A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.     

Influence of the extent of disruption of Bakers' yeast on protein adsorption in expanded beds

Expanded bed adsorption chromatography is used to capture the protein product of interest from a crude biological suspension directly, thereby eliminating the need for the removal of the cell debris. While this technique may replace three or four unit operations in a typical downstream process for biological product recovery, the adsorption process is influenced by the interaction between the microbial cells or cell debris and the adsorbent as well as the presence of contaminating solutes. The influence of the extent and nature of disruption of Bakers' yeast on the adsorption of the total soluble protein and alpha-glucosidase was investigated in this study. Two different techniques were used for cell disruption: high pressure homogenisation and hydrodynamic cavitation. Two different adsorbents were chosen: anionic Streamline DEAE and cationic Streamline SP. The settled bed height and the superficial velocity were constant across all experiments. The feedstock was characterised in terms of viscosity, pH, conductivity, particle size distribution of the cell debris and the extent of protein and alpha-glucosidase released. The performance of the adsorption process was found to be influenced by the electrostatic interactions of cell debris with the anionic adsorbent Streamline DEAE and the intraparticle diffusional resistance inside the pores of the adsorbent matrix. The increase in the intensity of disruption resulted in an increase in the dynamic binding capacity (10% feed) of both the total soluble protein and the alpha-glucosidase. However, the increase in the DBC of protein and alpha-glucosidase were not proportional. The amount of protein that could be adsorbed per ml of adsorbent from the samples subjected to a lower intensity of disruption was found to exceed that obtained at a higher disruption intensity on increasing the volume of feed suggesting multilayer adsorption. In this case, selective adsorption of the model protein alpha-glucosidase was reduced, illustrating the compromise of maximising protein recovery through non-specific binding. The study illustrates the need for an interrogation of the intensity of disruption needed and a rigorous understanding of the influence of cell debris and adsorbent-protein interaction, in optimising the selective recovery of intracellular products by EBA.     

Optimization of poly(β-hydroxybutyric acid) recovery from Alcaligenes latus: combined mechanical and chemical treatments

Recovery of the intracellular bioplastic poly(β-hydroxybutyric acid) or PHB from fed-batch cultured Alcaligenes latus, ATCC 29713, was examined using combinations of chemical and mechanical treatments to disrupt the cells. Chemical pretreatments used sodium chloride and sodium hydroxide. For salt pretreatment the cells were exposed to NaCl (8?kg?m^?3) and heat (60?°C, 1 h), cooled to 4?°C, and mechanically disrupted. For alkaline treatments, the cells were exposed to sodium hydroxide (0.025–0.8 kg?NaOH per kg biomass) and mechanically disrupted at ambient temperature. A combined treatment with sodium chloride (8 kg m^?3), heat (60?°C, 1 h), and alkaline pH shock (pH 11.5, 1?min) was also tested. Mechanical disruption employed a continuous flow bead mill (2,800 rpm agitation speed, 90?ml?min^?1 slurry flow rate, 512 m mean bead diameter, bead loadings of 80% or 85% of chamber volume). Disruption was quantified by protein release. Over most of the disruption period, the release of PHB was approximately proportional to protein release. Regardless of the pretreatment or bead load, the disruption obeyed first order kinetics; hence, the rate of protein release was directly proportional to the amount of unreleased protein. Relative to untreated biomass, pretreatment always produced earlier protein release during milling. Pretreatment with a minimum of 0.12?kg NaOH per kg biomass was necessary to enable complete disruption within three passes (85% bead load). Untreated biomass required more than twice as many passes. Irrespective of the chemical pretreatment, the bead loading strongly influenced the disruption rate which was higher at the higher loading. Alkaline hydrolysis associated PHB loss was observed, but it could be limited to insignificant levels by immediate neutralization of disrupted homogenates.     

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.     

Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability

Although Haematococcus pluvialis is one of the most importantnatural sources of the carotenoid astaxanthin as a pigmentor for theaquaculture industry, the thick sporopollenin cell wall in the cysts hindersastaxanthin extraction and its subsequent bio-availability to fish. A rangeof physical and chemical processes were tested to promote the disruptionof the encysted cells. The efficacy of these processes was evaluated interms of astaxanthin recovery, which was assessed by determining theextent of leaching of astaxanthin into an organic solvent. The processestested were: autoclave 30 min, 121 ^°C, 1 atm; HCl 0.1 M, 15min and 30 min; NaOH 0.1 M, 15 min and 30 min; enzymatictreatment with a mixture of 0.1% protease K and 0.5% driselase in aphosphate buffer, pH 5.8, 30 ^°C, for one hour; spray drying, inlet180 ^°C, outlet 115 ^°C; and mechanical disruption, with acell homogeniser developed for this purpose. The mechanical(homogenisation) and autoclave treatments were the most effective in termsof extraction and availability.     

A microscale yeast cell disruption technique for integrated process development strategies

Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus-like particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3-fold and further improved the comparability of the lysate to that of the laboratory-scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (< =8 degrees C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins.     

The disruption of Alcaligenes eutrophus by high pressure homogenisation: key factors involved in the process.

The disruption of the Gram-negative bacterium Alcaligenes eutrophus by high pressure homogenisation, using the APV Gaulin 15M 8BA and 30CD homogenisers is reported. The operating parameters such as operating pressure, number of passes, temperature and biomass concentration, mimicked trends previously reported for yeasts. Extension of the study to consider the effect of cell characteristics, including the growth rate, size and shape, illustrated the dominant effect of the growth phase. The improved disruption of bacterial cultures in the logarithmic phase with respect to stationary phase cultures was confirmed by an increased dependence of actively growing cultures on the operating pressure. An increase in size in excess of 30% on the accumulation of the storage product, PHB in the stationary phase caused little change in the ease of disruption. The use of transmission electron microscopy to directly monitor the disruption on multiple passes shed light on the two-stage nature of this disruption process.     

A novel technique for the measurement of disruption in high-pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20 degrees C to 5 degrees C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4 degrees C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.     

Study of physical and biological factors involved in the disruption of E. coli by hydrodynamic cavitation

Hydrodynamic cavitation results in flow restriction in a flow system causing rapid pressure fluctuations and significant fluid forces. These can be harnessed to mediate microbial cell damage. Hydrodynamic cavitation was studied for the partial disruption of E. coli and selective release of specific proteins relative to the total soluble protein. The effects of the cavitation number, the number of passes, and the specific growth rate of E. coli on the release of periplasmic and cytoplasmic proteins were studied. At the optimum cavitation number of 0.17 for this experimental configuration, 48% of the total soluble protein, 88% of acid phosphatase, and 67% of beta-galactosidase were released by hydrodynamic cavitation in comparison with the maximum release attained using multiple passes through the French Press. The higher release of the acid phosphatase over the total soluble protein suggested preferred release of periplasmic compounds. This was supported by SDS-PAGE analysis. The absence of micronization of cell material resulting in the potential for ease of solid-liquid separation downstream of the cell disruption operation was confirmed by TEM microscopy. E. coli cells cultivated at a higher specific growth rate (0.36 h(-1)) were more easily disrupted than slower grown cells (0.11 h(-1)). The specific activity of the enzyme of interest released by hydrodynamic cavitation, defined as the units of enzyme in solution per milligram of total soluble protein, was greater than that obtained on release by the French Press, high-pressure homogenization, osmotic shock, and EDTA treatment. The selectivity offered indicates the potential of enzyme release by hydrodynamic cavitation to ease the purification in the subsequent downstream processing.     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

Association of the cyclic AMP chemotaxis receptor with the detergent-insoluble cytoskeleton of Dictyostelium discoideum

