Current Biochemical Engineering (Discontinued) - Volume 2, Issue 1, 2015

The stringent requirements and high cost involved in the production of biomolecules from pharmaceutical, food and speciality chemical industries demand appropriate real-time process monitoring and control for downstream processes. Process analytical technology (PAT) is now an established concept for process monitoring and quality compliance in the pharmaceutical industries and is rapidly gaining importance in the bioprocessing industries. PAT tools of importance to biotechnological industries include optical spectroscopic methods such as Raman spectroscopy, near- and mid-infrared spectroscopy, attenuated total reflection Fourier transform spectroscopy, acoustic emission spectroscopy, coupled with spatial methods such as digital imaging and hyperspectral chemical imaging. These tools have potential application in monitoring and control of unit operations in downstream processing such as crystallisation, freeze-drying, centrifugation and filtration. In this review we provide an overview of the PAT tools, including spectroscopic methods and imaging technologies, followed by a critical analysis of their application in various unit operations, their potential and the challenges encountered. The ‘big data’ obtained from various PAT tools demands the application of multivariate data processing methods. We also provide a brief discussion of the multivariate statistical methods (chemometrics) applied to extract the rich information contained in typical data obtained from various PAT tools.

This article review strategies for Candida antarctica lipase B (CALB) production in Pichia pastoris expression system and the techniques used for separation and purification of CALB. Due to ease of purification, most researchers prefer to express CALB as extracellular product. Though, CALB is secreted into growth medium, it is significant to recover target protein with minimal loss. Protein purification involves sequence of steps and the extent of CALB purification depends on the final application of the product. Here, cell clarification techniques such as centrifugation, membrane filtration and chromatographic techniques like ion-exchange, affinity, hydrophobic interaction and gel permeation are discussed to purify CALB. As immobilized form of CALB offers numerous advantages over free enzyme, various immobilization techniques have also been discussed.

Compounds extracted from plants with biological activity are the subject of interest for their role in the pharmaceutical and nutraceutical industries. Extraction of these compounds has been traditionally done using conventional methods using organic solvents and applying stringent conditions such as high temperatures resulting in the decrease of yield and purity. Developing alternative ecofriendly extraction techniques with better selectivity and efficiency are highly desired. In the past decade supercritical fluid extraction (SFE) technique has gained lot of attention in isolation of bioactive compounds from plants. The present review focus on the principles of supercritical fluid extraction technique and recent developments in the extraction of functional ingredients from plants. Fundamental concepts emphasising the procedures needed for the supercritical extraction of bioactive compounds from plants have been discussed along with its applications, advantages and disadvantages. Based on the extraction capabilities and many advantages of SFE, this technology is likely to be a highly beneficial and useful for extraction of compounds from plant products.

Bioseparation and downstream processing occupy a complex and important position in biotechnology and biochemical engineering. Purification and concentration of biological materials requires new approaches that are gentle enough to protect the biological activity. Osmotic membrane distillation (OMD) is an interesting alternative for the concentration of thermoliable compounds at ambient temperature and atmospheric pressure without product deterioration. The main advantage of OMD process is maximum achievable concentration and can be easily integrated with other separation and purification processes. This review describes the operational principle, theoretical aspects and recent applications of osmotic membrane distillation process in biotechnology and allied fields.

Aqueous two-phase extraction has been effectively used for the separation and purification of several biological molecules. The main advantages of aqueous two-phase extraction are biocompatibility, scope for continuous operation, process integration and ease of scale up. In downstream processing, aqueous two-phase extraction has found its applications in extraction, purification and concentration of various biomolecules. In this review, some of the applications of aqueous two-phase systemsemployed in downstream processing besides some basic aspects and a few case studies are presented. Some of the recent developments with regard to aqueous two-phase extraction are reviewed and the future prospects are also briefly discussed.

Consumer demand has increased for processed products that keep more of their original characteristics. Downstream processing refers to the purification of biological products, generally in marketable quantities especially in the production of biomolecules such as antibiotics, hormones, antibodies, vaccines, industrial enzymes, natural fragrance, flavor compounds. There have been various advances in drying with respect to quality, rehydration, and energy minimization. The choice of an appropriate drying medium is very important during dehydration and subsequent storage. Freeze-drying is considered as a suitable dehydration process for bacteria, with the ultimate goal of achieving a solid and stable final formulation. Spray drying also allows preparation of stable and functional products and can be applied to stabilize heat sensitive materials such as enzymes and probiotic bacteria. This paper is a review of drying as a unit operation in downstream processing and discusses the innovative drying technologies, stages in downstream processing, applications in downstream processing of biomolecules and microorganisms. Freeze-drying of microorganisms, optimal spray drying of enzymes, eggs and probiotics were also considered. The paper concluded that as drying technologies become more diverse and complex, dryer selection becomes an increasingly difficult and demanding task as changes in operating conditions of the same dryer can affect the quality of the product.

Bioactive peptides are specific protein fragments that have a positive impact on functions and conditions and may ultimately influence health. Milk protein derived active biological constituents, bioactive peptides, has received much attention for the biological significances. Milk peptides have shown to possess multiple physiological and physicochemical properties and are therefore regarded as very important constituents for incorporation in functional and novel foods, dietary supplements and even pharmaceuticals with the purpose of targeting specific diseases. The current review will be focusing on the different processes used for fractionation and purification of the bioactive peptides derived from milk and milk products. At lab-scale, techniques usually followed to concentrate/purify bioactive peptides are Ultrafiltration membranes, Mass exclusion chromatography, Ion-exchange chromatography, Affinity chromatography and High-performance liquid chromatography and can be used in different combinations at the industrial scale.

Interactive effects of aeration rate and agitation speed on β-carotene production by Mucor azygosporus using waste whey filtrate in a laboratory fermenter were studied. The maximum β-carotene concentration (1045 µg/g dcw) was obtained at low agitation speed (150 rpm) and moderate aeration rate (1.0 vvm) after 72 h of incubation with a biomass of 7.9 g/L. The agitation and aeration provided better homogeneity in the culture medium with greater availability of nutrients and oxygen leading to a 2.8-fold increase in β-carotene production from that in the shake flask.

Endophytic fungi were isolated from the leaves of medicinal herb Phyllanthus sp. sourced from different areas of Pune and then screened for Trichothecin production. Out of 30 endophytic fungal cultures isolated and screened, only one AAP-PS-1 was found to produce Trichothecin extracellularly in appreciable amounts and was identified as Trichothecium sp. by morphological, cultural and molecular methods. Trichothecin, a sesquiterpene from endophytic fungus Trichothecium sp. was purified using prep TLC, RP-HPLC; quantified and completely characterised by UV-VIS, FTIR, ESI-MS, HRMS and 1D and 2D NMR techniques. One liter of Trichothecium sp. yielded 4.25 mg of Trichothecin. We screened Trichothecin for antifungal activity against filamentous fungi and yeast, apoptotic activity against B16F10 cells, anticancer activity against MDA-MB-231, HeLa and B16F10 cells as well as antimetastatic activity against MDA-MB-231 cell line. It was observed that Trichothecin inhibited MDA-MB-231 cell migration and arrested B16F10 cells at G2/M phase indicating its potential as antimetastatic and antiproliferative compound. Further, for selectivity, we checked for its cytotoxicity against mouse embryonic fibroblast (NIH3T3) and human peripheral blood mononuclear cells (PBMC) and found that it was not cytotoxic in the given concentration range of Tricothecin used against cancer cell lines. Assessment of toxicity towards human RBC revealed only less than 0.1% hemolysis as compared to Triton X-100 suggesting safe nature of Trichothecin. It was also found that Trichothecin exhibits anti-fungal activity against a range of filamentous fungi and yeast. Given its potent and selective anticancer activity, Trichothecin could be used as scaffold for chemical and biological transformations to generate analogues with better pharmacological properties.

Two contaminating fungi were isolated from a bioreactor containing diethylketone and Streptococcus equisimilis, subsequently characterized at molecular level and identified as belonging to the Alternaria and Penicillium genera. The ability of these fungi to biodegrade DEK is evaluated. The kinetic parameters are estimated using four growth kinetic models for biodegradation of organic compounds available in literature. The experimental data for Alternaria sp. and Penicillium sp. was found to be better fitted by the Haldane and the Luong respectively. Biodegradation rate kinetics was evaluated using zero-order, pseudo-first order, pseudo-second order and three-half order models. The pseudo-second-order model was found suitable for all the concentrations of DEK tested for the biodegradation assays using Penicillium sp. whereas for the Alternaria sp. this model just describes properly the assays with initial concentrations of DEK higher than 0.5 g/L. The percentage of biodegraded DEK were approximately 100%, for all the initial concentrations tested.

Microbial exopolysaccharides (EPS) may have technological applications in food and pharmaceutical industries, and in environmental health. EPS may be used in foods as natural thickening, stabilizing, bodying or gelling agent, and/or as emulsifiers or as fat replacer. In addition, EPS has been reported to have health augmenting features such as prebiotic, immunostimulatory, anti-tumor and hypocholesterolemic properties. EPS producing probiotics bacteria may help enhancing organoleptic properties of fermented foods. In present study, Enterococcus faecium strains viz. Enterococcus faecium 1.1, E. faecium 2.0 and E. faecium 4.0, which had several probiotically important attributes, were investigated for their EPS producing potential. E. faecium 4.0 produced highest yield of EPS (470 mg/L) and was followed by E. faecium 1.1 (220 mg/L) and E. faecium 2.0 (180 mg/L). FTIR analysis showed that EPS from all the three E. faecium strains was homopolymer of glucose. Degradation temperatures of EPS from three E. faecium strains varied in the range of 270.4°C-279.3°C as studied by thermal gravimetric analysis. Differential scanning calorimetry of EPS from E. faecium 1.1, E. faecium 2.0 and E. faecium 4.0 showed the melting point of 55.9°C, 88.6°C and 83.5°C; and enthalpy of 102.5, 56.03, and 213.9 J/g, respectively. EPS from all the three E. faecium strains expressed growth inhibition of E. coli. Purified EPS from all three E. faecium strains exhibited excellent emulsification properties i.e. 96.70 % (E. faecium 1.1), 96.78 % (E. faecium 2.0) and 97.77 % (E. faecium 4.0).