Choice Of Extraction Method Research Articles

Botanical Originand Extraction Methods of Philippine Stingless Bee (Tetragonula biroiFriese) Pollen and its Food Functionality: Phenolic and Flavonoid Content and Antioxidant Activity

With its high nutritional and bioactive content, bee pollen is recognized as a functional food. However, its composition is highly dependent on a range of factors, such as geographical location. In this research, we used DPPH to determine the antioxidant activity of several stingless bee (Tetragonula biroi) pollen samples sourced from five different areas in the Philippines and compare their phenolic and flavonoid content. Additionally, we compared two extraction methods: maceration (or soaking of samples) for three days and sonication for 30 minutes. Interestingly, our findings show that the phenolic content of bee pollen is weakly associated with antioxidant activity, indicating that other bioactive chemicals may play a more significant part in the antioxidant characteristics of bee pollen. Antioxidant properties based on the DPPH assay of pollen harvested from Calamba are higher compared to other samples from different geographic areas, i.e., Quezon Province, Sorsogon, Laguna, and Albay. When compared to the maceration approach, extracts generated from the sonication process had lower antioxidant activity. These findings suggest that Philippine stingless bee pollen is a potential source of bioactive compounds, and the choice of extraction method and geographic source are significant factors affecting its antioxidant activity.

Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

Solid-State 13C Nuclear Magnetic Resonance Study of Soluble and Insoluble β-Glucans Extracted from Candida lusitaniae.

β-glucans are widely known for their biological activities. However, the choice of extraction method can significantly influence their structural characteristics, thereby potentially impacting their biological functions. In this paper, three fractions of β-glucans were obtained from Candida lusitaniae yeast via alkali and hot-water extraction methods and were analyzed using solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Solid-state NMR spectroscopy was used as a nondestructive technique that preserves the structure of the analyzed molecules. The results suggest that differences in the β-glucan structure are affected by the choice of extraction method. The main difference occurred in the 82-92 ppm region with signal presence suggesting that β-glucans have a linear structure when hot-water-extracted, which is absent in alkali-extracted fractions resulting in the acquisition of β-glucans with an ordered, possibly helical structure. A hot-water extracted water-insoluble (HWN) fraction consists of linear β-1,3-glucans with other signals indicating the presence of β-1,6-linked side chains, chitin and small amounts of α-glucan impurities. For those that are alkali-extracted, alkali-insoluble (AN) and water-soluble (AWS) fractions are structurally similar and consist of an ordered β-1,3-glucan structure with β-1,6-linked side chains and a significant amount of α-glucan and chitin in both fractions.

Effect of extraction methods on the chemical, structural, and rheological attributes of fructan derived from Arctium lappa L. roots

The focus of this study was the evaluation of how extraction techniques impact the chemical, structural, and rheological attributes of fructans extracted from Arctium lappa L. roots. Three distinct extraction procedures were used, utilizing water as solvent, infusion extraction conducted at ambient temperature (25 °C for 5 min), thermal extraction employing reflux (100 °C for 2 h), and ultrasound-assisted extraction (50 °C for 1.38 h with a 158 W output). Chemical characterization by Nuclear Magnetic Resonance (NMR) and colorimetric analyses revealed the obtaining of inulin-type fructans (yield 83 %). The degree of polymerization (DP) was found to be the lowest for ultrasound-assisted extraction (14.38), followed by the room-temperature (20.41) and thermal (21.14) extraction techniques. None of the extraction techniques appeared to modify the molecular structure of the isolated compounds. In X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses, distinct crystallization patterns were observed for the room-temperature and thermal extraction techniques, though all fractions consistently exhibited characteristic bands of inulin-type fructan. Rheological assessments indicated a viscoelastic nature of the fractions, with those extracted thermally demonstrating a greater viscosity. This study shows that the choice of extraction method can influence the structural characteristics of inulin-type fructans derived from the burdock root.

Extraction of Polyphenols from Slovenian Hop (Humulus lupulus L.) Aurora Variety Using Deep Eutectic Solvents: Choice of the Extraction Method vs. Structure of the Solvent.