Treatment of 6-h differentiated Dictyostelium discoideum cells with the nonionic detergent Triton X-100 dissolves away membranes and soluble components, as judged by marker enzyme distributions, leaving intact a cytoskeletal residue that contains approximately 10% of the cell protein and 50% of the actin. Nitrobenzooxadiazo-phallacidin staining for F-actin and electron microscopy of detergent-extracted whole-mounts indicate that the cytoskeletons retain the size and shape of intact cells and contain F-actin in cortical meshworks. The cytoskeletons contain little if any remaining membrane material by morphological criteria, and the plasma membrane enzymes cyclic nucleotide phosphodiesterase and alkaline phosphatase are absent from the insoluble residue, which retains only 15% of the membrane concanavalin A-binding glycoproteins. This detergent-insoluble residue retains a specific [3H]cAMP-binding site with the nucleotide specificity, rapid kinetics and approximate affinity of the cAMP receptor on intact cells. Upon detergent extraction of cells, the number of cAMP-binding sites increases 20-70%. The binding site is attached to the insoluble residue whether or not the cAMP receptor is occupied at the time of detergent addition. The pH dependence for recovery of the insoluble cAMP-binding site is much sharper than that on intact cells or membranes with an optimum at pH 6.1. Conditions of pH and ionic composition that lead to disruption of the cytoskeleton upon detergent treatment also result in the loss of cAMP binding. During differentiation, the detergent- insoluble cAMP binding increases in parallel with cell surface cAMP receptors and chemotaxis to cAMP.     

Promotion of Xyloglucan Metabolism by Acid pH

Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H⁺ secretion theory of auxin action.     

High pressure disruption of yeast cells: The use of scale down operations for the prediction of protein release and cell debris size distribution

Experiments were carried out aimed at establishing the effects of equipment scale down on the disruption of Baker's yeast cells in high pressure homogenisers. Data are reported on the cell debris particle size distribution (PSD) and on total protein release as a function of the applied pressure for two valve geometries and three scales of operation covering flow rates of 28, 60 and 280 L/h. A comparison of the results from the experiments indicates that over the range of parameters investigated both the total protein release and the cell debris PSDs are independent of valve geometry and flow rate through the homogeniser. These observations are discussed in the light of relevant previous publications. The cell debris PSDs have been simulated by using a recently published model and the total protein release data are described by the well-established Hetherington expression (Hetherington et al., 1971). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 642-649, 1997.     

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.     

Oxidation of oat β-glucan in aqueous solutions during processing

The study investigated carbonyl group formation along the chain and the chain cleavage of cereal β-glucan during heat treatments, high pressure homogenisation, cold storage and ascorbic acid treatment of aqueous solutions of this soluble dietary fibre. The carbonyl group content and its distribution along the chain were simultaneously determined with the chain cleavage using a HPSEC/labelling method, originally developed for water-insoluble cellulose. Ascorbic acid treatment resulted in a relatively high degree of carbonyl content and extensive degradation of β-glucan, even in concentrations typically found in foods. The thermal oxidation of the β-glucan was considerable at 120 °C in a β-glucan solution with co-extracted compounds from oat ingredient, and in the highly purified solutions in presence of ferrous ions. Oxidation also probably contributed to the molecular properties during high pressure homogenisation, even thou the main degradation mechanism is the hydrolysis caused by mechanical energy. In addition to the cleavage of the β-glucan chain, the formation of compact, high molar mass species or molecule clusters were obtained in the study after ascorbic acid, heat (120 °C) and homogenisation treatments.     

Two types of haemolytic activity of detergents

The nonionic detergent Triton X-100 at concentrations of about 0.003 to 0.008% causes swelling followed by the haemolysis of erythrocytes suspended in 160 mM KCl. The rate of haemolysis increases with the increase in detergent concentration. Finally all the erythrocytes are haemolysed. The resistance of erythrocytes to this detergent decreases with an increase in temperature. The effect of Triton X-100 is explained by increased membrane permeability to KCl and colloid osmotic haemolysis. The anionic detergent, sodium dodecyl sulfate (SDS), at concentrations of about 0.003 to 0.001% causes the haemolysis of a certain number of erythrocytes. This number increases with an increase in detergent concentration. The resistance of erythrocytes to SDS increases with an increase in temperature. The effect of SDS is explained by direct disruption of membranes by the detergent.     

