Ultrasonic Cell Disruption Research Articles

Valorization of Spent Brewer’s Yeast Bioactive Components via an Optimized Ultrasonication Process

The increasing need for sustainable waste management and food fortification requires continuous agri-food biotechnological innovation. Spent brewer’s yeast (SBY) is a mass-produced underutilized by-product of the brewery industry and has elevated bioactive potential. The current study presents a streamlined ultrasonic SBY cell lysis method, with the main goal of bioactive compound valorization. The influence of selected ultrasonication parameters on protein release and, implicitly, on the cell disruption efficiency, was assessed. The SBY derivatives resulting from the ultrasonic cell lysis were SBY extracts (SBYEs) and cell walls (SBYCWs), which were evaluated in terms of protein content, antioxidant activity (AOA) and total polyphenol content. Scanning electron microscopy (SEM) and FT-IR spectroscopy were used to characterize SBYCWs in relation to the morphological and chemical transformations that follow ultrasonic yeast cell disruption. The optimal ultrasonication conditions of 6.25% SBY concentration, 40 °C and 33.33% duty cycle (DC) ensured the most efficient lysis. The SBY derivatives with the most elevated antioxidant activity were obtained at temperatures below 60 °C. SBYCWs had the highest polyphenol content and a relatively high content of β-glucan under these parameters. Optical microscopy and SEM confirmed the release of intracellular content and separation of SBYCWs.

Comparative Analysis of Volatile Compounds from Four Radish Microgreen Cultivars Based on Ultrasonic Cell Disruption and HS-SPME/GC-MS.

The ultrasonic cell disruption method was used to efficiently extract isothiocyanates and other volatile compounds from radish microgreens. A total of 51 volatiles were identified and quantified by headspace solid-phase micro-extraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS) in four radish microgreen cultivars, mainly including alcohols, aldehydes, isothiocyanates, sulfides, ketones, esters, terpenes, and hydrocarbons. The correlation between cultivars and volatile compounds was determined by chemometrics analysis, including principal component analysis (PCA) and hierarchical clustering heat maps. The aroma profiles were distinguished based on the odor activity value (OAV), odor contribution rate (OCR), and radar fingerprint chart (RFC) of volatile compounds. This study not only revealed the different flavor characteristics in four cultivars but also established a theoretical basis for the genetic improvement of radish microgreen flavors.

HVT-vectored H7 vaccine protects chickens from lethal infection with the highly pathogenic H7N9 Avian influenza virus

Since mid-2016, the highly pathogenic H7N9 subtype avian influenza virus (AIV) has threatened both public health and the poultry industry. Although a vaccination strategy has been deemed imperative to manage the virus, the most commonly used inactivated vaccines today are susceptible to interference from maternal antibodies and associated with an over-reliance on humoral immunity. In response, we developed a recombination vaccine with the herpesvirus of turkeys (HVT) as the vector to squeeze HPAI H7N9 and assessed its protective efficiency in immunized chickens. By inserting an enhanced green fluorescent protein (EGFP) expression cassette (i.e., MCMV+EGFP+SV40 polyA) into the HVT065 and HVT066 positions, we obtained the recombinant HVT expressing EGFP (i.e., rHVT-EGFP). Electroporation and EGFP tags improved the efficiency of transfection compared with transfection using expression plasmids without any fluorescent labeling and traditional liposomes. Using limiting dilution analysis and ultrasonic cell disruption techniques, we screened and purified a cell-bound herpes virus based on rHVT-EGFP and consequently constructed a recombinant HVT expressing the hemagglutinin (HA) of H7N9 (i.e., rHVT-H7HA), which was able to proliferate similarly to the parental strain, stably pass for at least 15 generations in vitro, and replicate stably in multiple organs in vivo. After chickens were immunized with rHVT-H7HA, the average antibody titers reached up to 3 log2 at 35 d post-vaccination and remained stable. Those results suggest that rHVT-H7HA can protect chickens against H7N9 with a dose-independent immune protection rate of 90% and significantly reduce the lung virus titer 4 d post-challenge.