Polyphenols from Slovenian hops (Humulus lupulus L.) of the Aurora variety were extracted by different methods and using classical solvents and several deep eutectic solvents (DES) based on choline chloride as the hydrogen bond acceptor component. The obtained extract solutions were analyzed by HPLC for the content of extracted α- and β-acids and extracted xanthohumol. It was found that choline chloride:phenol DES concentrated aqueous solution had an extraction efficiency close to that of diethyl ether, which is considered one of the best classical extraction solvents for polyphenols from hops. The comparison of the extraction efficiency with other choline chloride-based DESs showed that the chemical similarity of the phenol ring in the solvent DES with the polyphenols in hops may be crucial for a highly efficient extraction with choline chloride:phenol DES. On the other hand, the choice of extraction method and the viscosity of the solvents tested seem to play only a minor role in this respect. As far as we know, this is the first study to attempt to relate extraction efficiency in the extraction of hydrophobic solutes to the compressibility of the DES extractants, the latter of which may be correlated with the extent of hydrophobic hydration around the DES components. In addition, using the heating and stirring method for the preparation of choline chloride-based DES concentrated aqueous solutions we found no support for the occurrence of water in two different roles (in the structural and in the dilution role) in these solvents.

Characterization and Comparative Assessment of the Essential Oil from Lime (Citrus aurantifolia) Exocarp Using Maceration and Soxhlet Extraction Methods

Citrus fruits are a rich source of essential oils that have various applications in the cosmetic, food, and pharmaceutical industries. Lime is notable for its high essential oil yield, which contains active compounds that possess antimicrobial, antioxidant, and anticancer properties. This study aimed to compare the maceration and Soxhlet extraction methods for obtaining essential oil from lime exocarp as well as characterizing the compounds in the oil using Gas Chromatography-Mass Spectrometry (GC-MS). The study found that the Soxhlet extraction method had a higher yield of oil compared to the maceration method. However, the maceration method had a lower acid value and free fatty acid content, and a higher saponification value. The oil obtained using the Soxhlet extraction method was more acidic than that of the essential oil obtained using the maceration method. The study also found that limonene was the most prominent compound in both extraction methods. However, the percentage of β-ocimene and γ-terpinene were significantly higher in the maceration method compared to the Soxhlet extraction method. Limonene, β-ocimene, and γ-terpinene are important compounds found in essential oils and have various medicinal properties. These findings have significant implications for the essential oil industry. The choice of extraction method can influence the composition of the essential oil obtained, as well as its chemical and physical properties. Therefore, it is crucial to consider the intended use of the essential oil when choosing an extraction method.

Chemical and Bioactive Screening of Green Polyphenol-Rich Extracts from Chestnut By-Products: An Approach to Guide the Sustainable Production of High-Added Value Ingredients.

Opportunities for the valorisation of agro-industrial residues of the chestnut (Castanea sativa Mill.) production chain have been fostered with the production of multifunctional polyphenol-rich extracts with the potential to be introduced as natural additives or active components in several products. Nonetheless, it is crucial to explore the feasibility of different extracts from the various by-products for these applications through the exhaustive study of their composition and bioactivities without losing sight of the sustainable character of the process. This work aimed at the screening of the phenolic compound composition and bioactivities of different green extracts of chestnut burs, shells and leaves, as the first step to establish their potential application as natural ingredients, primarily as food preservatives. To this end, maceration (MAC) as a conventional extraction method besides ultrasound and microwave-assisted extractions (UAE and MAE) was employed to obtain the extracts from chestnut by-products using water (W) and hydroethanolic solution (HE) as solvents. Phenolic compounds were analysed by HPLC-DAD-(ESI-)MS/MS; the antioxidant capacity was assessed by colourimetric assays, and the antimicrobial activity was evaluated against several strains of food-borne bacteria and fungi. The leaf extracts obtained by MAC-HE and UAE-HE presented the highest concentration of phenolic compounds (70.92 ± 2.72 and 53.97 ± 2.41 mg.g-1 extract dw, respectively), whereas, for burs and shells, the highest recovery of total phenolic compounds was achieved by using UAE-HE and UAE-W (36.87 ± 1.09 and 23.03 ± 0.26 mg.g-1 extract dw, respectively). Bis-HHDP-glucose isomers, chestanin and gallic acid were among the most abundant compounds. Bur extracts (MAC-HE and UAE-HE) generally presented the highest antioxidant capacity as measured by TBARS, while the best results in DPPH and reducing power assays were found for shell extracts (MAE-W and MAC-HE). Promising antibacterial activity was noticed for the aqueous extracts of burs, leaves and hydroethanolic extracts of shells, with emphasis on the MAE-W extract of burs that showed bactericidal activity against E. cloacae, P. aeruginosa and S. aureus (MBC 5 mg.mL-1). Overall, it can be concluded that chestnut by-products, including burs, shells and leaves, are sources of polyphenolic compounds with significant antioxidant and antimicrobial activities. The choice of extraction method and solvent greatly influenced the composition and bioactivity of the extracts. These findings highlight the potential of chestnut by-products for the development of natural additives, particularly for food preservation, while also emphasizing the importance of sustainable utilization of agricultural waste materials. Further research is warranted to optimize extraction techniques and explore additional applications for these valuable bioactive compounds.

