Mechanism of cell disintegration in a high pressure homogenizer

The disintegration of baker's yeast (Saccharomyces cerevisiae) by a high pressure homogenizer, to a pressure of 25,000 psi. (172.37 MNm^?2) is described, together with details of the methods of measurement used to obtain information on the valve movement and pressure transients. The theory of the mechanism of cell disintegration is discussed.     

The Influence of Heat Processing and Mechanical Disintegration on Yeast for Single-Cell Protein

Yeast was processed by means of different technical drying procedures, heating in water suspension, and mechanical disintegration. The influence on the ultrastructure, the nutritive value and on the availability of the cell nitrogen-containing compounds to chemical extractants was studied. On micrographs no cell wall disrupture could be observed after any of the heat treatments. The internal cell structure was affected at the higher temperatures. After drum drying this structure was destroyed to a large extent. The heat treatments increased the nutritive value compared to unheated yeast cells but did not increase the availability of the cell content to chemical extractants. Mechanical disintegration increased both the nutritive value and the availability to chemical extractants. Heat processes and mechanical disintegration give high nutritive value to the yeast. Mechanical disintegration is advantageous when processing steps such as extraction with chemicals are necessary for obtaining specific protein products.     

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation.     

超声空化强度测量的研究进展

近几十年来,超声波已经广泛应用于生物学和医学领域,在医学领域中超声波可以作为信息载体用于探测人体的病变信息,并且可以用一定剂量的超声波作用于人体病变组织,并通过它对组织的作用达到一定的治疗目的。作为一种无创、非介入性外科技术,它的疗效和安全性越来越被人们所关注。超声波与人体组织的相互作用有三种,分别是机械机制,热学机制,和空化机制。对于机械机制和热机制人们比较熟悉,而对于空化机制则相对陌生。随着超声空化在医学中的应用越来越广泛,其安全性越来越受到人们的关心。要么是其强度迭不到治疗效果,要么是其强度过大损伤人体,因此其强度已成为人们关心的主要主题。本文主要介绍超声空化的主要探讨了几种测量方法及对超声空化有影响的几种参量,并对超声空化的发展进行了展望。     

Some methods for processing of single-cell protein

Methods for the production of protein concentrates, with a low content of nucleic acid, in kilogram quantities from yeast have been studied with the aid of equipment designed for operation on pilot-plant scale. The influence of drum drying and mechanical disintegration on the nutritive value of the yeast was also investigated. Drum drying and mechanical disintegration improved the nutritive value of the yeast but high extractability of protein and nucleic acid was only obtained after mechanical disintegration. Protein concentrates without and with cell walls were produced from mechanically disintegrated yeast. The different fractions which were obtained when separating cell walls and precipitating protein by heating at alkaline pH, were analyzed. After protein precipitation, about 90% of the RNA could be precipitated from the supernatant by addition of acid, giving a product containing 50% RNA of the dry weight. The protein precipitate obtained after cell wall separation had an RNA content of less than 2% and contained 70–l75% of the amino acids in the starting yeast material. Protein concentrates containing cell walls were produced by precipitating protein by heating at alkaline pH directly after mechanical disintegration. The content of RNA was about 2% and the yield of amino acids was 70–80%. It was found that the nutritive value of the protein concentrate was higher than that of the starting yeast material. To produce such a protein concentrate on a large scale, the process described can probably be employed.     

Detachment and sonoporation of adherent HeLa-cells by shock wave-induced cavitation

The interaction of lithotripter-generated shock waves with adherent cells is investigated using high-speed optical techniques. We show that shock waves permeabilize adherent cells in vitro through the action of cavitation bubbles. The bubbles are formed in the trailing tensile pulse of a lithotripter-generated shock wave where the pressure drops below the vapor pressure. Upon collapse of cavitation bubbles, a strong flow field is generated which accounts for two effects: first, detachment of cells from the substrate; and second, the temporary opening of cell membranes followed by molecular uptake, a process called sonoporation. Comparison of observed cell detachment with results from a theoretical model considering peeling cell detachment by a wall jet-induced shear stress shows reasonable agreement.     

Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves

Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering. Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid. When the cells were suspended in 12% (v/v) ethanol–water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system. How this behaviour alters the efficiency of retention/immobilisation was measured. As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy. Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex. The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured. Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects. Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation. The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range.     

Beitrag zur Optimierung des mechanischen Aufschlusses von Hefezellen

The influence of technological variables on the cell disintegration of yeast suspensions by means of a ball mill and a high pressure homogenizer has been studied by measuring the electrical conductivity. The rate constants and half-life periods are calculated. Regarding the production of protein isolates, the stipulation of optimal disintegration conditions requires a compromise between a degree of disruption as high as possible and a low destruction of the cell walls. By homogenizing, fragments of cell walls arise which are more uniform and better separable in comparison with the milling process. Therefore the mechanical breakage of yeast cells on a large scale should be carried out by the use of high pressure homogenizers.     

