Acid Isolation Methods Research Articles

Characteristics and Applications of Bionanosilica from Betung Bamboo Leaves

Nanoparticles are materials that are currently widely used in research due to their novelty and the growing number of suitable applications. Silica nanoparticles can be produced by synthesizing using several methods such as melting, coprecipitation, sol-gel, and ultrasonication. The aim of this study is to determine the most appropriate synthesis method for the production of SiO₂ nanoparticles to optimize the quality of physical properties of fast-growing wood. The synthesis of SiO2 nanoparticles used in this study utilized three different methods: acid isolation method (F1), sol-gel method (F2), and reflux method (F3). Characterization of SiO2-NPs was performed using particle size analyzer (PSA), X-ray diffraction analysis (XRD), and Fourier transform infrared spectroscopy (FTIR). The results of PSA analysis showed that the acid isolation method produced the smallest SiO2 particle size compared to the sol-gel and reflux methods. The zeta pontential value in each method shows that the particles produced are unstable because the potential zeta value produced is around -10 mV to -30 mV. The results of FTIR and XRD analysis show that the synthesized material is a SiO₂ compound with a cristobalite phase. Application of the material on jabon wood through impregnation showed an improvement in physical properties, including an increase in WPG, density, and BE, especially in the sol-gel method (F2).

Testing of the Effect of Food Additives on the DNA Isolation from Mackerel and Fish Products

Authentication of fish products by DNA analysis requires the extraction of high quality DNA without the presence of inhibitors. Many nucleic acid isolation methods are currently available; silicate centrifugal columns have become very popular due to their speed and ease of extraction. However, their disadvantage may be the principle based on DNA charge, which may be affected by food composition, or clogging due to a poor sample pretreatment. The aim of this work was to compare three DNA isolation methods using different principles (silicate centrifugal columns, modified magnetic beads, Cetrimonium bromide and chloroform extraction) and to evaluate their suitability for DNA isolation from fish muscle. The criteria assessed were the recovery, purity and amplifiability of the isolated DNA. Mackerel tissue was analysed without and with the addition of additives commonly used in the manufacture of fish products, namely diphosphates (E 450) and colorants (E 110 and E 124), and the selected method was subsequently applied to commercial fish products. The modified method using the detergent CTAB proved to be the most suitable.

Nutritional Value and Structure Characterization of Protein Components of Corylus mandshurica Maxim.

Alternative protein sources for the human diet may help overcome the growing food pressure. Plants with abundant resources and high protein content are potential sources. In this article, graded proteins and isolated proteins from Corylus mandshurica Maxim kernels were extracted by the Osborne procedure and the alkali-solution and acid-isolation method, respectively, and the contents of the five proteins, and the differences in nutritional value and structural properties of the main proteins, were investigated. Amino acid analysis revealed that the total essential amino acids in the five proteins ranged from 249.58 to 324.52 mg/g. The essential amino acid profiles in the proteins were similar to those of FAO/WHO except for the alcohol-soluble protein. The essential amino acid indices ranged from 58.59 to 72.19 and the biological values ranged from 52.16 to 66.99, and the highest nutritional indices were found for the isolate and water-soluble protein, which were 41.68 and 55.78, respectively. The molecular weight pattern distribution of the protein isolates of the Corylus mandshurica Maxim kernel was more similar to that of the water-soluble proteins by SDS-PAGE. The β-sheet and α-helix were the main secondary structures in the two protein fractions. The fluorescence spectra showed that the maximum fluorescence intensity of the two proteins and their λmax were also somewhat different. From the perspective of microscopic morphology, the two proteins are mainly compact and irregular lamellar structures, but the surface of the water-soluble protein is more flat and regular. Both proteins have good solubility, and the water-soluble protein has higher solubility. In general, the protein isolates of the Corylus mandshurica Maxim kernel and the water-soluble protein showed their potential as plant protein resources.