Preparation, characterization and properties of cinnamon essential oil nano-emulsion formed by different emulsifiers

Essential oil has unstable properties and low bioavailability. In order to improve these problems, with the help of ultrasonic wave, stable nano-emulsions were prepared with different emulsifiers. In this experiment, the formation of nano-emulsions with different emulsifiers at different concentrations was studied, including sodium caseinate (SC), whey glycoprotein (WGP), soy protein isolate (SPI) and Tween 80. Meanwhile, we studied the effect of ultrasonic cell disruption apparatus and homogenizer on the particle size of nano-emulsions. Cinnamon essential oil nano-emulsion prepared under the optimum emulsifier and its concentration has good microstructure and rheological properties. The stability of cinnamon essential oil nano-emulsion in different environments was studied, including centrifugation, freeze-thaw, heating-freezing and long-term (30 d) stability. The nano-emulsion of cinnamon essential oil is stable in emulsifiers. The nano-emulsions prepared by different emulsifiers has different particle sizes in different environments and their advantages and disadvantages are compared. Finally, how different nano-emulsion were digested in vitro was also studied. The in vitro digestion of nano-emulsion by different emulsifiers was simulated. Its properties include particle size, zeta potential and polymer dispersion index (PDI).

Preparation of Recombinant Formate Dehydrogenase from Thermotolerant Yeast Ogataea parapolymorpha and Crystallization of Its Apo- and Holo- Forms

NAD+-dependent formate dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaFDH, EC 1.2.1.2) with the additional N-terminal Gly residue and its double mutant OpaFDH_AD were overexpressed in E. coli cells. The enzyme yield was, respectively, 6000 and 6200 units per liter of the cultivation medium. Purified enzymes were obtained as homogeneous preparations with a yield of 62%. The purification procedure included ultrasonic cell disruption, heat treatment of cell free extracts at 55°C for 15 min, and hydrophobic chromatography on the Phenyl Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted in the preparation of crystals of the apo-form but not the holo-form. Crystals of the holo-form were prepared in the case of the OpaFDH_AD mutant in the presence of 7 mM NAD+ and 10 mM sodium azide. The size and quality of the crystals are sufficient to collect X-ray diffraction data and determine the enzyme three-dimensional structure.

The application of an aqueous two-phase system combined with ultrasonic cell disruption extraction and HPLC in the simultaneous separation and analysis of solanine and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit

An aqueous two-phase system was used in conjunction with ultrasonic cell disruption to extract and separate solanine (mainly solasonine and solamargine) and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit. The optimized conditions of the present study were determined by a single-factor experiment and a multifactor experiment. The concentration of ethanol was set at 60% and the duration of the ultrasonic cell disruption extraction was 50 min. In the ethanol-K2CO3 aqueous two-phase separation system, the concentration of ethanol was 36%, the concentration of K2CO3 was 0.21 mg·mL−1, and the temperature was 15 °C. The solasonine and solamargine were determined by high-performance liquid chromatography, and the Solanum nigrum polysaccharide was determined by an ultraviolet–visible spectrophotometer in accordance with the phenol–sulfuric acid method. xUnder optimized conditions, the average extraction efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 95.86%, 95.95% and 96.95%, respectively, and the average separation efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 2.07 mg·g−1, 2.05 mg·g−1 and 8.15 mg·g−1, respectively.

Simultaneous Cell Disruption Extraction and Purification Followed by Two-phase Derivatization Procedure for the Determination of Organotin Compounds in Wet Biological Samples by GC–MS