Degradation of proteins in canavanine-grown yeast

Degradation of normal as well as canavanine proteins in growing yeast is suppressed by glucose. This suggests that the same mechanism may be operating in the catabolism in both cases. Degradation of normal and canavanine proteins is increased by disintegration of cell structure. Proteins synthesized in the presence of an amino acid analogue may not be degraded preferentiallyin vivo even when they are rather sensitive to endogenous proteolytic enzymes. Participant of the UNESCO postgraduate course onModern Problems in Biology.     

Mechanical disintegration of microorganisms in an industrial homogenizer

Disintegration of microorganisms in a continuously working industrial homogenizer has been studied. The homogenizer consists of rotating discs in a cylinder filled with glass beads. Different parameters for disintegration of baker's yeast were investigated. The disintegration process is a first-order reaction and it is influenced by the flow rate of the suspension and by the agitator speed. At a flow rate of 200 liters/hr about 85% of the yeast cells can be disrupted in a single pass through the disintegrator. This type of disintegrator can be used for disruption of cells in order to produce single-cell protein, active enzymes and other valuable cell components.     

A hydrodynamic mechanism for the disintegration of Saccharomyces cerevisiae in an industrial homogenizer

Amongst the commercial type of homogenizers the Manton-GaulinAPV homogenizer (APV Company Ltd., Crawley, Surrey, England) which is generally being used for other purposes than cell disintegration processes, has recently been proved to be effective for the breakage of yeast cells. To understand fully the disintegration process occurring in such machines it becomes necessary to describe their functions through mathematical expressionsbased on a realistic hydrodynamic model. A mathematical expression describing the protein release at an applied pressure has been derived from an energy balance in the homogenizer combined with the size distribution function of yeast cell population. This expression has been confirmed experimentally under conditions where it shows that turbulence is the controlling factor in the system. Furthermore it indicates the area where more investigations are needed to improve the efficiency of the process of disintegration.     

Spatio-temporal dynamics of glycolysis in cell layers. A mathematical model

Glycolytic oscillations occur in many cell types and have been intensively studied in yeast. Recent experimental and theoretical research has been focussed on the oscillatory dynamics and the synchronisation mechanism in stirred yeast cell suspensions. Here we are interested in the spatio-temporal organisation of glycolysis in cell layers. To this end we study a grid of a few thousand compartments each containing a cell. The intracellular dynamics is described by a core model of glycolysis. The compartments can exchange metabolites via diffusion. The conditions for oscillatory dynamics in a single compartment are investigated by bifurcation analysis. The spatio-temporal behaviour of the cell layer is studied by simulations. The model predicts the propagation of repetitive wave fronts induced by a substrate gradient. The formation of these waves crucially depends on the diffusive exchange of the reaction product between cells. Depending on the kinetic parameters complex spatio-temporal behaviour such as periodic termination of waves can arise. In these cases the cellular oscillation characteristics depend on the location of the cell in the array.     

Effect of some quaternary benzylammonium salts on physiology of yeast

In order to determine the biological activity of eight compounds belonging to a group of quaternary ammonium salts, their influence on the active methionine transport, the integrity of cell membranes, respiration, and viability of Saccharomyces cerevisiae and some other yeast species has been investigated. The earliest effect observed during ammonium salts action on yeast cells is an immediate methionine transport abolishment followed by its fast leakage, which indicates increasing cell membrane degradation. Gradual decline of other biological functions such as respiration and viability is thus a result of disintegration and lack of tightness of the cell membranes. The studied compounds are characterized by a rather unspecific spectrum of action on yeast resulting in irreversible damage of cell walls and cell membranes, which in consequence leads to cell death.     

空化微流体在生物医学方面的应用

空化效应是发生在液体内部的一种极其复杂的流体物理现象,能产生极高的中心能量密度,并伴随发光、发热、冲击波、高速射流等极端物理现象,它的存在能使一些极端的反应得以实现。空化效应发生时形成的空化微流体在破坏细胞形貌、微操控、微混合等方面有广泛地应用。本文综述了空化微流体及其产生的强烈冲击波在生物医学方面的应用,包含空化微流体在破坏细胞形貌、微小元件的操控以及加快液体混合等三个方面。     

Experiences with a 20 litre industrial bead mill for the disruption of microorganisms

Suspensions of several yeast strains and bacterial species were disrupted in a continuously operating industrial agitator mill of 22.7 litre internal working volume. The influence of agitator speed, flow rate, concentration of microorganisms in the slurry, packing density of glass beads and bead diameter on the disruption process was studied using baker's yeast (Saccharomyces cerevisiae). Cell disintegration was followed by assaying the appearance of protein and the activities of d-glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate:NADP⁺ oxidoreductase, EC 1.1.1.49] and α-d-glucosidase [α-d-glucoside glucohydrolase, EC 3.2.1.20] in the soluble fraction. The best operating conditions for the disintegration of baker's yeast with respect to activity yield appeared to be at a rotational speed of 1100 rev/min, a flow rate of 100 litre h^?1 and a cell concentration of 40% (w/v). The location of the desired enzyme in the cell is of importance for the choice of bead diameter and packing density of the glass beads. Temperature increase and power consumption during disintegration are also strongly influenced by the bead loading in the mill. With optimized parameters, 200 kg baker's yeast can be processed per hour with a degree of disintegration >85%. The disruption process in the mill was found to be very effective for several yeast species tested, e.g. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Candida boidinii. The usefulness of the Netzsch LME 20-mill for the disruption of bacteria species was demonstrated with Escherichia coli, Brevibacterium ammoniagenes, Bacillus sphaericus and Lactobacillus confusus. As expected, the mill capacity for bacterial disruption was significantly smaller than for the yeast. Between 10 and 20 kg per h bacteria may be processed, depending on the organism.     

