Downstream Processing Steps Research Articles

Extended gene expression for Gag VLP production achieved at bioreactor scale

AbstractBACKGROUNDTransient gene expression has been widely used for virus‐like particles (VLP) production in both small‐ and large‐scale systems. The extended gene expression (EGE) technique is based on repeated medium exchanges and retransfections of cell culture to prolong the production phase. The aim of this study was to demonstrate scalability of the approach by operating EGE continuously in a controlled bioreactor to obtain similar results to those achieved with shake flask EGE for VLP production. A standard batch cultivation was also carried out in shake flask.RESULTSFor EGE in shake flasks and in a bioreactor, cell viability was comparable; however, the bioreactor enabled much higher cell densities and specific growth rates than the shake flasks. Owing to this increased cell growth in the bioreactor, the percentage of GFP‐positive cells was considerably lower at the end than in the shake flasks. GagGFP VLP titers were similar in both shake flasks and bioreactor. Nanoparticle tracking analysis revealed that the ratio of VLPs/total particles (VLPs and cell vesicles) was higher in the shake flasks than in the bioreactor, possibly due to higher cell densities achieved in the bioreactor.CONCLUSIONSIn this study, EGE methodology was carried out in a bioreactor system for the first time while maintaining GagGFP production titers. Overall, our findings call for further optimization and implementation of common downstream processing steps to improve yield and VLP quality. © 2018 Society of Chemical Industry

Direct recovery of mangostins from Garcinia mangostana pericarps using cellulase-assisted aqueous micellar biphasic system with recyclable surfactant

The α- and γ-mangostins from Garcinia mangostana pericarps (GMP) exhibit antioxidant, anti-bacterial, anti-inflammatory and anti-tumor properties. The extraction yields α- and γ-mangostins are often limited by the presence of the GMP cell walls. Therefore, the extraction and recovery of mangostins from GMP with an Aspergillus niger cellulase-assisted aqueous micellar biphasic system (CA-AMBS) was developed for enhanced yield of mangostins. Effects of the concentration of cellulase, the incubation time and the temperature of the system on the recovery of mangostins were investigated. The optimum condition for the recovery of α- and γ-mangostins was obtained with the addition of 0.5% (w/w) cellulase incubated at 40°C for 2h. High log partition coefficients of α-mangostins (log Kα 4.79±0.02) and γ-mangostins (log Kγ 4.02±0.02) were achieved. High yields of α-mangostins (73.4%) and γ-mangostins (14.0%) were obtained from the micelle-rich bottom phase with final concentrations of 3.67mg/mL and 0.70mg/mL, respectively. The back-extraction of mangostins was performed with the addition of 30% (w/w) of isopropanol and 0.05M of KCl at pH 9 to the bottom phase of the CA-AMBS. The yields of the α- and γ-mangostins from GMP were considerably enhanced with the CA-AMBS and the direct recovery of mangostins was demonstrated without additional downstream processing steps.

Calcium Alginate Bead-mediated Enhancement of the Selective Recovery of a Lead Novel Antifungal Bacillomycin Variant

In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF1 to AF5) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF4 was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF4 purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF4 lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO4 (2mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8mg/L was achieved with 44% selectivity at 30°C compared to unoptimized conditions (186.4 ± 6.1mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF4 variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF1 and AF2. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF4 variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans. Thus, endotoxin removal is important in protein purification for academic research and in GMP manufacturing of biopharmaceuticals. Several methods exist to remove endotoxin, but often require additional downstream-processing steps, decrease protein yield and are costly. These disadvantages can be avoided by using an integrated endotoxin depletion (iED) wash-step that utilizes Triton X-114 (TX114). In this paper, we show that the iED wash-step is broadly applicable in most commonly used chromatographies: it reduces endotoxin by a factor of 103 to 106 during NiNTA-, MBP-, SAC-, GST-, Protein A and CEX-chromatography but not during AEX or HIC-chromatography. We characterized the iED wash-step using Design of Experiments (DoE) and identified optimal experimental conditions for application scenarios that are relevant to academic research or industrial GMP manufacturing. A single iED wash-step with 0.75% (v/v) TX114 added to the feed and wash buffer can reduce endotoxin levels to below 2 EU/ml or deplete most endotoxin while keeping the manufacturing costs as low as possible. The comprehensive characterization enables academia and industry to widely adopt the iED wash-step for a routine, efficient and cost-effective depletion of endotoxin during protein purification at any scale.