A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.     

Disruption of Saccharomyces cerevisiae using enzymatic lysis combined with high-pressure homogenization

The disruption of commercially-available pressed Bakers' yeast (Saccharomyces cerevisiae) was studied using a relatively new high-pressure homogenizer (the Microfluidizer). Initial experiments using only mechanical disruption generally gave low disruption yields (i.e., less than 40% disruption in 5 passes). Consequently combinations of two disruption methods, namely enzymatic lysis and subsequent homogenization, were tested to identify achievable levels of disruption. The enzyme preparation employed was Zymolyase, which has been shown to effectively lyse the walls of viable yeast. Yeast cell suspensions ranging in concentration from 0.6 to 15 gDW/L were disrupted with and without enzymatic pre-treatment. Final total disruption obtained using the combined protocol approached 100% with 4 passes at a pressure of 95 MPa, as compared to only 32% disruption with 4 passes at 95 MPa using only homogenization. A model is presented to predict the fraction disrupted while employing this novel enzymatic pretreatment.Nomenclature a exponent of pressure (-) - b exponent of number of passes (-) - K disruption constant (MPa^-a) - N number of passes (-) - P pressure (MPa) - R total fraction of cells disrupted (-) - Ro fraction of cells disrupted after enzymatic pre-treatment (-) - X cell concentration (dry weight) (gDW/L) abbreviation DW dry weight     

The effect of bile, bile acids and detergents on calcium absorption in the chick

1. Bile from rachitic or normal chicks causes an immediate increase in the intestinal absorption of soluble calcium in rachitic and vitamin D(3)-treated chicks as tested in vivo by intestinal-loop and oral-dosing methods. 2. This effect is apparently solely due to the taurine-conjugated bile acids present in the bile and is independent of the action of vitamin D. 3. Chick bile and bile acids can increase the solubility and the absorption of calcium presented as sparingly soluble calcium hydrogen phosphate. 4. In addition, bile is necessary to some extent at least for the intestinal absorption of vitamin D(3) in the chick and this would indirectly enhance the absorption of calcium. 5. Thus bile is capable of a threefold action in the absorption of calcium in the chick. It is suggested that the direct action on sparingly soluble forms of calcium is of considerable physiological importance since most of the calcium in the normal bird's diet would be in this form. 6. Bile acids enhance the absorption of calcium in all regions of the small intestine of the chick. 7. Of a range of bile acids and detergents tested for enhancement of calcium absorption, various taurine-conjugated bile acids and sodium lauryl sulphate, an anionic detergent, are effective. A non-ionic detergent (Tween 80) and a cationic detergent (Zephiran) are without effect. 8. The ability of a substance to increase directly the intestinal absorption of soluble calcium appears to depend to some extent on an anionic detergent action, i.e. the ability to form a salt or complex soluble to some extent in both aqueous and lipid phases. 9. In chicks the immediate deposition of calcium ((45)Ca) in the bones closely reflects any increase in plasma calcium radioactivity regardless of the cause of the increase and regardless of the vitamin D(3) status. Although sodium lauryl sulphate can increase markedly the calcium absorption from the gut and the immediate deposition in the bones it has no significant effect on rickets. 10. Some of the implications of these findings are discussed.     

Release of aminopeptidase from Lactobacillus casei sp. casei by cell disruption in a Microfluidizer

Cell disruption in a Microfluidizer was a function of both operating pressure and number of passes. The operating pressure had greater effect on the disruption than did the number of passes as indicated by the magnitude of the constants from the cell disruption equation. Protein release correlated with aminopeptidase release by Lactobacillus casei sp. casei. The optimum operating pressure for enzyme extraction was 76 MPa with loss of enzyme activity about 15 to 20%.