Freeze-dried biological samples used for the determination of organotin compounds (OTCs) involve complicated or tedious pretreatment (e.g., freeze-drying) that may result in loss of analyst or contamination. In the present study, a rapid and accurate method for determination of five kinds of OTCs—monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), diphenyltin (DPhT) and triphenyltin (TPhT) in fresh biota samples—was developed and further compared with freeze-dried tissues. It involved a sample preparation method of simultaneous extraction and purification of OTCs in fresh biological samples, which was developed with ultrasonic cell disruption and primary secondary amine (PSA) adsorption, and followed by two-phase (organic solvent and cell membrane phases) derivatization before the analysis by gas chromatography–mass spectrometry. The extraction efficiency from biota matrices was relatively better with the acetate/acetic acid and tropolone into methanol as extraction agents and primary secondary amine as grease-removing agents. The retention of cell membrane-like materials as well as 25% of ethyl acetate as co-extraction reagents used after centrifugation improved the targeted OTCs’ recoveries. The proposed method, under the optimal condition, has shown satisfactory performance in wet biological samples. The limits of detection obtained were less than 10 ng Sn g−1 ww (wet weight) for all analytes. Spiked recoveries were within the range 89–118% and the relative standard deviations were less than 7% (n = 5) when quantified by matrix-matched calibration curves. Finally, the applicability of this new method was further verified by comparing the results from wet and freeze-dried oyster tissues collected from two coastal cities in China.

Aggregate formation affects ultrasonic disruption of microalgal cells

Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K+, Ca2+ and Al3+ to Chlorella vulgaris cultured in Bold’s Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K+>Ca2+>Al3+ samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency.

Cell disruption of S. cerevisiae by scalable high-intensity ultrasound

Ultrasonic disruption of yeast and other microbial cell cultures is commonly used for laboratory-scale protein preparations because it is rapid, efficient and simple to use compared to such methods as high-pressure homogenization (HPH) and bead milling. Lysing by sonication is also more effective than other cell disruption methods for the recovery of periplasmic, membrane-bound, or insoluble recombinant proteins. Until recently, however, due to amplitude limitations of conventional-design pilot and industrial-size sonication equipment, ultrasonic cell disruption was only feasible on the laboratory scale. In this study, we show that Barbell Horn Ultrasonic Technology (BHUT) can be successfully used for the disruption of Sacharomyces cerevisiae (S. cerevisiae) cells on a large scale. In particular, we show that by using pilot-scale BHUT-based equipment, total protein and alkaline phosphatase can be efficiently extracted from S. cerevisiae cells, achieving about an order of magnitude productivity increase factor with respect to laboratory-scale results. Since the size of BHUT-based ultrasonic processors can be increased further, ultrasonic cell disruption now has the ability to develop into a valuable commercial-scale method, potentially superior to HPH and bead milling techniques in this area of application.

Ultrasonic disruption of yeast cells: Underlying mechanism and effects of processing parameters

During the ultrasonic treatment of yeast cells, the damage to the cell walls and membranes was monitored by assessing the release of cell wall polysaccharides and intracellular proteins, respectively. At a low acoustic intensity (10 W/cm2), the polysaccharides were released faster than the proteins, whereas at higher acoustic intensities (24 and 39 W/cm2), this trend was reversed. At elevated temperatures, additional cell wall polysaccharides were released, whereas fewer intracellular proteins were released. Increasing processed volumes, initial cell concentrations, and salt concentrations led to the decrease of both fractions. However, the total releases per ultrasound treatment remained constant regardless of the processed volumes, and increased with the increase of initial cell concentrations. The results suggest that the ultrasonic disruption of yeast cells begins with the breakdown of the cell wall before continuing to the cell membrane. These findings may offer new avenues for exploring more efficient cell disruption or microbial inactivation processes. Ultrasonic technology has been intensively studied for microbial inactivation or cell disruption. It is believed that the inner cell membrane is the target of the ultrasonic damage. The mechanism of microbial inactivation via ultrasound involves the thinning of the cell membranes. In this study, we monitored the ultrasonic damage to yeast cell walls and membranes by monitoring the release of cell wall polysaccharides and intracellular proteins, respectively. Our results demonstrate that the ultrasonic disruption of yeast cells begins with the breakdown of the cell wall before continuing to the cell membrane. Increasing the temperature weakens the cell wall and thermally coagulates the intracellular proteins. Increasing the processed volumes, initial cell concentrations, and salt concentrations reduces the releases of cell wall polysaccharides and intracellular proteins from the viewpoint of yeast cells. However, the total releases of each fraction per ultrasound treatment remain constant regardless of the processed volumes, and increase with increasing initial cell concentrations. Our results may offer new insights towards the exploration of more efficient industrial processes for microbial inactivation or cell disruption, and in elucidating the effects of processing parameters.

Ultrasonic cavitation for disruption of microalgae

Challenges with mid-stream fractionation steps in proposed microalgae biofuel pathways arise from the typically dilute cell density in growth media, micron scale cell sizes, and often durable cell walls. For microalgae to be a sustainable source of biofuels and co-products, efficient fractionation by some method will be necessary. This study evaluates ultrasonic cell disruption as a processing step that fractionates microalgae. A range of species types with different sizes and cell wall compositions were treated. The initial seconds of sonication offered the most significant disruption, even for the more durable Nannochloropsis cells. Following this initial period, diminishing effectiveness was attributed, by acoustic measurements, to attenuation of the ultrasound in the ensuing cloud of cavitating bubbles. At longer exposure times, differences between species were more pronounced. Processing higher concentrations of Isochrysis slowed cell disintegration only marginally, making the expenditure of energy more worthwhile.

A Novel Dextran Dextrinase from Gluconobacter oxydans DSM-2003: Purification and Properties

Dextran has already been widely applied in food, pharmaceutical, and chemical industries. In this study, a novel intracellular dextran dextrinase (DDase, EC 2.4.1.2) from Gluconobacter oxydans DSM-2003 exhibiting catalytic activity to synthesize dextran from maltodextrin was purified to homogeneity by ultrasonic cell disruption, ion exchange chromatography, and gel filtration. This procedure showed 187.5-fold purification from the cell-free extract with 41.9% yield. And the apparent molecular weight was estimated to be 62kDa by SDS-PAGE. It was different from the reported literatures, which found that the molecular weight of intracellular and extracellular DDase of G. oxydans ATCC-11894 was 300 and 152kDa, respectively. Otherwise, it showed different physicochemical characteristics (optimal temperature and pH, thermal, pH stability, effect of metal ions) from the DDase of G. oxydans ATCC-11894. This indicated that DDase of G. oxydans DSM-2003 was a novel one compared to the reported literatures.

Large-Scale Optimization-Based Classification Models in Medicine and Biology

We present novel optimization-based classification models that are general purpose and suitable for developing predictive rules for large heterogeneous biological and medical data sets. Our predictive model simultaneously incorporates (1) the ability to classify any number of distinct groups; (2) the ability to incorporate heterogeneous types of attributes as input; (3) a high-dimensional data transformation that eliminates noise and errors in biological data; (4) the ability to incorporate constraints to limit the rate of misclassification, and a reserved-judgment region that provides a safeguard against over-training (which tends to lead to high misclassification rates from the resulting predictive rule); and (5) successive multi-stage classification capability to handle data points placed in the reserved-judgment region. To illustrate the power and flexibility of the classification model and solution engine, and its multi-group prediction capability, application of the predictive model to a broad class of biological and medical problems is described. Applications include: the differential diagnosis of the type of erythemato-squamous diseases; predicting presence/absence of heart disease; genomic analysis and prediction of aberrant CpG island meythlation in human cancer; discriminant analysis of motility and morphology data in human lung carcinoma; prediction of ultrasonic cell disruption for drug delivery; identification of tumor shape and volume in treatment of sarcoma; discriminant analysis of biomarkers for prediction of early atherosclerois; fingerprinting of native and angiogenic microvascular networks for early diagnosis of diabetes, aging, macular degeneracy and tumor metastasis; prediction of protein localization sites; and pattern recognition of satellite images in classification of soil types. In all these applications, the predictive model yields correct classification rates ranging from 80 to 100%. This provides motivation for pursuing its use as a medical diagnostic, monitoring and decision-making tool.