The Effect of the Drying Process on the Properties of Bacterial Cellulose Films from Gluconacetobacter hansenii

Bacterial cellulose (BC) is a polymeric material that presents unique structural and mechanical properties, high crystallinity, biocompatibility, and biodegradability. All these excellent properties make BC attractive for various biotechnological applications, ranging from biomedical to electronic devices. However, the cultivation conditions to produce BC as well as the downstream processing steps can affect the properties of BC films by modifying the microstructure of the material. This paper reports on the effect of drying method on BC films produced by Gluconacetobacter hansenii using two different procedures (oven drying at 50°C and freeze- drying). Structural changes in the BC films were evaluated using scanning electron microscopy (SEM), thermogravimetric analyses (TGA), X-ray diffraction (XRD), and Fourier-Transform Infrared (FT-IR). The two samples were visually different, as the oven dried BC was transparent while the freeze-dried BC was whitish. SEM micrographs showed that the samples had similar interweaving, but the freeze-dried material presented a higher porosity while oven dried presented collapsed fibers, leading to a volume reduction of the film. The analysis of the thermal stability showed that the films have a similar degradation profile, starting the process of degradation at 319 °C for the oven and at 325 °C for the freeze-dried samples. The BC films showed also similar crystallinities (85%), although their diffratograms exhibit different peaks suggesting that the drying process changed the percentage of Ia/Is polymorphs of the films, which was corroborated by the FT-IR results. These differences in the BC films characteristics submitted to different drying procedures can have an impact on their mechanical properties and water absorption capability, thus potentially influencing the type of possible applications.

AN INSIGHT ON ALGAL CELL DISRUPTION FOR BIODIESEL PRODUCTION

Objective: This review article deals with the effect that various cell disruption techniques have on the efficiency of lipid extraction. We have reviewed existing algal cell disruption techniques that aid the biodiesel production process.Methods: Current rise in demand for energy has led the researcher to focus on the production of sustainable fuels, among which biodiesel has received greater attention. This is due to its larger lipid content, higher growth rate, larger biomass production, and lower land use. Extraction of lipid from algae (micro and macro) for the production of biodiesel involves numerous downstream processing steps, of which cell wall disruption is a crucial step. Bead milling, high-pressure homogenization, ultra-sonication, freeze-drying, acid treatment, and enzymatic lysis are some methods of cell disruption. The cell disruption technique needs to be optimized based on the structure and biochemical composition of algae.Result: The lipid extraction efficiency varies depending on the algal species and the cell disruption technique used.Conclusion: In-depth research and development of new techniques are required to further enhance the cell disruption of the algal cell wall for the enhanced recovery of lipids. In addition, the operating costs and energy consumption should also be optimized for the cost-effective recovery.

Optimization of enzymatic hydrolysis for effective lipid extraction from microalgae Scenedesmus sp.

Abstract Cell wall disruption is an essential downstream processing step for improving the efficiency of lipid extraction from microalgae. Enzyme-assisted extraction of lipid from microalga Scenedesmus sp. with cellulase, xylanase and pectinase, using various parameters, such as enzyme concentration, temperature, pH and incubation time, was optimized by central composite design (CCD) coupled with response surface methodology (RSM). Both the lipid extraction from microalgae and the fatty acid methyl esters (FAMEs) production under optimal conditions showed a ∼twofold in the yields compared to the control group with no enzymatic treatment. SEM images, FTIR measurement, XPS and HPLC analysis showed that the enzymatic pretreatment caused significant alterations in the cell wall structure of microalgae. And the disruption of microalgal cell walls was primarily attributed to the breakage of β-glucosidic linkages in cellulose and hemicellulose. The study showed a promising approach can lead to an improvement in the lipid extraction yield from microalgae and further provide valuable information for the use of enzymes in microalgal processes.

Identification of process conditions influencing protein aggregation in Chinese hamster ovary cell culture.

Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells. Furthermore, rapid and efficient bioprocess optimization has been performed using design of experiments (DoE) strategies. In this work, we describe a DoE-based approach for the analysis of process parameters and cell culture additives influencing protein aggregation in Chinese hamster ovary (CHO) cell cultures. Important bioprocess variables influencing the aggregation of mAb and host cell proteins were identified in initial screening experiments. Response surface modeling was further applied in order to find optimal conditions for the reduction of protein aggregation during cell culture. It turned out that a temperature-shift to 31 °C, osmolality above 420 mOsm/kg, agitation at 100 rpm and 0.04% (w/v) antifoam significantly reduced the level of aggregates without substantial detrimental effects on cell culture performance in our model system. Finally, the aggregation reducing conditions were verified and applied to another production system using a different bioprocess medium and another CHO cell line producing another mAb. Our results show that protein aggregation can be controlled during cell culture and helps to improve bioprocessing of mAbs, by giving insights into the protein aggregation at its origin in mammalian cell culture.

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate.

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 23 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37°C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33°C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media.

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process‐related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ‐650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141–149, 2018

Accessing New Monomers from Biopriviledged Molecules By Coupling Metabolic Engineering and Electrosynthesis

Recent progress in metabolic engineering has generated new microorganisms able to convert carbohydrates and waste lignin into biopriviledged molecules.1 One of these molecules, muconic acid, can be further diversified to adipic acid and terephthalic acid, which are central to the production of Nylon-6,6 and polyethylene terephthalate (PET). Its semi-hydrogenation also leads to new molecules such as trans-3-hexenedioic acid (3HDA), a compound that enables the synthesis of bio-advantaged Nylon-6,6. However, to achieve economically sustainable conversion, it is crucial to streamline the fermentation and downstream catalytic processing steps. This generates new challenges as biogenic impurities inherent to the fermentation broth can deactivate Pt-group metal catalysts. An alternative approach would be to utilize novel conversion schemes using robust base metals that are stable in the presence of biogenic impurities.2 We show that electrosynthesis represents a promising approach for the direct processing of unpurified broths.2,3 The water, salts, and acidic impurities already present in the medium provide a suitable electrolyte, while protons in the solution provide the hydrogen required for the desired transformation. Muconic acid was converted to 3HDA with a 94% yield and 100% faradaic efficiency using lead (Pb) electrodes.4 Under these conditions, 3HDA can be produced at a competitive price of $2.00 kg-1. The reaction mechanism and the origin of the high selectivity to 3HDA will be discussed. 1. B.H. Shanks and P. Keeling, Bioprivileged Molecules: Creating Value from Biomass, Green Chem. 2017, advance article. https://dx.doi.org/10.1039/C7GC00296C2. M. Suastegui, J. E. Matthiesen, J. M. Carraher, N. Hernandez, N. Rodriguez Quiroz, A. Okerlund, E. W. Cochran, Z. Shao, J.-P. Tessonnier, Combining Metabolic Engineering and Electrocatalysis: Application to the Production of Polyamides from Sugar, Angew. Chem. Int. Ed., 2016, 55, 2368-2373. http://dx.doi.org/10.1002/anie.2015096533. J. E. Matthiesen, J. M. Carraher, M. Vasiliu, D. A. Dixon, J.-P. Tessonnier, Electrochemical Conversion of Muconic Acid to Biobased Diacid Monomers, ACS Sustainable Chem. Eng., 2016, 4, 3575-3585. http://dx.doi.org/10.1021/acssuschemeng.6b006794. J. E. Matthiesen, M. Suástegui, Y. Wu, M. Viswanathan, Y. Qu, M. Cao, N. Rodriguez-Quiroz, A. Okerlund, G. Kraus, D. R. Raman, Z. Shao, J.-P. Tessonnier, Electrochemical Conversion of Biologically Produced Muconic Acid: Key Considerations for Scale-up and Corresponding Technoeconomic Analysis, ACS Sustainable Chem. Eng., 2016, 12, 7098-7109. http://dx.doi.org/10.1021/acssuschemeng.6b01981

Adenovirus Particle Quantification in Cell Lysates Using Light Scattering.

Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand. Light scattering is an established process to measure virus particles' size, though due to cell impurities, adequate quantification of adenovirus particles in cell lysates by light scattering has been impossible until today. This report describes a new method using light scattering to measure virus concentration in nonpurified cell lysates. Here we report application of light scattering, a routine method to measure virus particle size, to virus quantification in enzymatically conditioned crude lysates. Samples are incubated with phospholipase A2 and benzonase and filtered through a 0.22 μm filter cartridge prior to quantification by light scattering. Our results show that this treatment provides a precise method for fast and easy determination of total adenovirus particle numbers in cell lysates and is useful to monitor virus recovery throughout all downstream processing.

Comparison of different inactivation methods on the stability of Indian vaccine strains of foot and mouth disease virus

In this study, the efficiency of binary ethyleneimine (BEI) in combination with formaldehyde (FA) and glutaraldehyde (GTA) in inactivating the Indian FMDV vaccine strains is compared. The acceptable safety of virus inactivation was faster and the inactivation rates were increased many-folds with combination of inactivants than BEI alone. FMDV A was inactivated rapidly than the other two serotypes with BEI + FA combination. Inactivation plots were linear for all the serotypes irrespective of inactivation process. Further, the integrity studies on 146S using serotype specific ELISA indicated no significant change in the antigenic mass of all the serotypes throughout the inactivation process. However, the loss of 146S antigen occurred in the subsequent steps of downstream processing. Further, the studies on intactness of viral RNA using real time PCR indicated the amplification of 1D gene sequences in all the preparations of timed samples irrespective of serotypes/inactivation process. Further, inactivated virus preparation (146S) was more stable at lower temperatures for all the serotypes/inactivation process. Among the combinations of inactivants, BEI + FA out performed compared to BEI + GTA and BEI in terms of inactivation rates, 146S yield and its storage stability, irrespective of the serotypes.

Continuous Heterogeneous Crystallization on Excipient Surfaces

A novel continuous heterogeneous crystallization process is developed in which the active pharmaceutical ingredient (API) is crystallized directly on the surface of an excipient within the crystallizer. The product is subsequently dried and formed into tablets without the need for complex downstream processing steps, such as milling, sieving, granulation, and blending. The aim is to eliminate many steps of the particle processing in drug product manufacturing. The APIs and excipients systems were selected by investigating heteroepitaxial mechanisms. The effects of various process parameters, such as temperature, residence time, and mode of operation, on drug loading were studied. Three different process designs—mixed suspension mixed product removal with a traditional impeller, Viscojet mixing, and a fluidized bed crystallizer—were utilized for direct crystallization of the API on the surface of the crystalline excipient. The excipient selection and process design parameters have a significant impact on d...

Techno-economic assessment of hydrogen production based on dual fluidized bed biomass steam gasification, biogas steam reforming, and alkaline water electrolysis processes

In this paper, three CO2-neutral H2 production processes were investigated. The three employed technologies were dual fluidized bed (DFB) biomass steam gasification, biogas steam reforming (BSR), and alkaline electrolysis (AEL) powered by renewable electricity with their necessary downstream separation and purification process steps. Aspen Plus process simulations were carried out in order to calculate the mass and energy balances of the three processes. In addition, a techno-economic assessment was carried out for a fictitious business producing H2 at a rate of 90kgh−1 in Austria in 2016. This assumption was used so that the economic feasibility of these investigated processes could be directly compared.The simulation results show that the DFB biomass steam gasification process has a higher H2 conversion rate (51.4%) but a lower fuel based H2 production efficiency (38.9%) than the BSR process (27.2% and 47.0%, respectively). Moreover, the alkaline electrolysis process shows the highest energy based H2 conversion efficiency at about 66%.The results of the economic assessment show that the DFB biomass steam gasification process has investment costs of 12.1 MEUR followed by the biogas steam reforming process with investment costs of 9.9 MEUR. The alkaline electrolysis process has investment costs of 4.4 MEUR. However, the after tax H2 break-even price of the DFB process is the lowest with 0.148EURkWh−1. The BSR process has an after tax H2 break-even price of 0.152EURkWh−1 and the AEL process has an after tax H2 break-even price of 0.191EURkWh−1. The net present value (NPV) calculations reveal that the BSR process has the highest NPV, followed by the AEL process and the DFB biomass steam gasification process. However, the NPV of all three processes are very similar. In general, all three H2 production processes perform at the same level based on the results of the process simulation and the chosen economic assumptions.