Determination of inorganic As, DMA and MMA in marine and terrestrial tissue samples: a consensus extraction approach

Environmental context Arsenic can be found in all environmental compartments in a large number of chemical forms of varying toxicity. We performed an inter-laboratory comparison study focusing on quantitation of some of the most toxic arsenic forms in seven different biological materials and found very good agreement among the submitted results. Certification of the studied materials will provide suitable quality control samples for environmentally relevant concentrations of arsenic in food products and biota.Rationale Arsenic (As) speciation analysis in biological matrices has been performed for several decades; however, there are very few matrix certified reference materials available for the validation of analytical methods. The literature data on the mass fractions of As species in the existing certified reference materials are inconsistent and suggest method extraction dependency.Methodology In the present study, an international round-robin study was organised to identify the possible sources of discrepancies in quantitation of several As species in different matrices of biological reference materials: one plant tissue, three marine and three terrestrial biological tissues. Each participating laboratory was provided with a set of identical calibration standard solutions, and analysed the samples by following a common group extraction method as well as using an in-house protocol.Results The results showed that significant biases can be introduced by insufficient verification of the analyte’s mass fractions in the calibration standard solutions. The choice of extraction method seems to have very little impact on the quantitation of As species in the studied plant and terrestrial biological tissues. However, following a prescribed extraction method led to significant reduction of uncertainties in more complex samples such as marine animal tissues.Discussion Some differences in the mass fractions of As species extracted from marine animal tissues in water (with and without H2O2) were observed and should be further investigated. Despite the variety of extraction methods used, very good agreement between reported mass fractions was achieved and the combined consensus values will be published as certified reference values of As species in the studied materials.

Efektivitas Jenis Pelarut dan Metode Ekstraksi dari Daun Talas (Colocasia esculenta L. Schoot)

Taro leaves (Colocasia esculenta L. Schoot) are widely used as vegetables and herbal medicines. In proving taro leaves as herbal medicine, taro leaves are changed in the form ofextract preparations. The choice of extraction method will affect the results of flavonoid levelsand antioxidant activity. Extraction methods that are widely used to extract taro leaves aremaceration and soxhletation. Maceration extraction method, the sample was immersed in 1000ml of ethanol and 1000 ml of n-hexane for 3 x 24 hours. Every 24 hours replaced with a newsolvent. Extracts were made by soxhletation method using ethanol as solvent and the other usingn-hexane as solvent. The material that has been submerged in petroleum ether is wrapped infilter paper and then tied with thread. Then it is put into the socket extractor. Furthermore, theflavonoid and antioxidant levels were tested. The highest flavonoid content in taro leaves wasobtained by maceration method with ethanol solvent, as much as 12.836 mg QE/g. The highestantioxidant activity was 91.812% by maceration method and n-hexane solvent. The macerationmethod with n-hexane solvent can obtain taro leaf extract which has high antioxidant activity. Kata Kunci—Antioksidan, Colocasia esculenta, Ekstraksi, Flavonoid, Pelarut

Lentil protein: impact of different extraction methods on structural and functional properties

Plant proteins with improved solubility, foaming, and emulsifying properties are required to meet the demand for plant-based foods. This study evaluated the influence of alkaline extraction combined with enzyme- and ultrasound-assisted extraction on lentil protein structure and functionality. Enzyme- and ultrasound-assisted extractions were not capable of increasing the protein yield compared to alkaline extraction alone. However, the purity of isolated protein was dependent on the extraction process, ranging from 82.7% to 90%. Although the molecular mass, zeta potential profiles, and denaturation temperature were not dependent on the extraction method, the enthalpy of denaturation for protein obtained solely by alkaline extraction was significantly lower than that for assisted processes, indicating that protein denaturation is caused by an alkaline process. Changes in protein structure were also suggested by solubility analyses that showed that lentil proteins obtained by enzyme-assisted and ultrasound-assisted extraction have better solubility at pH 7 when compared to alkaline extraction alone. The surface activity of lentil protein was evidenced by interfacial and surface analysis, and it was influenced by the extraction process applied. We demonstrated that combining alkaline extraction with assisted processes, especially ultrasound technology, results in concentrates/isolates with higher solubility as compared to ones obtained solely by the traditional alkaline method, even though the choice of extraction method should depend on the desired functionality.

Preanalytical Variables and Sample Quality Control for Clinical Variant Analysis.