Optimization of Deoxyribonucleic Acid (DNA) isolation methods from several types of cosmetic samples for molecular-based halal tests

Bagi seorang muslim status halal suatu produk pangan maupun non-pangan harus terpenuhi secara mutlak, begitu juga dengan produk kosmetik. Kosmetik terdiri dari berbagai bahan dan yang menjadi titik kritis status kehalalan suatu produk kosmetik adalah kompleksitas komposisi produk kosmetik yang memungkinkan penggunaan bahan dasar yang berasal dari babi seperti gelatin, asam lemak, gliserin dan kolagen yang mana bahan-bahan tersebut adalah bahan yang sangat umum digunakan dalam pembuatan produk kosmetik. Tujuan dilakukannya penelitian ini adalah untuk untuk mengoptimasi metode isolasi DNA pada beberapa jenis sampel kosmetik untuk uji halal berbasis molekuler. Penelitian dilakukan dalam tiga tahap, yaitu ekstraksi DNA, amplifikasi dan analisis rt-PCR, dan tahap terakhir adalah visualisasi dengan media elektroforesis gel agarose. Optimasi isolasi DNA dilakukan dengan tiga metode yang berbeda, yaitu dengan menggunakan kit isolasi DNA QIAamp® DNA Mini Kit (QIAGEN), isolasi dengan laruta phenol-chlororoform GENEzol™ (Geneaid), dan menggunakan campuran resin Chelex-TE 10%. Hasil menunjukkan bahwa metode isolasi terbaik dari ketiga metode yang dilakukan adalah metode isolasi DNA dengan menggunakan QIAamp® DNA Mini Kit (QIAGEN) yang mana berdasarkan visualisasi elektroforesis metode ini dapat mengisolasi DNA dari semua sampel.

Preparation and quality of okara protein gel gummies

Okara protein (OP) was extracted by alkali solution and acid-isolation method using bean dregs as raw materials. With OP as the main raw material, the mixed gelatin and modified starch as the gelling agent, xylitol as the sweetener, and buffered lactic acid as the sour agent, the elasticity, hardness, stickiness and chewiness of the texture analyzer were analyzed by the single-factor test and orthogonal test. Combined with sensory evaluation, the optimal formula for the preparation of OP gel gummies was comprehensively obtained as: the ratio of gelatin to modified starch was 9:1; the xylitol addition was 18 g; the addition of buffered lactic acid was 0.7mL, and the addition of OP solution was 16mL, respectively. The gel gummies prepared under this formula have the advantages of suitable sweetness and sourness, good taste, good stickiness and uniform color. It also has special physiological functions, such as immune regulation and antihypertensiveness.

Implementation of <i>in-house</i> Methods for Isolating Fungal DNA of Clinical Samples

Over 200 species of fungi are responsible for a variety of infections that can occur in various parts of the human body. There are several phenotypic methods for identifying these fungal elements; however, these approaches have limitations. Molecular methods are now routinely used in well-equipped mycology laboratories. However, the first step in isolating genetic material can often be costly, can suffer from external DNA contamination and some components have toxicity for personnel. The general objective of this work was to identify the best local method for isolating genetic material from fungi based on cost, yield and time-consuming criteria. A total of ten (10) different nucleic acid isolation methods were tested. Those tests using thermal or mechanical shock for cell lysis delivered better quality than those using chemical lysis. Thus, based on our criteria, the best methods for nucleic acid isolation and purification of fungal elements were cetyltrimethylammonium bromide (CTAB) combined with sterilized sea sand (less expensive) and the chelator Chelex® coupled with glass beads (faster).

Easy Express Extraction (TripleE)-A Universal, Electricity-Free Nucleic Acid Extraction System for the Lab and the Pen.