Improved cavitational cell disruption following pH pretreatment for the extraction of β-galactosidase from Kluveromyces lactis

The efficient release of intracellular β-galactosidase by ultrasonic disruption of cells treated with aqueous solutions of different pH has been investigated to improve the cavitational efficacy of the cell disruption process. Pretreatment of the cells at different pHs for various time intervals was studied. The maximum yield (95 ± 3 U/ml) was obtained by pretreatment of the cells at pH 4.4 for 6 h and subsequent ultrasonic cell disruption for 40 min. The maximum yield on ultrasonic disruption without pretreatment was 7.2 ± 0.6 U/ml. The energy efficiency of the disruption process for releasing β-galactosidase using different pre-treatments has been calculated and compared.

Effectiveness of ultrasonic cell disruption as a function of the suspension concentration

Our goal was to explore the interactions between the acoustic phenomena taking place in the ultrasound field, the concentration of the biological particles of the ultrasound field, and the cell biology effects of these interactions. Using output power of 9 W cm-2 and frequency of 1.117 MHz, the concentration was determined, expressed in g l-1, of lyophilized Saccharomyces cerevisiae baker's yeast needed for stopping cavitation in the sound field. Then by using multiples of the aforementioned concentration, we monitored the acoustic phenomena occurred in the sound field and, simultaneously, we examined the survival dynamics of the cells. Examined acoustic phenomena were the following: acoustic streaming, standing wave, and cavitation. Physical parameters (suspension density, dissolved oxygen, temperature) of the sound field had essential effect on the acoustic phenomena formed in the ultrasound field and on the threshold levels of their formation. The phenomena affected the composition of the material in t...

Rapid and selective extraction of phycocyanin from Spirulina platensis with ultrasonic cell disruption

A study was conducted on the efficiency of phycocyanin extraction from Spirulina platensis (Arthrospira platensis) cells disrupted by ultrasonic irradiation. Extraction followed first-order kinetics with respect to the length of time for irradiation. The first-order rate constant increased linearly with the output of ultrasonic irradiation. In order to extract phycocyanin there was an appropriate range of ultrasonic frequency, fu. In addition the most important finding is that the purity of phycocyanin in its crude extract depended on fu. For example, phycocyanin was extracted with higher purity at fu = 28 kHz than at fu = 20 kHz. It is suggested that rapid and selective extraction of phycocyanin from S. platensis may be possible if an optimized ultrasonic application is developed for a given suspension.

Ultrasonic cell disruption of stabilised sludge with subsequent anaerobic digestion

The world-wide increasing environmental awareness and its subsequent regulations have led to the application of improved technologies in wastewater purification plants. This has resulted in higher wastewater and sludge productions. Sludge is the by-product of such plants and it is not only rich in organic carbon and pathogens but also in heavy metals and other environmental pollutants. In Europe, agricultural application of dried sludge (bio-solids) is confronted with negative reactions from the citizens, governmental organisations, farmers and the food industry. Ultrasonic disruption of sludge is a popular mechanical disruption process in sludge treatment. During ultrasonic treatment, high frequency acoustic signals are used to initiate the cavitation process. The applied ultrasonic field leads to a breakdown of cohesive forces of the liquid molecules resulting in the generation of cavitation bubbles. A shock wave is released by the collapse of the cavitation bubbles and propagates in the surrounding medium forming jet streams that cause the disruption of cells in sludge. Disruption of sludge cells enables the release of light organic substances into the sludge water thereby exposing them for further anaerobic digestion. This paper presents results on the disruption of conventionally stabilised sludge through the application of the ultrasonic field. In order to reduce the specific energy input (i.e. ratio of the consumed energy during ultrasonic disruption to the input sludge mass) and improve biogas production, the total solids content of the stabilised sludge was increased before disruption. The anaerobic digestion of sludge samples was carried out in a set of specially constructed laboratory anaerobic digesters. Results showed that subsequent anaerobic digestion of the ultrasonically disrupted sludge could improve biogas production with reduced sludge quantity that is vital to the economic consideration of the wastewater treatment plants. This process encourages the exploitation of valuable materials and energy from stabilised sewage sludge just before its final disposal. The negative effects of mixing disrupted sludge with its separated sludge water are also shown in this paper. This expresses the microbiological instability of the anaerobic process caused by the mixing process.