83 - Manufacturing solutions for autologous cell therapies

The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that are robust and are compatible with limited downstream processing steps. As more cell therapeutics progress through clinical testing, current in vitro culture methods lack efficiency and reproducibility. Moreover, single-use devices and high quality animal origin-free reagents support the future implementation of manufacturing solutions that will be required following clinical success. Here, we describe the implementation of single use bioreactors and high quality media for expansion of cell therapies. We focus on an example of autologous T cells. The presentation will review solutions addressing animal origin-free expansion of cells within the context of different upstream process development steps with good cell yield, high viability and maintenance of identity. Start to finish solutions for manufacturing, including high quality reagents, are key enabling technologies for success in commercializing cell therapies.

Electrofiltration improves dead-end filtration of hyaluronic acid and presents an alternative downstream processing step that overcomes technological challenges of conventional methods.

Hyaluronic acid (HA) dispersion obtained from the bacteria Streptococcus equi was concentrated by electrofiltration. In the conventional downstream processing of HA, extraction and precipitation lead to increase in environmental issues, structural changes, and time and energy related costs. Using electrofiltration as an alternative technology delivers solutions to these limitations. Experiments were conducted in order to test the applicability of electrofiltration to downstream processing of the negatively charged HA. The structural changes and molecular weight distributions, often a consequence of the employed separation method, were tested by analysis of the initial dispersions and final products. In comparison to the conventional filtration, concentration factors were increased up to almost four times without any detectable structural change in the final product.

Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass.

Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an ∼20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level.IMPORTANCE We show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives.

Conventional optimization of aqueous extraction of pullulan in solid-state fermentation of cassava bagasse and Asian palm kernel

Abstract Solid-liquid extraction (leaching) is an important downstream processing step for the recovery of pullulan from solid state fermentation. In this study, pullulan was produced in a laboratory scale solid state fermentor (working volume 1.9 L) by Aureobasidium pullulans using cassava bagasse and Asian palm kernel as solid substrates. Pullulan was recovered from the substrate after fermentation by solid-liquid extraction. The effects of different factors, such as volume ratio of solvent and solid, pH of solvent, temperature of the system and agitation speed on the recovery of pullulan by Aureobasidium pullulans from solid state fermentation using cassava bagasse and Asian palm kernel as solid substrate. The leaching kinetics was followed and the first order rate constants for cassava bagasse and Asian palm kernel were found to be 0.167 min −1 and 0.144 min −1 respectively.

Electrotechnologies, microwaves, and ultrasounds combined with binary mixtures of ethanol and water to extract steviol glycosides and antioxidant compounds from Stevia rebaudiana leaves

The aim of this work was to investigate the potential use of non-conventional technologies (microwaves, ultrasounds, pulsed electric fields, and high voltage electrical discharges) to enhance the extraction of high-added value compounds (mainly stevioside and rebaudioside A), and antioxidants (total phenolic compounds, flavonoids, chlorophylls, and carotenoids), from Stevia rebaudiana leaves. After treatment at equivalent energy, a supplementary diffusion was performed until 1 h. Results demonstrated that when water (100%) was used as solvent, the maximum recovery of stevioside (50.8 ± 0.1 mg/g) was obtained after ultrasound-assisted extraction, while microwave allowed the highest yields of rebaudioside A (22.7 ± 0.1 mg/g). However, pulsed electric field technology was the most efficient to recover stevioside (44.2 ± 0.1 mg/g) and rebaudioside A (22.4 ± 0.3 mg/g) when using 50% ethanol-water. Results obtained through this work pave the way toward the possible scaling-up of some of these non-conventional technologies for a multistep selective extraction of valuable compounds from Stevia leaves. Practical applications Recovering high added value compounds (i.e. stevioside and rebaudioside A) from stevia leaves in food industry is usually performed using thermal extraction under agitation after drying and grinding. Although efficient, this technique generates an extract not only rich in targeted compounds but also in impurities, which complicates the downstream processing steps. Selective extraction of stevioside and rebaudioside A using non-conventional extraction technologies (i.e. pulsed electric field, etc.) may allow reducing the processing steps (grinding, downstream purification, etc.), and thus an alternative to the conventional method. For this reason, investigating the impact of these technologies at laboratory scale to recover target molecules from stevia leaves is of paramount importance before scaling up the process.