The complexity of the current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method (easy express extraction, called TripleE) as a centrifugation-free and electricity-free nucleic acid isolation method. The procedure is based on the well-established magnetic-bead extraction technology using an in-house self-made magnetic 8-channel and a rod cover. With this extraction system, nucleic acids can be isolated with two simple and universal protocols. One method was designed for the extraction of the nucleic acid in resource-limited “easy labs”, and the other method can be used for RNA/DNA extraction in the field for so-called molecular “pen-side tests”. In both scenarios, users can extract up to 8 samples in 6 to 10 min, without the need for any electricity, centrifuges or robotic systems. In order to evaluate and compare both methods, clinical samples from various viruses (African swine fever virus; lumpy skin disease virus; peste des petits ruminants virus; bluetongue virus), matrices and animals were tested and compared with standard magnetic-bead nucleic acid extraction technology based on the KingFisher platform. Hence, validation data were generated by evaluating two DNA viruses as well as one single-stranded and one double-stranded RNA virus. The results showed that the fast, easy, portable and electricity-free extraction protocols allowed rapid and reliable nucleic acid extraction for a variety of viruses and most likely also for other pathogens, without a substantial loss of sensitivity compared to standard procedures. The speed and simplicity of the methods make them ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings.

Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.

Preliminary study on protein isolation process of walnut dregs by single factor experiments and response surface method

The production of protein by alkali-solution and acid-isolation method will lead to the increase of salt in the feed liquid and affect its quality. In order to simplify the production of protein by the alkali acid precipitation process and obtain protein with high content and quality, the cation exchange resin was used to separate the walnut dreg protein. The desalination rate, protein content and recovery rate were used as indicators. Single factor experiments and response surface method were used to optimize the time used to isolate the protein from the cation exchange resin, the speed and temperature of the water bath shaker. Our results showed that the optimal time was determined at 62 min; the optimal speed of the water bath shaker was 120 rpm, and the optimal temperature was 49 °C. Under these conditions, the optimal protein recovery was 95.22% and the optimal protein content was 78.12%.

Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks.

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.

Optimizing the Synthesis of Lignin Derivatives from &lt;em&gt;Acacia mangium &lt;/em&gt;to Improve the Enzymatic Hydrolysis of Kraft Pulp Sorghum Bagasse

The present study is aimed at optimizing the synthesis of Amphipilic lignin derivatives (A-LD) from the isolated lignin of A.mangium black liquor (BL), using the one and two step acid isolation method, and commercial lignin (LS) was used as comparison. The experimental design was conducted using Taguchi method, which consisted of four parameters and two level factors, with reference to the matrix orthogonal array, L8, including temperature, reaction time, amount of polyethylene glycol diglycidylethers (PEGDE) and Kraft lignin (KL). Furthermore, the kraft pulp of sweet sorghum bagasse (SSB) was used as substrate in the enzymatic hydrolysis (NREL method), with addition of A-LD, whose functional group and surface tension were then characterised using ATR-FTIR and surface tension equipment. Conversely, an improvement in the reducing sugar yield (RSY) compared to the control was observed after adding various A-LDs to the substrate during enzymatic hydrolysis. This product was more prospective for L2S than others products under milder circumstances, due to the fact that it possesses the lowest surface tension. Also, Taguchi analysis demonstrated the treatment at 60 °C for 1 h with 3.0 g and 1.0 g of PEDGE and lignin, respectively as the optimum condition, while the amount of lignin present was included as a factor with the propensity to significantly affect A-LD L1S and LS. Therefore, it was established that the A-LDs from A. mangium kraft lignin require milder synthesis conditions, compared to other existing methods and despite the differences in optimum experimental condition for L2S and LS, the functional groups in the IR spectra possessed very identical characteristics.

Синтез молочной кислоты грибом Rhizopus oryzae F-1030 на питательных средах из сульфитных щелоков

