The effects of starch granule size and pH conditions of dry heated octenyl succinic anhydride (OSA) modification on physicochemical, morphological, thermal and pasting properties of both the starch fractions were investigated. Amylose content and swelling power of OSA modified both starch fractions at different pH's were significantly (p < 0.05) lower as compared to their native counterpart starches. OSA modification at different pH's resulted in decrease in light transmittance of both starch pastes. Morphological investigations revealed that the modification caused drastic changes on the surface of starch granules. OSA modification at different pH's resulted in decrease in gelatinization temperatures for both SGF and LGF potato starches as compared to their native counterpart starches. The variations in pH during modification had great effects on the pasting characteristics of both starch fractions. When OSA modified potato starch (both LGF and SGF) was reacted at pH 4 and pH 6, the peak viscosity decreased significantly (p < 0.05) as compared to their native counterpart starches.

Reduced glutathione disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization

We previously reported that treatment of B16 melanotic melanoma cells with reduced glutathione (GSH) converts them to amelanotic cells without any significant down-regulation of tyrosinase activity. To characterize the cellular mechanism(s) involved, we determined the intracellular distribution of melanocyte-specific proteins, especially in melanin synthesis-specific organelles, termed melanosomes by subcellular fractionation followed by Western blotting and confocal laser microscopy (CFLM). In the melanosome-rich large granule fraction and in highly purified melanosome fractions, while GSH-induced amelanotic B16 cells have significantly diminished levels of protein/activity of tyrosinase and tyrosinase-related protein-1 compared with control melanized B16 cells, there was substantially no difference in the distribution and levels of dopachrome tautomerase and the processed isoform of Pmel17 (HMB45) between control melanized and GSH-induced amelanotic B16 cells. Analysis of merged images obtained by CFLM revealed that whereas tyrosinase, Pmel17 and dopachrome tautomerase colocalize with each other in the control melanized B16 cells, tyrosinase does not colocalize with Pmel17 or its processed isoform and with dopachrome tautomerase in GSH-induced amelanotic B16 cells. The sum of these findings suggests that reduced glutathione selectively disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization, probably serving as a putative model for oculocutaneous albinism type 4.

Exploring the surface morphology of developing wheat starch granules by using Atomic Force Microscopy

AbstractWheat starch granules from 7, 14, 21, 28, 35, 42, and 49 days after anthesis (DAA) were imaged using AFM. Starches were scanned in their native form as well as following exposure to iodine vapour in‐situ at 100% humidity. Starch at 7 DAA exhibited only one granule population, while starches at other maturities had two size populations. Starches from all stages of maturation exhibited blocklet structures. Larger fuzzy blocklets were observed at early stages while they became smaller and less fuzzy at subsequent stages of maturity. Furthermore, at all stages, small granules had larger fuzzy blocklets and higher surface roughness than the large granule counterpart; however the surface roughness of both small and large granule fractions decreased gradually with maturation. Amorphous growth ring of starch granules with ∼1 × 1 µm to ∼3 × 6 µm size islands were also observed. In‐situ iodine exposure demonstrated that iodine interacted with glucan polymers in the amorphous background first and then spread over to polymers on top of blocklets. Iodine‐glucan polymer interaction further increased the surface roughness.

Granule Sizes of Canna (Canna edulis) Starches and their Reactivity Toward Hydration, Enzyme Hydrolysis and Chemical Substitution

AbstractSmall and large granule fractions were isolated from canna starch (Canna edulis, green leaf cultivar), and their morphology, physicochemical properties, susceptibility towards granular starch hydrolyzing enzymes and chemical reaction with propylene oxide were investigated. Canna starch consisted of a mixed population of large, medium and small granules; the mean of granule diameter was 47.4 μm. The small granules presented round and polygonal shapes, whereas the large granules had oval and elliptical shapes. Significant variations in digestibility of the various granules size by granular starch hydrolyzing enzymes were observed. During the first 24 h, the hydrolysis rate of small granules was higher than that of native and large granule starches. After 72 h, however, the degree of hydrolysis of small granule, large granule and native starches had reached the extent of 19.6%, 32.0% and 27.2%, respectively. The larger the granule size, the higher the MS obtained when modified with propylene oxide, which was due to the higher swelling power of the large granules. The results obtained from this study suggest that small granules had lower water and chemical affinity when compared with the bigger ones. The difference in the reactivity of small and large granules could be presumably attributed to the starch components (amylose and amylopectin) and their organization of glucan chains in ordered and/or less ordered structure of these two fractions.

Relationship between Starch Granule Size Distribution and Selected Malting Parameters

The relationship between malting parameters and the ratio of large and small starch granules was studied in the endosperm of caryopses of 12 varieties of spring and winter barley (Hordeum vulgare L.) of Czech, German, and British origin. Starch granule size distribution was measured using two principally different methods, low-angle laser light scattering (LALLS) and gravitational field-flow fractionation (GFFF), over a 3-year period. An increase in a large starch granule fraction in the nonmalted caryopses of spring barley varieties studied was accompanied by a statistically significant decline in the values of extract, saccharide extract, relative extract at 45°C, Kolbach index, modification (Carlsberg method), wort color, starch content, and β-glucans in nonmalted caryopses. The higher ratio of large starch granules in two- and six-row winter varieties was not used in creation of extract, probably due to a lower activity of cytolytic and proteolytic enzymes. However, statistical correlations found between the studied malting parameters and starch granule size distributions were rather low. Statistically highly significant effects of varieties and variety–year interactions on large and small starch granules were determined.

Physico-chemical, rheological and structural properties of fractionated potato starches

In an attempt to reveal whether behaviour differs between various granule size classes of potato starches, small (SGF), medium (MGF) and large (LGF) granule fractions were separated from the native starches of three potato cultivars, and some of their physico-chemical and functional properties studied. There was a significant variation in the granule size distribution of the native starches and their separated fractions, when studied using particle size analysis and scanning electron microscopy. The granule size ranges for LGF, MGF and SGF were 40–65, 20–40, and 1–20 μm, respectively. The granule sizes in the fractions separated from the native starch of the cultivar Kufri Ashoka were larger than in the corresponding fractions separated from the other two cultivars (Kufri Kunden and Kufri Dewa). For all three cultivars, LGF had higher amylose content and a lower swelling power than the corresponding MGF and SGF. The light transmittance and solubility of the native starches and their three fractions increased, while enzymatic digestibility decreased with the increase in granule size. Among the three fractions, pasting properties such as peak and final viscosities were observed to be lower for SGF, while peak viscosity temperatures were lower for LGF. The breakdown and setback in viscosity were observed to be highest for LGF and lowest for SGF, for all three cultivars. The lowest values of dynamic mechanical properties such as G′, G″, G ∗, η ∗ and η′ were recorded for gels of native Kufri Dewa starch and its fractions during frequency sweep testing on a dynamic rheometer. The textural attributes of the gels obtained from the native starches and their fractions showed a relationship with their respective pasting behaviours. A significant and progressive change was observed in the texture of gels during 7 days storage at 4 °C.

Pasting properties and (chemical) fine structure of acetylated yellow pea starch is affected by acetylation reagent type and granule size

Yellow pea starch was fractionated into small and large size granule fractions and then modified with acetic anhydride and vinyl acetate (acetylation after sieving) or first acetylated in the same way and then fractionated into small and large size fractions (acetylation before sieving). Acetylation with different procedures (acetylation before or after sieving) resulted in different degrees of substitution for small size granule fractions when acetic anhydride was used as reagent. Modification with vinyl acetate gave products with much higher peak viscosities than modification with acetic anhydride for the same granule size and same degree of substitution (DS). The location and distribution of acetyl groups was investigated by analyzing the α-amylase hydrolysates of isolated amylose and amylopectin with chromatography and mass spectrometry. Mass spectra showed that the substituent distribution mainly depended on the type of reagent and was not affected by the granule size. Both amylose and amylopectin were modified in an heterogeneous way and the reactions took place in different regions of the granule. It is postulated that acetylation occurs more homogeneously throughout the granule when vinyl acetate is used as reagent, while the reaction with acetic anhydride to a large extent takes place in the outer lamellae of granule.

Gelatinisation related structural aspects of small and large wheat starch granules

Wheat starches from four European varieties (Charger, Estica, Skirlou and Soissons), and their small and large granule fractions were characterised on a structural level. Gelatinisation properties of starches and fractions thereof were compared. Isoamylase debranching revealed only limited differences between amylopectin chain lengths of the various starches and their fractions. Wide-angle X-ray diffraction, which showed predominant A-type crystallinity for all starches, revealed that large granules had systematically higher B-type and total crystallinity than small granules. Small-angle X-ray scattering (SAXS) indicated structural differences between amylopectins of distinct granule classes. At 42% moisture, the SAXS patterns of small granules were more intense than those of large granules, which, along with their higher gelatinisation peak temperature, suggested denser crystalline lamellae. Upon hydration from 42 to 66% moisture, small granules showed decreased lamellar repeat distances. As small granules displayed narrower helix–coil transition endotherms than large granules, shorter single stranded chains are envisaged to connect longer double helices to the amylopectin backbone in small wheat granules.

