Sucrose Purification Research Articles

Generation of quality-controlled SARS-CoV-2 variant stocks.

One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research-for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controlled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5-8 d are required when virus is isolated from clinical swab material, and another 6-7 d is needed for sucrose-purifying the stocks.

Anionic bio-flocculants from sugarcane for purification of sucrose: An application of circular bioeconomy

In sugar production, polyacrylamide-based anionic flocculants are added for juice treatment, the main objective being to remove impurities that affect the quality of the sugar. However, if they remain in the final product, those polymers can present carcinogenic and neurotoxic actions besides contaminating the soils where the waste is discharged. To overcome this problem, the present study proposes, for the first time, natural flocculants based on cellulose obtained from sugarcane bagasse (residue from sugarcane processing) as substitutes for the flocculants based on polyacrylamide, normally used in sugar cane juice purification. Additionally, cellulose-based flocculants obtained from Acacia wood, developed in a previous study, have also been tested for sugar juice treatment. Acacia wood and sugarcane bagasse were first treated with a choline chloride/levulinic acid solution in a molar ratio of 1:2, at 160 °C, for 4 h. Subsequently, the cellulose-rich samples were modified by a two-stage process (oxidation with sodium periodate followed by reaction with sodium metabisulfite), and polyelectrolytes with different characteristics were produced. The final products obtained were characterized, and their performance in the treatment of sugarcane juice, at different concentrations (10, 50, 100, 250, and 500 mg kg−1), was evaluated and compared to the synthetic commercial flocculant (Flonex, based on polyacrylamide) usually used by the sugarcane industry in Brazil. The substitution of petrol-based flocculants by natural-based ones, obtained from sugarcane residues, is presented for the first time in this study, with very relevant performance of the new flocculants. Overall, it was possible to produce anionic flocculants, modifying the cellulose obtained from different raw materials, which showed good results in the purification of sucrose, when compared with the commercial polyacrylamide normally used. It is also important to stress that, for the first time, a residue from sugarcane industry could be used with success in the purification of the sugar juice itself, which constitutes a major novelty.

Optimized Regeneration of Petunia Protoplast and Its Association with Tissue Identity Regulators

The popular ornamental plant Petunia is also a valuable model plant in tissue culture. Cellular conversions during differentiation and regeneration have been investigated using various combinations of phytohormones; however, studies on genes for reprogramming toward desired tissue identities have been limited. In this study, we isolated Petunia protoplasts and cultured them in the callus, rooting, or shooting stages, which were used to establish the optimal protoplast culture conditions and to identify genes that epigenetically function as tissue identifiers. The optimal conditions for plasmolysis and enzyme digestion to obtain healthy protoplasts were compared, in which combinations of Viscozyme, Celluclast, and Pectinex (VCP) enzymes were more efficient in isolating protoplasts when followed by 21 to 25% sucrose purification and washing processes. The filtered and washed protoplasts started to divide at 1 day and developed into colonies after 3 weeks of culture, which showed higher efficiency in the Murashige and Skoog (MS) salt culture media compared to that in the Kao and Michayluk (KM) salt media. The pluripotent colonies formed calli on the solid medium supplemented with 3% sucrose after 4 weeks, and were destined to the same cell mass, rooting, or shooting on the regeneration medium. Three epigenetic controllers, ATXR2, ATX4A, and ATX4B, were highly expressed in calli, shoots, and organs of shoots and roots, respectively, confirming that dedifferentiation and regeneration of tissue identity is plastic.

Purification of Sucrose in Sugar Beet Molasses by Utilizing Ceramic Nanofiltration and Ultrafiltration Membranes.

Molasses is a sugar mill by-product with low value that today is used primarily for animal feed. However, molasses contains large amounts of sucrose which, if purified, could be used for other purposes. In this study, purification by membrane filtration using ceramic tubular ultrafiltration (UF) and nanofiltration (NF) was examined. NF purifies sucrose by removing small compounds, whereas UF removes larger compounds. Based on our results, high filtration fluxes could be obtained, and it was possible to clean the membranes sufficiently from fouling compounds. Sucrose was separated from other compounds, but the separation efficiency was generally higher with diluted molasses compared with concentrated molasses. This could be explained by more severe fouling when filtering dilute molasses or potentially due to aggregate formations in the molasses as our analysis showed. Overall, this study shows the potential of ceramic UF and NF membranes for sucrose purification from molasses.

Sucrose purification and repeated ethanol production from sugars remaining in sweet sorghum juice subjected to a membrane separation process.

The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2gL-1 sucrose, 8.5gL-1 glucose, and 4.5gL-1 fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9gL-1) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5gL-1 and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.

Cryptosporidium genotypes and subtypes distribution in river water in Iran.