Broad molecular profiling by next-generation sequencing of solid tumors has become a critical tool for clinical decision-making in the era of precision oncology. In addition to many already approved targeted therapies, more than half of ongoing oncology-related clinical trials are biomarker-driven. Therefore, accurate and reliable assays are needed to assess the genetic make-up of tumor cells and guide clinicians in the therapy decision process. In order to obtain high-quality NGS data for variant detection, certain preanalytical steps and quality metrics should be followed. These include assessment of sample types, choice of extraction method, library preparation technology, sequencing platform, and finally sequencing quality control. Each of these steps has certain challenges and pitfalls that need to be addressed and overcome, respectively. In this chapter, we address the preanalytical quality control and how each of the involved steps may influence the final result. Following these guidelines and QC metrics may help in obtaining optimalresults that will allow the precise and robust assessment of genetic variants in a clinical setting.

Application of environmental DNA for monitoring and management of aquatic biological invasions: Emerging trends and advancements towards best practice

Introduction Aquatic Invasive Species (AIS) are a growing concern for global biodiversity as humans continue to accelerate the transport of non-indigenous species beyond their natural range. These species may possess traits that allow them to thrive in new environmental conditions such as non-selective feeding and high reproductive output, causing ecological harm through competition with native species for limited local resources. Consequently, environmental DNA (eDNA) has come to the forefront of AIS management in recent years as a promising method to detect or monitor invasive species using rapid and non-invasive sampling to complement traditional surveying. As eDNA’s potential is explored and beginning to be adopted for a variety of applications around the world, it is increasingly important to synthesize the trends in field and laboratory protocols from different working groups to establish guidelines that will allow greater comparability between studies and improve experimental design. Methodology and Results This meta-analytic study collated and reviewed information from previously published eDNA studies that targeted AIS in freshwater and marine environments to recognize current patterns in sampling techniques, laboratory protocols, and potential geographic or taxonomic biases. A total of 492 records from 192 full-text articles were used in the analysis, composed of 408 species-specific and 84 metabarcoding records. With regards to sampling procedures, many studies were not explicit enough for true replicability, lacking critical information such as the volume of filtered water and details of storage conditions. There was no observable trend for eDNA extraction methods in either species-specific or metabarcoding approaches, with choice of extraction method being mostly arbitrary among laboratories as well as influenced by the recent emergence of dedicated commercial kits . Discussion This analysis revealed a wide variety of choices for collecting and processing eDNA samples, so it is recommended that there should be some sort of standardized workflow diagram or decision tree for every stage of the experimental design in order for researchers to determine what approaches best meet their research objectives. There is also a clear need for improving metadata reporting guidelines; although the relevance of some criteria depends on the goals and limitations of specific projects, there should be a standardized minimum set of parameters to be reported for each eDNA study, from environmental variables to decontamination practices to PCR conditions. This will increase consistency and transparency through all stages of eDNA research, which is key to collectively improving methodologies and moving forward in this field.

Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Comparison of extraction methods for per- and polyfluoroalkyl substances (PFAS) in human serum and placenta samples\u2014insights into extractable organic fluorine (EOF)

Since the detection of per- and polyfluoroalkyl substances (PFAS) in humans and different environmental media in the last two decades, this substance group has attracted a lot of attention as well as increasing concerns. The fluorine mass balance approach, by comparing the levels of targeted PFAS after conversion to fluorine equivalents with those of extractable organic fluorine (EOF), showed the presence of unidentified organofluorine in different environmental samples. Out of the thousands of PFAS in existence, only a very small fraction is included in routine analysis. In recent years, liquid chromatography coupled with tandem-mass spectrometry (LC-MS/MS) has demonstrated the ability to analytically cover a wide spectrum of PFAS. In contrast, conventional extraction methods developed 10 to 15 years ago were only evaluated for a limited number of PFAS. The aim of the present study was to evaluate the advantages and disadvantages of three different extraction methods, adapted from the literatures without further optimization (ion-pair liquid-liquid extraction, solid-phase extraction (SPE), using hydrophilic-lipophilic (HLB) or weak anion exchange (WAX) sorbents), for human biomonitoring of 61 PFAS in serum and placental tissue samples. In addition, levels of EOF were compared among these extraction methods via spiked samples. Results showed that performance, in terms of recovery, differed between the extraction methods for different PFAS; different extraction methods resulted in different EOF concentrations indicating that the choice of extraction method is important for target PFAS and EOF analysis. Results of maternal serum samples, analyzed in two different laboratories using two different extraction methods, showed an accordance of 107.6% (± 21.3); the detected perfluoroalkyl acids (PFAAs) in maternal and cord serum samples were in the range of 0.076 to 2.9 ng/mL.Graphical abstract