Lactic acid is an industrially important product with an expanding consumer market. However, lactic acid production and isolation methods used at the present time are not effective enough, lead to the formation of large amounts of polluting waste and their recycling is economically unproftable. At the same time, a half of the worldʼs lactic acid production is carried out by the microbiological method based on the fermentation of such costly sugarcontaining substrates as sucrose, molasses, treacle, sugar syrup, etc. These sugar-containing substrates usage signifcantly increases the fnal product cost. In order to solve the economicand environmental problems of lactic acid production it is necessary to revise the current raw material source and put cheaper and readily available sources of carbohydrates, such as sulphite liquor formed during sulphite pulping, into the lactic acid production. In turn to enhance the economic efciency of the Russian pulp and paper production it is necessary to use such paper production by-products as sulphite liquor to the fullest extent possible. Sulphite liquor is a chemical complex of inorganic and organic compounds such as monoand oligosaccharides. The article considers the dependence of the output of lactic acidsynthesized on the sulphite liquor medium by the fngus R. oryzae F-1030 on the used method of cultivation. In case of the semicontinuous culture method, the culture liquid was replaced with the similar volume of the sterile growth medium with the fungus biomass saving when the sugars in the medium were depleted. In case of the batch culture method, the synthesized lactic acid was precipitated with calcium hydroxide and the reducing substances recovery in the culture liquid was achieved by adding concentrated sulphite liquor when the sugars in the medium were depleted. The study demonstrates that the largest amount of synthesized lactic acid is obtained when using the semicontinuous method for cultivation of the fngus on the sulphite liquor medium prepared according to the technology recommended in the industry during preparation growth media for yeast cultivation. If it is impossible to carry out a full industrial pre-treatment of sulphite liquor, it is recommended to use the batch culture method for the fungus in order to obtain more synthesized lactic acid. For citation: Mingazova L.A., Kanarsky A.V., Kryakunova E.V., Kanarskaya Z.A. Lactic Acid Synthesis by the Fungus Rhizopus Oryzae F-1030 on Growth Media Based on Sulphite Liquors. Lesnoy Zhurnal [Russian Forestry Journal], 2020, no. 2, pp. 146–158. DOI: 10.37482/0536-1036-2020-2-146-158

The synthesis of titanium dioxide nanoparticles from titanium slag and its use for low temperature SCR catalyst

The titanium dioxide nanoparticles with high purity and specific surface area were synthesized through an alkali-solution and acid-isolation method with titanium slag as raw material. As a catalyst support of V2O5-WO3-MoO3/TiO2 selective catalytic reduction catalyst, the high specific surface area TiO2 nanoparticles can improve the low temperature activity of the catalyst and enhance its resistance to sulfur poisoning.

Recyclable One-Step Extraction and Characterization of Intact Melanin from Alpaca Fibers

Melanin, a ubiquitous natural pigment, has attracted much attention in energy and nanotechnology areas in recent years. With the increasing demand for high-quality melanin materials and sustainable experimental environment, this study was conducted to achieve melanin with intact structure through recyclable extraction method using ionic liquid (IL). Melanin extracted from base dissolution and acid isolation (BA) method and acid hydrolysis (AH) method were also used for comparison. SEM and 13C-NMR characterizations indicated that IL treatment has retained the original structure of melanin while other methods caused particle damage to various degrees. In addition, the recycled IL (up to five times) showed good reproducibility, and the performance of extracted melanin at the 5th cycle was well-maintained.

Automated nucleic acid isolation methods for HDV viral load quantification can lead to viral load underestimation.

HDV infection is a cause of severe liver disease. Diagnosis and monitoring of HDV RNA are important to patient management. Since 2012, a WHO standard for HDV RNA quantification has been available; however, the impact of RNA extraction methods on HDV viral load quantification has never been evaluated. The aim of this study was to compare four commonly used automated nucleic acid (NA) extraction methods (AmpliPrep, MagNA Pure, QIAcube QBK and QIAcube VRK) with a manual RNA extraction method (Instant Virus RNA/DNA kit) and evaluate the possible effect of each method on HDV RNA yield with subsequent amplification with the Robogene HDV assay. Serum samples from HDV-positive patients taken before treatment with pegylated interferon-α2a and at treatment weeks 12 and 48 were studied. The automated extraction methods MagNA Pure, Ampliprep and QIAcube VRK extraction led to about 10-fold lower HDV RNA values compared with the manual method of NA extraction, while the difference was smaller with QIAcube QBK (about 6-fold lower). The median viral load was 10,665 IU/ml for the manual method, 445 IU/ml for AmpliPrep, 3,209 IU/ml for MagNA Pure, 2,060 IU/ml for QIAcube QBK and 3,568 IU/ml for QIAcube VRK. Use of MagNA Pure led to misclassification of two on-treatment samples with low viral load as being false negative. The NA extraction method had a significant impact on the measured HDV viral loads determined by the commonly used Robogene assay, with the manual RNA method yielding consistently higher values of viral load. ClinicalTrials.gov Identifier: NCT00932971.