Intracellular distribution of copper and zinc in the liver of copper-exposed cattle from northwest Spain

The intracellular distribution of copper (Cu) and zinc (Zn) in the livers of normal and moderately Cu-exposed cattle was investigated with the aim of improving understanding of the pathophysiology of Cu accumulation in cattle. At total liver Cu concentrations within the generally accepted normal range (25–100 mg/kg fresh weight) the large-granule fraction was the main subcellular compartment for Cu accumulation, followed by the cytosol and the nucleus, whereas in the microsomal fraction Cu accumulation was very low. With increasing Cu exposure, the capacity of the large-granule fraction to accumulate Cu decreased, proceeding towards a plateau (estimated at about 80 mg/kg at a projected total liver Cu concentrations of 450 mg/kg), accompanied by progressively greater Cu accumulation in the nucleus and cytosol. Total liver Cu concentration had little influence on subcellular Zn distribution, with hepatic Cu concentrations being only moderately correlated with Zn concentration in the large-granule fraction. There was a strong association between the proportion of total Cu bound to metallothionein (MT) and the proportion of total Zn in the large-granule fraction, suggesting that Zn displaced from MT is taken up by the lysosomes for excretion. This pattern of Cu accumulation, as in sheep, may be due to the limited capacity for metallothionein binding of Cu and excretion in bile.

Differently sized granules from acetylated potato and sweet potato starches differ in the acetyl substitution pattern of their amylose populations

Acetylated potato and sweet potato starches were fractionated according to granule size. From the fractions obtained amylose and amylopectin were isolated and characterized with respect to degree of substitution (DS) and degradability with α-amylase, β-amylase and amyloglucosidase. The DS of the amylose populations of differently sized granule fractions was quite constant. In contrast, the DS of the amylopectin populations originating from the differently sized fractions increased with decreasing granule size. The acetylation was confirmed to occur throughout the amorphous regions, and only take place in the outer lamellae of crystalline regions of starch granules. The amylose populations isolated from small size granule fractions of the acetylated starches tested were less susceptible to all the enzyme digestions than the amylose originating from the large granule fractions, even though the DS was similar. The acetyl populations groups over the amylose molecules are more heterogeneously distributed and located more closely to the non-reducing ends for amylose originating from small size granule fractions when compared to amylose from lager sized granules.

The inhibitory effect of glycolic acid and lactic acid on melanin synthesis in melanoma cells.

Alpha-hydroxy acids (AHAs) such as glycolic acid (GA) and lactic acid (LA) have been reported to be effective in treating pigmentary lesions such as melasma, solar lentigines, and postinflammatory hyperpigmentation. The mechanism of this effect might be due to epidermal remodeling and accelerated desquamation, which would result in quick pigment dispersion. However, the direct effect of AHAs on melanin synthesis has not yet been well studied. To elucidate such a direct effect of AHAs on melanogenesis, we performed melanin assays, growth curve determinations, Northern and Western blotting for melanogenic proteins [tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2], and tyrosinase and, 4-dihydroxyphenylalaninechrome tautomerase enzyme activity assays using mouse B16 and human melanoma cells. GA or LA (at doses of 300 or 500 microg/ml) inhibited melanin formation in similar dose-dependent manner, without affecting cell growth. Although the mRNA and protein expression or molecular size of tyrosinase, TRP-1 and TRP-2 were not affected, tyrosinase activity was inhibited. To see whether GA and/or LA directly inhibit tyrosinase catalytic function, the effect of GA and LA on human tyrosinase purified from the melanosome-rich large granule fraction of human melanoma cells was performed. GA or LA were shown to inhibit tyrosinase enzyme activity directly, but this effect was not due to the acidity of GA or LA, because adjusting the pH to 5.6 (the pH of GA and LA at concentrations of 2500 microg/ml), did not affect tyrosinase activity. Taken together, these results show that GA and LA suppress melanin formation by directly inhibiting tyrosinase activity, an effect independent of their acidic nature. GA and LA might work on pigmentary lesions not only by accelerating the turnover of the epidermis but also by directly inhibiting melanin formation in melanocytes.

Damage to subcellular structures evoked by lipid peroxidation.