L-tyrosine and nicotine induce synthesis of l-Dopa and norepinephrine in human lymphocytes

Catecholamines (CA) were studied in peripheral human lymphocytes in basal conditions as well as after l-tyrosine and/or acetylcholine (ACh) stimulation. Nicotinic and muscarinic receptor activation and blockade were assessed. CA were determined after ultrasonic cell disruption in peripheral lymphocytes after incubation (1 h at 37°C) with the chemicals employed. l-tyrosine significantly increased ( P<0.01) l-Dopa and norepinephrine (NE) content of lymphocytes. ACh in the low μM range did not modify, whereas ACh (60 μM) and (120 μM) significantly increased ( P<0.01), both l-Dopa and NE intracellular levels. l-tyrosine plus ACh (60 μM) or (120 μM) significantly increased ( P<0.01) intracellular l-Dopa and NE versus control, versus l-tyrosine alone and versus ACh alone. The increase was higher than the algebraic sum of the individual increases. Nicotine (250 μM), but not muscarine (50 μM), significantly increased l-Dopa and NE in lymphocytes. Tetraethylammonium (500 μM) (nicotinic blocker), but not atropine (100 μM) (muscarinic blocker), inhibited the ACh-mediated increase of intracellular l-Dopa and NE. These data show that lymphocyte synthesis of CA is under nicotinic control. Since intracellular l-Dopa after l-tyrosine plus ACh increased 6-fold versus basal, 2-fold versus l-tyrosine alone and 3-fold versus ACh alone, it is concluded that ACh might regulate CA synthesis in lymphocytes through an activation of the rate limiting enzyme tyrosine hydroxylase.

Catecholamine content and in vitro catecholamine synthesis in peripheral human lymphocytes.

We studied the catecholamine (CA) content in peripheral human lymphocytes and the ability of these cells to synthesize CA in vitro. CA were separated by high performance liquid chromatography (HPLC) and determined in the supernatant by electrochemical detection as well as being determined after ultrasonic cell disruption in mononuclear leukocytes, adherent cells (monocytes/macrophages), total lymphocytes, and B- and T-cell enriched fractions. T lymphocytes contained L-Dopa and norepinephrine (NE), whereas B lymphocytes contained only L-Dopa. Lymphocytes seem to be able to synthesize NE from both L-tyrosine and L-Dopa added to the incubation medium in concentrations similar to the peripheral venous plasma (i.e. 5 x 10(-5) m and 10(-8) m, respectively). The addition of D-Dopa did not increase intracellular NE. alpha-methyl-p-L-tyrosine, benserazide, disulfiram, and fusaric acid (which are inhibitors of the enzymatic pathway) all decreased the synthesis of NE. After the addition of [3H]-L-Dopa (10(-8) m and 10(-7) m) to the incubation medium, [3H]-NE and [3H]-dopamine appeared. By increasing the concentration of L-Dopa in the medium (< 10(-6) m), CA were detected in the supernatant as well. These data show that peripheral human T lymphocytes contain and are able to synthesize CA from normal precursors in physiologic concentrations, i.e. a CA synthetic pathway is shown in nonneural cells. These data seem to support the hypothesis of autocrine and paracrine loops in the regulation of lymphocyte activity in lymphocytes taken from human cerebrospinal fluid (as suggested by other authors).

Description of ultrasound transducers through wave parameters

High energy ultrasonic waves cause cavitation in liquids. Since many years, a great number of low frequency cavitation effects is well known such as ultrasonic cleaning and biological cell disruption. Recently the interest in high frequency applications has been rising considerably, e.g. in the formation of peroxide in destilled water or the cleaning of wafers. These and other applications require efficient and reliable high frequency transducers. Therefore we are working on the design and optimization of transducers with frequencies of about 100 kHz up to 3 MHz. According to the specific demands of this process, computer simulations are being used in the course of the optimization. The Finite Elements Method, for example, is applied to investigate the forms of vibration. In addition to this, Network Analysis Methods are used to optimize the power transformation and the band width. By these means an average efficiency of about 80 percent is reached. These transducers are developed for laboratories as well as for technical scale applications.