Little is known about the diversity and public health significance of Cryptosporidium species in river waters in Iran. In the present study, we determined the genotype and subtype distribution of Cryptosporidium spp. in river water samples in Iran. A total of 49 surface water samples were collected from rivers and surface water in Guilan and Tehran provinces during 2009-2010. Water samples were filtrated through a 1.2-μm pore size membrane filter or by Filta-Max filter followed by immunomagnetic separation or sucrose purification methods. Genotype and subtype of Cryptosporidium were identified by sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively. A total of 24 (48.97%) water samples were positive for Cryptosporidium species by the 18sRNA-based polymerase chain reaction (PCR)-sequencing technique. DNA sequencing revealed the presence of five species of Cryptosporidium (C. parvum, C. hominis, C. muris, C. andersoni, and C. canis) in the water samples of the study area and, to our knowledge, the first report of C. muris in Iran. The results of GP60 gene analysis showed that all C. parvum and C. hominis isolates belonged to the IId and Id subtype families, respectively. The investigated river water supplies were heavily contaminated by pathogenic species of Cryptosporidium from humans and livestock. There is potential risk of waterborne cryptosporidiosis in humans and animals.

The effect of purification method on the completeness of the immature HIV-1 Gag shell

Elucidating the structure of the immature HIV-1 Gag core is an important aspect of understanding the biology of this virus. In doing so, preservation of the fragile Gag lattice is essential. In this study, the effects of purification methods on the structural and mechanical integrity of immature HIV-1 are examined. The results show that the morphological and mechanical properties of the virion are preserved to a significantly higher degree by Iodixanol (OptiPrep) purification compared to the standard sucrose method. In conclusion, these results indicate that OptiPrep instead of sucrose purification should be employed when conducting structural studies on the HIV-1 virion.

Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria

The aim of the present study was to investigate water supplies in southern Russia and Bulgaria, in order to estimate the occurrence of Giardia and Cryptosporidium in drinking water resources from these countries. A total of 166 water samples of different origin (surface, tap, bottled, well, spring and waste water) were collected from Rostov (southern Russia), Sofia and Varna (Bulgaria) Greater Areas and screened for the detection of Giardia cysts and Cryptosporidium oocysts. The method incorporated concentration of water samples by filtration and flocculation, sucrose purification, (oo)cyst detection/identification by immunofluorescence test and differential interference contrast. Sixteen out of 166 samples (9.6%) were positive for Giardia and 30 (18.1%) positive for Cryptosporidium. Both Giardia and Cryptosporidium were detected in tap, river, well and waste waters. Giardia cysts were additionally detected in bottled water. Particularly some river, waste and well water samples were highly contaminated with (oo)cysts. This study has shown that drinking water supplies in Russia and Bulgaria are subject to contamination with Giardia and Cryptosporidium, with potential hazards for the public health.

Purification and Characterization of the Enzymes of Fructan Biosynthesis in Tubers of Helianthus tuberosus Colombia (II. Purification of Sucrose:Sucrose 1-Fructosyltransferase and Reconstitution of Fructan Synthesis in Vitro with Purified Sucrose:Sucrose 1-Fructosyltransferase and Fructan:Fructan 1-Fructosyltransferase).

Sucrose:sucrose 1-fructosyltransferase (1-SST), an enzyme involved in fructan biosynthesis, was purified to homogeneity from tubers of Helianthus tuberosus that were harvested in the accumulation phase. Gel filtration under native conditions predicted a molecular mass of about 67 kD. Electrophoresis or gel filtration under denaturing conditions yielded a 27- and a 55-kD fragment. 1-SST preferentially catalyzed the conversion of sucrose into the trisaccharide 1-kestose (GF2). Other reactions catalyzed by 1-SST at a lower rate were self-transfructosylations with GF2 and 1,1-nystose (GF3) as substrates yielding GF3 and 1,1,1-fructosylnystose, respectively, as products. 1-SST also catalyzed the removal of the terminal fructosyl unit from both GF2 and GF3, which resulted in the release of sucrose and GF2, respectively, and free Fru. The purified enzyme did not display [beta]-fructosidase activity. An enzyme mixture of purified 1-SST and fructan:fructan 1-fructosyltransferase, both isolated from tubers, was able to synthesize fructans up to a degree of polymerization of at least 13 with sucrose as a sole substrate.

Use of dodecyl-β- d-maltoside in the purification and stabilization of RNA polymerase from brome mosaic virus-infected barley

The activity and specificity of RNA-dependent RNA polymerase (replicase) isolated from brome mosaic virus-infected barley was enhanced by extraction with the nonionic detergent dodecyl-β- d-maltoside. The enzyme was stable for at least 8 weeks when stored at −70°. A further 100-fold purification was obtained by centrifugation through sucrose in the presence of detergent. The polymerase activity was associated with the pellet fraction; the template dependence and specificity were similar to those of the enzyme before sucrose purification. SDS-PAGE analysis revealed a 110-kd protein in the purified pellet fraction from infected leaves that was absent from a similar fraction from healthy leaves. The protein had an identical electrophoretic mobility to that of protein la, the product of brome mosaic virus RNA 1 translation in vitro, and the profile of its tryptic polypeptides was very similar to that of protein 1a. These results support data obtained by inoculation of protoplasts with separated BMV RNA components ( Kiberstis, et al. (1981) , Virology 112, 804–808) that are consistent with the notion that RNA 1 codes for the viral replicase, or a subunit thereof.