Cellulose Nanostructures Obtained from Waste Paper Industry: A Comparison of Acid and Mechanical Isolation Methods

The paper industry primary waste is usually deposited in landfills or incinerated. This material has a high content of lignocellulosic components, which could be converted to cellulose nanostructures (CNS). This study aimed to compare compositional (Fourier transform infrared spectroscopy), thermal (thermogravimetric analysis), morphological (scanning electron microscopy) and dimensional (dynamic light scattering and atomic force microscopy) properties of CNS obtained through chemical and mechanical isolation processes. The FTIR results showed similar chemical bonding for both nanostructures, which indicated presence of cellulose and lignin. Their average size was 170 nm and 209 nm for chemical and mechanical processes, respectively. The morphology of SEM images showed a compact structure and the chemical isolation presents smaller CNS agglomeration than mechanical. TGA results showed higher thermal stability for CNS-chemical than CNS-mechanical samples and AFM images indicate the CNS morphologies, which showed nanoroads for CNS-chemical and nanoneedles for CNS-mechanical.

A novel tertiary prep-HPLC method for the isolation of single amino acids for AMS-radiocarbon measurement.

AMS-radiocarbon measurements of amino acids can potentially provide more reliable radiocarbon dates than bulk collagen analysis. Nonetheless, the applicability of such an approach is often limited by the low-throughput of existing isolation methods and difficulties in determining the contamination introduced during the separation process. A novel tertiary prep-HPLC amino acid isolation method was developed that relies on the combustion of eluted material without requiring any additional chemical steps. Amino acid separation was carried out using a gradient mix of pure water and phosphoric acid with an acetonitrile step in-between runs to remove hydrophobic molecules from the separation column. The amount of contaminant carbon and its 14C content were determined from two-point measurements of collagen samples of known 14C content. The amount of foreign carbon due to the isolation process was estimated at 4±1μg and its 14C content was 0.43±0.01 F14C. Radiocarbon values corrected for carbon contamination have only a minor increase in uncertainties. For Holocene samples, this corresponds to an added uncertainty typically smaller than 10 14Cyears. The developed method can be added to routine AMS measurements without implying significant operational changes and offers a level of measurement uncertainty that is suitable for many archaeological, ecological, environmental, and biological applications.

Effect of ribonucleic acid (RNA) isolation methods on putative reference genes messenger RNA abundance in human spermatozoa.

Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.

Transcriptomic profiling of intestinal epithelial cells in response to human, bovine and commercial bovine lactoferrins.

Lactoferrin (Lf) is an iron-binding glycoprotein present in high concentration in human milk. It is a pleiotropic protein and involved in diverse bioactivities, such as stimulation of cell proliferation and immunomodulatory activities. Lf is partly resistant to proteolysis in the gastrointestinal tract. Thus, Lf may play important roles in intestinal development. Due to differences in amino acid sequences and isolation methods, Lfs from human and bovine milk as well as commercially available bovine Lf (CbLf) may differ functionally or exert their functions via various mechanisms. To provide a potential basis for further applications of CbLf, we compared effects of Lfs on intestinal transcriptomic profiling using an intestinal epithelial cell model, human intestinal epithelial crypt-like cells (HIEC). All Lfs significantly stimulated proliferation of HIEC and no significant differences were found among these three proteins. Microarray assays were used to investigate transcriptomic profiling of intestinal epithelial cells in response to Lfs. Selected genes were verified by RT-PCR with a high validation rate. Genes significantly regulated by hLf, bLf, and CbLf were 150, 395 and 453, respectively. Fifty-four genes were significantly regulated by both hLf and CbLf, whereas 129 genes were significantly modulated by bLf and CbLf. Although only a limited number of genes were regulated by all Lfs, the three Lfs positively influenced cellular development and immune functions based on pathway analysis using IPA (Ingenuity). Lfs stimulate cellular and intestinal development and immune functions via various signaling pathways, such as Wnt/β-catenin signaling, interferon signaling and IL-8 signaling.

Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells

Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12–20% of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells.