Bead Homogenization Research Articles

Optimization of lung tissue pre-treatment by bead homogenization for subsequent culturomics

The discovery that the lung harbors a diverse microbiome, as revealed by next-generation sequencing, has significantly altered our understanding of respiratory health and disease. Despite the association between the lung microbiota and disease, the nature of their relationship remains poorly understood, and culture isolation of these microorganisms could help to determine their role in lung physiology. Current procedures for processing samples from the lower respiratory tract have been shown to affect the viability of microorganisms, so it is crucial to develop new methods to improve their survival. This study aimed to improve the isolation and characterization of lung microorganisms using a bead-beating homogenization method in a mouse model. Microsphere diameter and bead-beating time affected the survival of the microorganisms (E. coli, S. aureus and C. albicans). Using 2.3 mm diameter microspheres for 60 s of bead-beating promoted the survival of both bacteria and yeast strains. After intratracheal instillation of these microorganisms in mice, approximately 70% of the cells were recovered after the tissue homogenization. To assess the efficiency of the proposed method, the diversity of bacteria was compared between the homogenate and lung tissue samples. Ninety-one genera were detected in the lung tissue, and 63 in the homogenate. Bacterial genera detected in the homogenate represented 84% of the total abundance of the microbiota identified in the lung tissue. Taken together, these results demonstrate that the tissue homogenization process developed in this study recovered the majority of the microorganisms present in the lung. This study presents a bead-beating homogenization method for effective cultivation of lung tissue microorganisms, which may help to improve the understanding of host–microbe interactions in the lung.

OpenCell: A low-cost, open-source, 3-in-1 device for DNA extraction.

High-cost DNA extraction procedures pose significant challenges for budget-constrained laboratories. To address this, we introduce OpenCell, an economical, open-source, 3-in-1 laboratory device that combines the functionalities of a bead homogenizer, a microcentrifuge, and a vortex mixer. OpenCell utilizes modular attachments that magnetically connect to a central rotating brushless motor. This motor couples to an epicyclic gearing mechanism, enabling efficient bead homogenization, vortex mixing, and centrifugation within one compact unit. OpenCell's design incorporates multiple redundant safety features, ensuring both the device's and operator's safety. Additional features such as RPM measurement, programmable timers, battery operation, and optional speed control make OpenCell a reliable and reproducible laboratory instrument. In our study, OpenCell successfully isolated DNA from Spinacia oleracea (spinach), with an average yield of 2.3 μg and an A260/A280 ratio of 1.77, demonstrating its effectiveness for downstream applications such as Polymerase Chain Reaction (PCR) amplification. With its compact size (20 cm x 28 cm x 6.7 cm) and lightweight design (0.8 kg), comparable to the size and weight of a laptop, OpenCell is portable, making it an attractive component of a 'lab-in-a-backpack' for resource-constrained environments in low-and-middle-income countries and synthetic biology in remote field stations. Leveraging the accessibility of 3D printing and off-the-shelf components, OpenCell can be manufactured and assembled at a low unit cost of less than $50, providing an affordable alternative to expensive laboratory equipment costing over $4000. OpenCell aims to overcome the barriers to entry in synthetic biology research and contribute to the growing collection of frugal and open hardware.

Immunoassay testing forbarbiturates using alternative matrices inpostmortem tissues from cats anddogs.

The barbiturate drug pentobarbital is commonly used by veterinarians for the euthanasia of domestic animals. During the veterinary forensic autopsy, it is sometimes necessary to determine whether the animal was chemically euthanized with pentobarbital. The use of a human immunochromatographic test for barbiturate screening utilizing dog or cat urine has been previously validated; however, the use of alternative matrices for this purpose is yet to be explored when urine is not available. Postmortem heart, liver, spleen, skeletal muscle, blood and/or urine samples from 20 dogs and 26 cats with a reported chemical euthanasia status were processed using two different methods, bead homogenization and sonication, and screened for barbiturates using a human immunochromatographic test. There was 100% agreement of the immunochromatographic test results using the sonication method with the reported euthanasia status of both dogs and cats. Using the bead homogenization method, agreement with the reported euthanasia status was 93.3% and 96.7% for dogs and cats, respectively, due to invalid test results from four dog and two cat samples. A subset of liver samples (10 canine and 10 feline) was analyzed via gas chromatography-mass spectrometry, and there was 100% agreement between the immunochromatographic test results and gas chromatography-mass spectrometry results for both cats and dogs. Overall, our results support the use of a variety of alternative matrices for barbiturate screening in cats and dogs.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Evaluation of extraction methods for untargeted metabolomic studies for future applications in zebrafish larvae infection models

Sample preparation in untargeted metabolomics should allow reproducible extractions of as many molecules as possible. Thus, optimizing sample preparation is crucial. This study compared six different extraction procedures to find the most suitable for extracting zebrafish larvae in the context of an infection model. Two one-phase extractions employing methanol (I) and a single miscible phase of methanol/acetonitrile/water (II) and two two-phase methods using phase separation between chloroform and methanol/water combinations (III and IV) were tested. Additional bead homogenization was used for methods III and IV (III_B and IV_B). Nine internal standards and 59 molecules of interest (MoInt) related to mycobacterial infection were used for method evaluation. Two-phase methods (III and IV) led to a lower feature count, higher peak areas of MoInt, especially amino acids, and higher coefficients of variation in comparison to one-phase extractions. Adding bead homogenization increased feature count, peak areas, and CVs. Extraction I showed higher peak areas and lower CVs than extraction II, thus being the most suited one-phase method. Extraction III and IV showed similar results, with III being easier to execute and less prone to imprecisions. Thus, for future applications in zebrafish larvae metabolomics and infection models, extractions I and III might be chosen.

Characterizing the Host Coral Proteome of Platygyra carnosa Using Suspension Trapping (S-Trap).

Stony corals form the foundation of coral reefs, which are of prominent ecological and economic significance. A robust workflow for investigating the coral proteome is essential in understanding coral biology. Here we investigated different preparative workflows and characterized the proteome of Platygyra carnosa, a common stony coral of the South China Sea. We found that a combination of bead homogenization with suspension trapping (S-Trap) preparation could yield more than 2700 proteins from coral samples. Annotation using a P.carnosa transcriptome database revealed that the majority of proteins were from the coral host cells (2140, 212, and 427 proteins from host coral, dinoflagellate, and other compartments, respectively). Label-free quantification and functional annotations indicated that a high proportion were involved in protein and redox homeostasis. Furthermore, the S-Trap method achieved good reproducibility in quantitative analysis. Although yielding a low symbiont:host ratio, the method is efficient in characterizing the coral host proteomic landscape, which provides a foundation to explore the molecular basis of the responses of coral host tissues to environmental stressors.

Current Pretreatment/Cell Disruption and Extraction Methods Used to Improve Intracellular Lipid Recovery from Oleaginous Yeasts.

The production of lipids from oleaginous yeasts involves several stages starting from cultivation and lipid accumulation, biomass harvesting and finally lipids extraction. However, the complex and relatively resistant cell wall of yeasts limits the full recovery of intracellular lipids and usually solvent extraction is not sufficient to effectively extract the lipid bodies. A pretreatment or cell disruption method is hence a prerequisite prior to solvent extraction. In general, there are no recovery methods that are equally efficient for different species of oleaginous yeasts. Each method adopts different mechanisms to disrupt cells and extract the lipids, thus a systematic evaluation is essential before choosing a particular method. In this review, mechanical (bead mill, ultrasonication, homogenization and microwave) and nonmechanical (enzyme, acid, base digestions and osmotic shock) methods that are currently used for the disruption or permeabilization of oleaginous yeasts are discussed based on their principle, application and feasibility, including their effects on the lipid yield. The attempts of using conventional and “green” solvents to selectively extract lipids are compared. Other emerging methods such as automated pressurized liquid extraction, supercritical fluid extraction and simultaneous in situ lipid recovery using capturing agents are also reviewed to facilitate the choice of more effective lipid recovery methods.

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Retrospective Validation of a 168-Gene Expression Signature for Glioma Classification on a Single Molecule Counting Platform.

Simple SummaryMolecular classification of cancers has the potential to automate and decrease errors in cancer classification. We previously showed that transcriptomic classification is comparable to methylomic and mutation methods for glioma classification and may provide benefit in predicting survival prognosis. Here we validate the transcriptomic classification method on a single molecule counting gene expression platform using formalin-fixed paraffin embedded samples.Gene expression profiling has been shown to be comparable to other molecular methods for glioma classification. We sought to validate a gene-expression based glioma classification method. Formalin-fixed paraffin embedded tissue and flash frozen tissue collected at the Augusta University (AU) Pathology Department between 2000–2019 were identified and 2 mm cores were taken. The RNA was extracted from these cores after deparaffinization and bead homogenization. One hundred sixty-eight genes were evaluated in the RNA samples on the nCounter instrument. Forty-eight gliomas were classified using a supervised learning algorithm trained by using data from The Cancer Genome Atlas. An ensemble of 1000 linear support vector models classified 30 glioma samples into TP1 with classification confidence of 0.99. Glioma patients in TP1 group have a poorer survival (HR (95% CI) = 4.5 (1.3–15.4), p = 0.005) with median survival time of 12.1 months, compared to non-TP1 groups. Network analysis revealed that cell cycle genes play an important role in distinguishing TP1 from non-TP1 cases and that these genes may play an important role in glioma survival. This could be a good clinical pipeline for molecular classification of gliomas.

Improved quantification of lipid mediators in plasma and tissues by liquid chromatography tandem mass spectrometry demonstrates mouse strain specific differences

A liquid chromatography tandem mass spectrometry-based method for the quantitation of 39 lipid mediators in four sample types and in two mouse strains is described. The method builds upon existing methodologies for analysis of lipid mediators by A) utilizing a bead homogenization step for tissue samples; this eliminates the need for homogenization glassware and improves homogenization consistency, B) optimizing the isolation and purification of lipid mediators with polymeric reverse phase SPE columns with lower sorbent masses; this results in lower solvent elution volumes without loss of recovery and C) utilizing an on-column enrichment method to improve analyte focusing before chromatographic separation. The method is linear from 0.25-250 pg on column for low level lipid mediators and from 5-5000 pg on column for high level lipid mediators. The addition of a methyl formate elution step to a previously published method dramatically improved precision and recovery for the cysteinyl leukotrienes. Accuracy and precision for 4 different sample types including human plasma, mouse lung, mouse spleen and mouse liver is demonstrated. Liver samples had extremely high levels of a tentatively identified bile acid which interfered with quantitation of resolvin E1, 11B-prostaglandin F2a and thromboxane A2. Results from 2 different tissue sources from untreated mice (C57BL/6 versus BALB/c) showed dramatically different concentrations of lipid mediators.

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment.

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

Study of Top-down and Bottom-up Approaches by Using Design of Experiment (DoE) to Produce Meloxicam Nanocrystal Capsules.

In order to investigate the correlation among energy input-related, drug-related, and stabilizer-related aspects for both top-down and bottom-up nanocrystal production, meloxicam nanosuspensions (NS) were produced by using three different methods (low-energy wet milling, high-pressure homogenization, and precipitation) and each method was optimized by using design of experiment (DoE). Box-Behnken design of 3 factors and 3 levels was applied for the optimization of each method. All the three models were found to be significant and the optimized process parameters were used for production of NS, respectively. Interestingly, by comparison of the top-down and bottom-up approaches, the influence of energy input (homogenization pressure or milling speed) from the instruments seemed not significant for top-down compared with bottom-up for this drug. Different mechanisms of homogenization (relatively high energy zone) and milling (relatively low energy zone) led to obtained various significant correlations for each method. Capsules containing nanocrystals were successfully produced by using a novel method applying NS (after wet bead milling and homogenization processes) as wetting agent for direct capsuling and showed superiority regarding as dissolution rate compared with the traditional two-step method (freeze-dried powder used for capsuling as the first step). Different NS preparation methodologies proved to have a direct influence on the following capsuling process and consequently, in the dissolution rate. This study also proved that residual DMSO in nanosuspension after precipitation process could affect the freeze-drying process, which might further alter the redispersion and influence the downstream processes.

The comparing of homogenization methods for oxidative and antioxidative analyses of sperm

Pretreatment of sperm for oxidative and antioxidative analysis has been shown to be different in studies. These differences can affect the result of analyses. The aim of this study was to compare the analyses of sperm homogenization methods. The semen of four rams were used in the study. Pretreatment procedures included the rotary, bead, and ultrasonic homogenization methods. Oxidative and antioxidative status of sperm samples was assessed by measuring serum lipid hydroperoxide, total oxidant status, free sulfhydryl groups (SH total thiol), ceruloplasmin, and total antioxidant capacity levels. As a result, the homogenization methods before oxidative and antioxidative status analyses significantly affected the result (P < 0.05), and the ultrasonic homogenization method was the most effective. It is recommended to use the ultrasonic homogenization as a pretreatment method to determine the oxidative and antioxidative status analyses in sperm.

Stability analysis of the Cold Metal Transfer (CMT) brazing process for galvanized steel plates with ZnAl4 filler metal

In order to achieve lower CO2 emissions and lower fuel consumption, modern motor vehicle industries have been reducing automotive weight with an increasing degree in the past years by replacing steel structures with brazed and welded components of steel and lightweight metals. In this context, the combination of galvanized steel and aluminum admits considerable application potential. Joints of the two cited different types of materials can be produced by partial brazing, through the Cold Metal Transfer (CMT) process with addition of zinc-based wire alloys. However, stability analysis for this process is still not well understood. Most papers focus on metallurgical characteristics such as intermetallic phase formation and joint strength. In order to assess stability, bead on plate samples made of ZnAl4 deposits on an automotive DX56D + Z140 galvanized steel was statistically correlated to current and voltage oscillograms as well as high-speed images, after variation of the IBoost parameter from 30 to 150 A. Short-circuit and arc burning times were acquired and used to compute the Vilarinho Regularity Index for Short-Circuit Transfer (IVsc), which is based on variation coefficients. The lower the IVsc, the more stable is the metal transfer. The index was used to assess bead homogeneity. The results pointed that bead homogeneity was found only for IBoost values below 97 A. However, stability was reached for IBoost levels of 97 A (with homogeneous bead) and 150 A (without homogeneous bead). This means that to determine homogeneity, the metal transfer stability, determined through IVsc, is an indicator, but other factors such as current level and its effects over the melt pool must also be considered.

Selective extraction of proteins from bacterial cells by electroporation, sonoporation or glass bead homogenization

Déterminer le coût des pancréatectomies et identifier les facteurs associés à une majoration des coûts hospitaliers après résection pancréatique.Les patients opérés de résection pancréatique entre janvier 2008 et décembre 2014 ont été inclus. Les données de morbimortalité durant l’hospitalisation ont été recueillies ainsi que les coûts globaux relatifs à toutes les dépenses médicales durant l’hospitalisation initiale et les réhospitalisations dans les 90 jours.Cent vingt-sept patients opérés dans notre département ont été inclus dans cette étude. La majorité des patients ont été opérés de tumeurs malignes (70 % vs. 30 %). La médiane du coût hospitalier était de 21 392 euros [15 998–29 667]. L’âge (p = 0,011) et l’ictère (p < 0,001) préopératoire étaient associés à un coût hospitalier plus élevé. En peropératoire, la durée opératoire et les pertes sanguines étaient corrélées aux coûts d’hospitalisation (respectivement, p = 0,001 et p = 0,002). La duodénopancréatectomie céphalique était associée à un coût statistiquement plus élevé par rapport aux résections gauches (21 770 vs. 15 422, p = 0,001). En postopératoire, les complications postopératoires graves (Clavien-Dindo grade ≥ 3) (p = 0,001), infectieuses (p = 0,002) et hémorragiques (p = 0,001) augmentaient significativement le coût hospitalier. En analyse multivariée, les complications infectieuses (p = 0,003) et les complications graves (p = 0,01) étaient prédictives d’une augmentation des coûts hospitaliers.La chirurgie pancréatique est associée à un coût hospitalier élevé en rapport avec un taux de complication non négligeable.To define the cost of pancreatectomies and to identify factors associated with increased hospital costs after pancreatic resection.All patients undergoing pancreatic surgery in our department between January 2008 and December 2014 were included. All complications occurring during hospitalization or in the 90-day period after discharge were documented. The hospital costs were analyzed and predictive factors of increased hospital costs were determined.One hundred and twenty seven patients were identified. Most patients underwent pancreatectomy for malignant tumors (70%). Median hospital costs were 21,392 [15,998–29,667] euros. Age (P = 0.011) and preoperative jaundice (P < 0.001) were associated with higher hospital costs. Intra-operative surgical time and blood loss were correlated with increased costs (P = 0.001 and P = 0.002, respectively). Pancreatoduodenectomy was associated with statistically significantly higher costs compared to distal pancreatectomy (21,770 vs. 15,422 euros, P = 0.001). Severe post-operative complications (Clavien-Dindo grade ≥ 3) (P = 0.001), septic complications (P = 0.002) and hemorrhage (P = 0.001) statistically significantly increased costs. In multivariate analysis, septic (P = 0.003) and severe complications (P = 0.01) were statistically significantly associated with increased hospital costs.Pancreatic surgery is associated with high hospital costs, essentially related to post-operative complications.

Improving weld bead homogeneity in short arc GMAW processes applying low power diode lasers

Using laser and arc power in equal parts, laser-arc hybrid welding processes are successfully adapted by the industry. This paper shows the stabilizing effects of diode lasers on short arc GMAW (gas metal arc welding) which deliver just a fraction of the supplied arc power. In a comprehensive comparison the stabilized and the sole GMAW processes are confronted and differences regarding process behavior and weld bead properties are pointed out. The tests were carried out with DC01 (1.0330) plates of 1 mm thickness deploying lasers using a mean laser intensity of 1,1 x104 W/cm². Adding this low amount of intensity provides stability to the weld process and results in weld beads of higher homogeneity. The process behavior is characterized by the transient current and voltage curves which are analyzed in regards to consistency and uniformity. Metallographic analysis is employed to compare the weld bead dimensions at different energy ratios.

Standardized microgel beads as elastic cell mechanical probes.

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acrylamide (PAAm) microgel beads mimicking size and overall mechanics of biological cells. We produced mono-disperse beads at rates of 20-60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads' elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors.

Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations. Graphical abstract The steroid hormone profile is quantifiable from a single sample of bottlenose dolphin blubber using liquid chromatography tandem mass spectrometry. This profile can be applied to remotely collected dart biopsies and be used to determine reproductive or stress status of a wild-living dolphin.

Evaluation of cell disruption method for lipase extraction from novel thraustochytrids

Marine microorganisms represent a rich source of enzymes with the potential industrial application. In this study, we screened novel thraustochytrids for a range of enzymatic activities. The similarity between isolates based on enzyme activities was investigated using cluster analysis. Thraustochytrids were observed to possess different enzymatic activities, however, we focused on lipase activity. Two strains AMCQ-4b27 (34IU/g) and AMCQS1-9 (36IU/g) exhibited highest intracellular lipase activity among the isolates. To extract lipase, four different cell disruption methods such as bead vortexing, grinding, sonication and homogenization, were evaluated for their efficiency on lipase extraction yields and quality. Sonication was found to be the best method for enhancing lipase extraction yields from Thraustochytrium sp. AMCQ-4b27 (0.903IU/μg) and Schizochytrium AMCQS1-9 (1.44IU/μg).

カキからのノロウイルス抽出法の検討

To keep the safety of oyster intended for raw eating, monitoring of norovirus (NoV) contamination by rapid and sensitive method is important. We had developed a rapid method using bead homogenization to recover NoV particles from oyster tissues followed by RNA detection using reverse-transcription, real-time PCR. But there were still remains a need to improve the sensitivity. Therefore we re-evaluated bead homogenization conditions and studied the usefulness of enzymatic digestion after the bead homogenization. In the comparison of homogenization condition (speed and time), the mildest condition (3,500 rpm for 15 sec) showed the highest positive rate (60%) of NoV. On the contrary, when proteinase K digestion was added after bead homogenization, high intensity homogenization at 4,500 rpm for 15 sec showed high positive rate compared with the low intensity homogenization. In the comparison of digestion with two different enzymes, proteinase K and α-amylase, that digest organic compounds in digestive tissues after the bead homogenization, α-amylase digestion showed higher NoV GII RNA copy number (p<0.01) in average. At least 60-min digestion time by α-amylase was required. These results show that the bead homogenization method coupled with α-amylase digestion is useful to recover NoV particles in oyster digestive tissues.