Development of RT-qPCR assays for the detection of three latent viruses of pome

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

Choice of Extraction Method for Receiving Extraction from Seeds of Payne Hay with the High Content of Biologically Active Substances

Introduction. According to the literature, modern technologies, in particular, extraction in an ultrasonic field, make it possible to obtain concentrates of biologically active substances (BAS) with almost complete preservation of the chemical composition inherent in natural raw materials and high yield of extractive substances. At the same time, the ability to regulate the concentration of recoverable active substances during the technological process opens up prospects for the use of natural components as the main pharmaceutical substance.Aim. The purpose of this study was to select the optimal extraction method for obtaining extracts with a high content of BAS from fenugreek seeds. Materials and methods. Extracts were obtained at a temperature of 60±2 °С, by the method of dynamic maceration using an ultrasonic device. Ultrasonic extraction was performed using an ultrasonic installation I100-6/4. In both cases, purified water and ethanol solutions in various concentrations from 40 to 90% were used as extractants. The ratio of raw materials: extractant was as 1:10 (by weight). A portion of vegetable raw materials (10 g) was placed in a glass and poured 100 cm3 of extractant. Next, maceration or sonication was performed. Maceration was performed with magnetic stirring, the rotation speed was 100 rpm. Extraction was carried out at a temperature of 55–60 °C for 5 hours. Ultrasonic effects on solid plant materials were carried out with an intensity in the range from 17 to 22 kHz for 30–60 minutes.Results and discussion. Studies have allowed to determine the amount of extractives in the seeds of fenugreek hay, and choose the most promising method of extraction to obtain extracts with a high content of biologically active substances. A comparison was made between dynamic maceration and ultrasonic extraction. The most promising was the method of ultrasonic extraction at an oscillation frequency of 22 kHz. For 1 hour of extraction, it was possible to achieve depletion of raw materials. It is established that the content of extractable substances directly proportional depends on the duration of treatment and the frequency of ultrasonic waves. The correlation coefficient was 0.78.Conclusion. Ultrasonic extraction when exposed for 60 minutes allows you to get a greater amount of extractives compared with dynamic maceration. To achieve the same level of extractive substances by the method of dynamic maceration, the raw materials must be processed within 5 hours.

Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis.

BackgroundIn recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies.ResultsIn this study we conducted a systematic comparison of the DNA extraction methods used by the two major collaborative efforts: The European MetaHIT and the American Human Microbiome Project (HMP). Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol.ConclusionsWhereas it is comforting that the inter-individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies.

A comparison of molecular methods for hepatitis B virus (HBV) DNA detection from oral fluid samples

The objective of the present study was to evaluate four commercial DNA extraction methods and three PCR protocols for hepatitis B virus (HBV) detection in artificially contaminated oral fluid samples. The extraction protocols were selected based on ease of use and cost, and were also compared with respect to sensitivity and cost. Prior PCR optimization was conducted, in which the sample volume for DNA extraction and the concentrations of DNA and Taq DNA polymerase in the PCR were adjusted. One-round PCR, used to amplify the core region of the HBV genome, achieved high levels of sensitivity in comparison with nested and semi-nested PCR experiments that were designed for the amplification of HBV surface protein genes. Of the four extraction protocols evaluated, the RTP DNA/RNA Virus Mini kit and the QIAamp DNA Mini kit gave the highest recovery rates, presenting 20 copies of HBV DNA ml(-1) as the limit of detection. These results suggest that HBV DNA can be detected from oral fluid samples but that the optimization of the PCR assays and the choice of extraction methods must be determined by laboratories before the implementation of this method in routine diagnostics.

An optimised sample preparation method for NMR-based faecal metabonomic analysis

Faecal metabonomic NMR analysis plays an essential role in investigating the interactions between mammalian metabolism and symbiotic gut microbiota. However, the faecal metabolite extraction method remains to be optimised and standardised to take into consideration signal-to-noise ratios, pH and chemical shift consistency. In the current investigation, we compared extraction consistency of three homogenisation methods including manual ultrasonication, automatic homogenization with tissuelyser and their combination, and systematically optimised faecal metabolite extraction parameters, including the faeces-to-buffer ratio (W(f) : V(b)), extraction repetition times and duration. We found that automatic homogenisation with tissuelyser was the choice of extraction method owning to its good metabolite extraction consistency and high throughput. We also recommend W(f) : V(b) of 1 : 10 (mg microl(-1)) and use of the combined first two extracts as the resultant samples to represent faecal metabolite composition. Such recommendation is based on considerations of maximisation of the spectral signal-to-noise ratio, pH and chemical shift consistency, completeness of metabolite extraction and sample preparation throughput so that the method is suitable for analysing a large number of samples especially in human population studies.