Subcellular Structures, Lipid Peroxidation, Free Radical Scavengers The influence of lipid peroxidation (LP) on the rate of disruption of rat liver and kidney subcellular structures was studied under two experimental conditions. Damage to cell organelles was found only when peroxidation process carried out into large granule fraction suspensions. Exogenous thiobarbituric acid positive products were noneffective as membrane labilizers. Age, organ and cell organelle-linked differences in the response towards LP produced damage were observed. Rat liver peroxisomes showed higher stability than those of kidney with respect to injury induced by peroxidation process. In addition, in rat kidney and neonatal rat liver samples the lysosomes were found to be more sensitive than mitochondria to the damaging effect of this process. Thiourea, an inhibitor of diene conjugate formation as well as manitol and ethanol known as hydroxyl radical scavengers were tested as terminators of LP and as membrane protectors. Effectiveness was demonstrated only for thiourea.

Giardia intestinalis, a eukaryote without hydrogenosomes, produces hydrogen.

The microaerophilic flagellated protist Giardia intestinalis, the commonest protozoal agent of intestinal infections worldwide, is of uncertain phylogeny, but is usually regarded as the earliest branching of the eukaryotic clades. Under strictly anaerobic conditions, a mass spectrometric investigation of gas production indicated a low level of generation of dihydrogen (2 nmol x min(-1) per 10(7) organisms), about 10-fold lower than that in Trichomonas vaginalis under similar conditions. Hydrogen evolution was O2 sensitive, and inhibited by 100 microM metronidazole. Fluorescent labelling of G. intestinalis cells using monoclonal antibodies to typical hydrogenosomal enzymes from T. vaginalis (malate enzyme, and succinyl-CoA synthetase alpha and beta subunits), and to the large-granule fraction (hydrogenosome-enriched, also from T. vaginalis) gave no discrete localization of epitopes. Cell-free extracts prepared under anaerobic conditions showed the presence of a CO-sensitive hydrogenase activity. This first report of hydrogen production in a eukaryote with no recognizable hydrogenosomes raises further questions about the early branching status of G. intestinalis; the physiological characterization of its hydrogenase, and its recently elucidated gene sequence, will aid further phylogenetic investigations.

The influence of starch pore characteristics on pasting behaviour

SummaryPotato, wheat and corn starches were segregated into large and small granule fractions. The specific surface area, volume, and average diameter of pores, as well as the pasting characteristics of starch dispersions in water were determined. In all the starches investigated the small granule fractions were characterized by the largest specific surface area, pore volume and pore diameter in comparison to fractions of large granules and the initial starches. The small granule fraction of potato starch gave better pasting characteristics (apart from maximum viscosity) than fractions of large granules and the initial potato starch. On the other hand, the large granule fractions of corn and wheat gave the highest maximum viscosity.

The depigmenting effect of alpha-tocopheryl ferulate on human melanoma cells.

Oral vitamin E (alpha-tocopherol, alpha-T) supplementation has been reported to improve facial hyperpigmentation. alpha-Tocopheryl ferulate (alpha-TF) is a compound of alpha-T and ferulic acid connected by an ester bond; ferulic acid is also an antioxidant, and could scavenge free radicals induced by ultraviolet (UV) radiation, and thus maintain the long-lasting antioxidative effect of alpha-T. Our aim was to see whether alpha-TF might be useful as a whitening agent and an antioxidant to improve and prevent facial hyperpigmentation following UV exposure. In this study, the inhibitory effect of alpha-TF on melanogenesis was examined biochemically using human melanoma cells in culture. The results show that alpha-TF, solubilized in ethanol or in 0.5% lecithin, inhibited melanization significantly, as did alpha-T at a concentration of 100 microg/mL, without inhibiting cell growth. This phenotypic change was associated with inhibition of tyrosinase and 5, 6-dihydroxyindole-2-carboxylic acid polymerase activities, and the degree of inhibition was dose dependent. No significant effect on DOPAchrome tautomerase activity was observed. alpha-TF did not directly inhibit tyrosinase activity of the large granule fraction extracted from human melanoma cells, and Western blotting revealed that there were no changes in protein content or in molecular size of tyrosinase, tyrosinase-related protein (TRP)-1 or TRP-2. Therefore, the inhibition of tyrosinase activity by alpha-TF might be due to effects at the post-translational level, and possibly by a secondary molecule activated by alpha-TF. These results suggest that alpha-TF is a candidate for an efficient whitening agent which suppresses melanogenesis and inhibits biological reactions induced by reactive oxygen species.