Bioprocess challenges to the isolation and purification of bioactive peptides

Food protein-derived bioactive peptides (BPs) have been reported to trigger certain physiological responses in the body, thereby influencing health positively. These peptides have attracted high research and consumer interests due to their huge potential of use in functional foods and other dietary interventions of disease control and health promotion. However, successful product development is limited by the fact that current manufacturing processes are either difficult to scale up, high in cost, or have the potential to affect the structure-activity properties of these peptides. To overcome these challenges, we have proposed in this review, the use of an integrated ‘-omics’ approach comprising in silico analysis and ‘-omics’ techniques (such as peptidomics) to respectively forecast and validate the biological and physico-chemical properties of the peptides. This information is then used for the rational design of suitable purification steps for peptides of interest. Downstream purification could also be undertaken by liquid chromatography using monolithic adsorbents physico-chemically engineered (using results of in silico analysis) for rapid isolation of peptides. By coupling the high throughput and predictive capability of ‘-omics’ to the enhanced convective hydrodynamics of monolithic columns, it becomes feasible, even at preparative scale, to produce BPs that meet the requirements of high purity, potency, and cost-effectiveness.

Recent insights into microbial catalases: isolation, production and purification

Catalase, an oxidoreductase enzyme, works as a detoxification system inside living cells against reactive oxygen species formed as a by-product of different metabolic reactions. The enzyme is found in a wide range of aerobic and anaerobic organisms. Catalase has also been employed in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to its other applications in textile, paper, food and pharmaceutical industries. New applications for catalases are constantly emerging thanks to their high turnover rate, distinct evolutionary origin, relatively simple and well-defined reaction mechanisms. The following review provides comprehensive information on isolation, production and purification of catalases with different techniques from various microbial sources along with their types, structure, mechanism of action and applications.

Isolation and characterization of microsatellite loci to DNA fingerprint the powerful owl (Ninox Strenua)

An enriched microsatellite library was constructed for the Powerful Owl (Aves; Strigiformes: Ninox strenua) from which 14 polymorphic microsatellite markers were characterized. Forty individuals (32 unrelated and four pairs of siblings) were genotyped to determine the application of these markers for genetic profiling. The mean observed and expected heterozygosity for unrelated individuals was 0.53 and 0.59, respectively. We demonstrate that this suite of markers is sufficient to unequivocally identify individuals and will be beneficial in assessing the population genetics and reproductive ecology of this species.

Isolation and restraint stress results in differential activation of corticontrophin-releasing hormone and arginine vasopressin neurones in sheep

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Activation of the hypothalamo-pituitary-adrenal axis by isolation and restraint stress during lactation in ewes : effect of the presence of the lamb and suckling

We investigated the effect of the presence and absence of lambs and suckling by lambs to attenuate activation of the hypothalamo-pituitary-adrenal (HPA) axis to isolation and restraint stress in lactating sheep. In experiment 1, blood samples were collected every 10 min from nonlactating (n = 5) and lactating (n = 5) ewes for 4 h before and during stress. In experiment 2, ewes (n = 6) were allocated to 1) nonlactating, 2) lactating with lambs absent, 3) lactating with lambs present but unable to suckle, and 4) lactating with lambs present and able to suckle. Blood samples were collected over 8 h with no stress (control day) and for 4 h before and 4 h during stress (stress day). In experiment 1, the mean (±SEM) cortisol concentrations increased significantly (P < 0.05) in nonlactating ewes during stress but did not change in lactating ewes. In experiment 2, cortisol did not vary on the control day or pretreatment of the stress day but increased (P < 0.05) during stress in all groups except lactating ewes with lambs present and able to suckle. The greatest cortisol response occurred in nonlactating ewes followed by lactating ewes with lambs absent and lactating ewes with lambs present but unable to suckle. During stress, the ACTH concentrations increased (P < 0.05) in nonlactating ewes and lactating ewes with lambs absent but not in lactating ewes with lambs present. We conclude that the activity of the HPA axis during isolation and restraint is reduced in lactating ewes and that the presence of lambs increases this level of attenuation.

Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species : screening of strains and optimization of omega-3 production

An isolation program targeting Thraustochytrids (marine fungoid protists) from 19 different Atlantic Canadian locations was performed. Sixty-eight isolates were screened for biomass, total fatty acid (TFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) content. Analysis of fatty acid methyl ester results discerned four distinctive clusters based on fatty acid profiles, with biomass ranging from 0.1 to 2.3 g L−1, and lipid, EPA, and DHA contents ranging from 27.1 to 321.14, 2.97 to 21.25, and 5.18 to 83.63 mg g−1 biomass, respectively. ONC-T18, was subsequently chosen for further manipulations. Identified using 18S rRNA gene sequencing techniques as a Thraustochytrium sp., most closely related to Thraustochytrium striatum T91-6, ONC-T18 produced up to 28.0 g L−1 biomass, 81.7% TFA, 31.4% (w/w biomass) DHA, and 4.6 g L−1 DHA under optimal fermentation conditions. Furthermore, this strain was found to produce the carotenoids and xanthophylls astaxanthin, zeaxanthin, canthaxanthin, echinenone, and β-carotene. Given this strain’s impressive productivity when compared to commercial strains, such as Schizochytrium sp. SR21 (which has only 50% TFA), coupled with its ability to grow at economical nitrogen and very low salt concentrations (2 g L−1), ONC-T18 is seen as an ideal candidate for both scale-up and commercialization.

Immunostimulatory principles from Chlorella pyrenoidosa – part 1 : isolation and biological assessment in vitro

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin™), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin™ is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin™ is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

First isolation and structural determination of cyclic β-(1→2)-glucans from an alga, Chlorella pyrenoidosa

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.

Seasonal differences in the effect of isolation and restraint stress on the luteinizing hormone response to gonadotropin-releasing hormone in hypothalamopituitary disconnected, gonadectomized rams and ewes

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

The isolation and characterisation of humic acids using a variety of alkaline extractants

The investigation into humic acid; chemistry examined the effect the extraction technique used to isolate humic material from the sediment had on the chemical/structural composition and yield of the acid; compared the various isolation techniques used in the literature and developed an extraction technique which minimises the solubilisation of the heavy metals from the inorganic sediment and, examined the complexation capacity of humic acids derived from a sediment source in relation to the heavy metal content and extraction technique.

Isolation and characterisation of Arabidopsis RAD30 and ERCC1

This thesis describes the isolation and characterisation of two plant genes, AtERCC1 and AtRAD30. Evidence from protein homology comparisons, and functional complementation, in vitro mutagenesis, or interaction assays suggests the involvement of these genes in the repair or tolerance, respectively, of UV-induced DNA change.

The male germ unit of Rhododendron : quantitative cytology, three-dimensional reconstruction, isolation and detection using fluorescent probes

The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.

Is psychotherapy a vector for isolation and anomie?

A key traditional question the client learns in the conventional psychotherapies is ‘Am I getting what I want?’. But can this question incite a mindset that does not align with the ‘give and take’ essence of sustainable everyday relations? Is it possible that the psychotherapies—if these practices can be bundled together—might teach clients to become more self-centred and relationally illiterate? MARK FURLONG suggests that well-intentioned practitioners can inadvertently de-empathise, ignore or even disrupt their clients’ intimate networks. Findings from his research support the proposition that the action of the mainstream therapies tends to undermine the service users’ prospects for sustainable personal relationships. Exceptions were found in the specialist settings of paediatric and aged care, and in narrative and family therapy practice.

Isolation and characterisation of polymorphic microsatellite loci for Noisy Miners Manorina melanocephala, with successful cross-amplification in Bell Miners M. melanophrys

We characterized 15 polymorphic microsatellite loci identified from a Noisy Miner (Manorina melanocephala) blood sample using 454 whole genome shotgun sequencing. Levels of polymorphism were assessed using 15 Noisy Miners. The average number of alleles per locus was 5.1. These loci were then cross-amplified to assess their suitability in a single population of Bell Miners (M. melanophrys). Given the landscape level impact that these species are having on the health of vegetation and biodiversity of a range of vertebrates throughout much of south-eastern Australia, these primers will help identify colony dispersal patterns and thus aid in modeling predictions of miner presence and tenure length in threatened ecosystems.