Disintegration of Microorganisms and Preparation of Yeast Cell Walls in a New Type of Disintegrator

Microbial cells were disintegrated in a new type of rotary disintegrator with a disc stirrer by a combination of shear force layers, collisions, and rolling of glass beads which were brought into motion by the stirrer. The rate of disintegration at a given dry bed volume of Ballotini beads and a given volume of cell suspension is proportional to the peripheral velocity of the stirrer up to 18 m/sec. Horizontal arrangement of the stirrer increases the effectiveness about five times; 100% disintegration of yeast cells was achieved under optimal conditions within 72 sec at a concentration of 3.5g (dry weight)/100 ml of suspension, and within 96 sec at a concentration of 10.5g (dry weight)/100ml. At 17.5 g (dry weight)/100 ml, the stirrer began to slip. The cell walls of yeast were obtained at the desired degree of crushing and the course of purification was determined by infrared spectral analysis.     

An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes

Application of ultrasound transiently permeabilizes cell membranes and offers a nonchemical, nonviral, and noninvasive method for cellular drug delivery. Although the ability of ultrasound to increase transmembrane transport has been well demonstrated, a systematic dependence of transport on ultrasound parameters is not known. This study examined cell viability and cellular uptake of calcein using 3T3 mouse cell suspension as a model system. Cells were exposed to varying acoustic energy doses at four different frequencies in the low frequency regime (20-100 kHz). At all frequencies, cell viability decreased with increasing acoustic energy dose, while the fraction of cells exhibiting uptake of calcein showed a maximum at an intermediate energy dose. Acoustic spectra under various ultrasound conditions were also collected and assessed for the magnitude of broadband noise and subharmonic peaks. While the cell viability and transport data did not show any correlation with subharmonic (f/2) emission, they correlated with the broadband noise, suggesting a dominant contribution of transient cavitation. A theoretical model was developed to relate reversible and irreversible membrane permeabilization to the number of transient cavitation events. The model showed that nearly every stage of transient cavitation, including bubble expansion, collapse, and subsequent shock waves may contribute to membrane permeabilization. For each mechanism, the volume around the bubble within which bubbles induce reversible and irreversible membrane permeabilization was determined. Predictions of the model are consistent with experimental data.     

Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.     

Mechanisms of palatal epithelial seam disintegration by transforming growth factor (TGF) beta3

TGFbeta3 signaling initiates and completes sequential phases of cellular differentiation that is required for complete disintegration of the palatal medial edge seam, that progresses between 14 and 17 embryonic days in the murine system, which is necessary in establishing confluence of the palatal stroma. Understanding the cellular mechanism of palatal MES disintegration in response to TGFbeta3 signaling will result in new approaches to defining the causes of cleft palate and other facial clefts that may result from failure of seam disintegration. We have isolated MES primary cells to study the details of MES disintegration mechanism by TGFbeta3 during palate development using several biochemical and genetic approaches. Our results demonstrate a novel mechanism of MES disintegration where MES, independently yet sequentially, undergoes cell cycle arrest, cell migration and apoptosis to generate immaculate palatal confluency during palatogenesis in response to robust TGFbeta3 signaling. The results contribute to a missing fundamental element to our base knowledge of the diverse roles of TGFbeta3 in functional and morphological changes that MES undergo during palatal seam disintegration. We believe that our findings will lead to more effective treatment of facial clefting.     

The effect of ultrasound cavitation on endothelial cells

Acoustic cavitation has been widely explored for both diagnostic and therapeutic purposes. Ultrasound-induced cavitation, including inertial cavitation and non-inertial cavitation, can cause microstreaming, microjet, and free radical formation. The acoustic cavitation effects on endothelial cells have been studied for drug delivery, gene therapy, and cancer therapy. Studies have demonstrated that the ultrasound-induced cavitation effect can treat cancer, ischaemia, diabetes, and cardiovascular diseases. In this minireview, we will review the impact of ultrasound-induced cavitation on the endothelial cells such as cell permeability, cell proliferation, gene expression regulation, cell viability, hemostasis interaction, oxygenation, and variation in the level of calcium ions, ceramide, nitric oxide (NO) and nitric oxide synthase (NOS) activity. The applications of these effects and the cavitation mechanism involved will be summarized, demonstrating the important role of acoustic cavitation in non-invasive ultrasound treatment of various physiological conditions.