Extractive Fermentation Employing Ion-Exchange Resin to Enhance Cell Growth and Production of Metabolites Subject to Product or By-Product Inhibition

In recent years, commercial production of proteins and metabolites from microbial fermentation for industrial applications has increased significantly. Innovative approaches are directed towards the improvement of the conventional batch fermentation method and the segregated downstream processing of target product to improve the overall process efficiency and to ensure that the process is economically viable. Feedback inhibition is a common problem faced during fermentation process when the concentration of end-product/by-product reaches a certain level. The excessive accumulation of end-product/by-product in the culture may inhibit the growth of cell and represses the secretion of target metabolite. In the production of many fermentative products such as antibiotics, amino acids, and fungal metabolites, a serious problem of feedback inhibition is often encountered. Cultivation of lactic acid bacteria and recombinant bacteria is usually subjected to by-product inhibition. Hence, extractive fermentation via in situ ion-exchange-based adsorptive technique is a possible approach to be used industrially to mitigate feedback inhibition, aimed at enhancing fermentation performance. In this chapter, advances in this area were presented. Strategies to overcome problem related to product/by-product inhibitions by this technique via dispersed, external, and internal resin system, and the general methodology in the implementation of the technique were also discussed

Purification of a bacteriocin‐like inhibitory substance derived from pediococcus acidilactici Kp10 by an aqueous micellar two‐phase system

A polymer–salt aqueous two-phase system (ATPS) consisting of polyethylene–glycol (PEG) with sodium citrate was developed for direct recovery of a bacteriocin-like inhibitory substance (BLIS) from a culture of Pediococcus acidilactici Kp10. The influences of phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and sodium chloride (NaCl) on the partition behaviour of BLIS was investigated. Under optimum conditions of ATPS, the purification of BLIS was achieved at 26.5% PEG (8000)/11% sodium citrate with a TLL of 46.38% (w/w), VR of 1.8, and 1.8% crude load at pH 7 without the presence of NaCl. BLIS from P. acidilactici Kp10 was successfully purified by the ATPS up to 8.43-fold with a yield of 81.18%. Given that the operation of ATPS is simple, environmentally friendly and cost-effective, as it requires only salts and PEG, it may have potential for industrial applications in the recovery of BLIS from fermentation broth

Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10

An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30min and FR of 20mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10-15kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10

Reverse micellar system in protein recovery - a review of the latest developments

Reversed micellar system (RMS) is an innovative technique used for the isolation, extraction and purification of proteins and enzymes. Studies have demonstrated that RMS is an efficient purification technology for extracting proteins and enzymes from natural plant materials or fermentation broth. Lately, reverse micelles have wider biological applications and the ease of scaling up and the possibility for the continuous process have made RMS a vital purification technique in various fields. In this study, an extensive review of RMS with the current application in biotechnology is examined. This review provides insights into the fundamental principles, key variables and parameters of RMS. In addition, a comparative study of RMS with other liquid-liquid extraction techniques is also included. The present review aims to provide a general overview of RMS by summarising the research works, since the introduction of the technology to current development

Protein separation using surfactant precipitation

Protein precipitation using a variety of surfactants has been shown to have considerable potential as a protein separation technique, and considerable work on using anionic surfactants has been carried out by previous researchers. However, anionic surfactants are only suitable for high pI proteins due to concerns about protein stability. Therefore, the first aim of this work was to develop a surfactant precipitation method for low pI proteins based on using cationic surfactants. The effect of important parameters such as the molar ratio of TOMAC to protein (Rp), and pH on the precipitation of bovine serum albumin, α-amylase, and trypsin inhibitor were examined. Recovery of the TOMAC-protein complex by solvent extraction and counter-ionic surfactant (AOT) was also studied. Varied results were obtained for the three proteins, and were correlated with protein properties, and it was found that the protein’s hydrophobicity and molecular weight were the best predictors for precipitation efficiency and recovery. The second aim of this research was to examine the feasibility of using a biocompatible surfactant – methyl ester sulphonate (MES) as a precipitating-ligand for target proteins in this surfactant precipitation technique. This work was a major breakthrough in the application of a new generation of ‘green’ surfactants for protein extraction. Lysozyme was used as a model protein in a single component system, and the influence of Rp, and pH were examined. Similarly, the recovery of the precipitate using solvent extraction and a counter-ionic surfactant, AOT, was studied. The performance of MES in precipitation was compared to a conventional surfactant, AOT, and it was found that their performance was comparable. This further highlighted its potential to be used as precipitant in protein purification. The third aim of this work was to apply the surfactant precipitation method to the purification of a target protein from a real industrial sample. Bacteriocin produced by Pediococcus acidilactici Kp10 was chosen as a target protein for this purpose. With a concentration of 11.56 mM of AOT (pH 4), precipitate recovery by acetone (0.99 mM NaCl), and a final recovery phase of 20 mM PBS (pH 7), about 86.3% of overall activity recovery, and a purification factor of about 53.8 was obtained. Further, this separation technique was shown to be better than reverse micellar extraction, and aqueous two-phase extraction in terms of performance. Hence, the surfactant precipitation technique was proven to be an effective and a viable separation method.Open Acces

Simple surface plasmon resonance bio-sensor for the quantification of recombinant human epidermal growth factor (rhEGF) in expanded bed recovery

The conventional quantification assays used monitor the performance of protein downstream processing are typically not user friendly as it is laborious, time consuming and costly. At the same time, the emergence of multi-modal adsorbent, streamline Direct HST (1.8 g/cm3) to handle high ionic strength feedstock can potentially improve the recovery of protein in expanded bed adsorption (EBA). Hence, the objectives of the study are to develop a reliable quantification assay for recombinant human epidermal growth factor (rhEGF) by Surface Plasmon Resonance and to examine its application for the monitoring of rhEGF recovery by HST adsorbent in EBA. It was developed based on the BIAcore 3000 instrument with anti-rhEGF antibody immobilized on the CM5 chip. The performances of the assay were: assay linearity (25 to 250 ng/mL), accuracy (within 10% recovery of target), precision (below 3.4% CV), intra- and inter-assay precision (less than 20% CV) and rhEGF onto Streamline HST was at pH 4 in acetate buffer without the NaC1 or at high (1.8 M) NaC1 concentration as determined from the batch binding experiment. The electrostatic interaction, hydrophobic interaction and the hydrogen bonds responsible for this. As for the elution, potassium buffer with pH 12 used exploiting the effect of electrostatic repulsion. The effect of cells (Escherichia coli and Pastoris pastoris) on the performance of HST adsorbent in capturing rhEGF was also being examined by fitting the adsorption into the langmuir isotherm. The thermodynamic parameters: maximum binding capacity (qm) and equilibrium dissociation constant (Kd) are little affected as the concentration of these biomass increased from 0 to 4.73 % w/v. the result showed that the adsorption level was not a strong function of the biomass concentration. Next, the feasibility of using the HST adsorbent to capture rhEGF in EBA was finally tested using the single component sample (only rhEGF) with the presence of biomass (4.73 % w/v E. coli or P. pastoris). Fastline 10 column (Upfront, Denmark) was used to handle 20 mL of sample volume with adsorbent settled bed height of 2 cm (1.6 mL). the results demonstrated that a consistent yield can be obtained even with the presence of cells (97.38% for control, 96.94% for P. pastoris and 96.43% for E. coli) and a stable bed can be achieved, suggesting that no interference of adsorption by biomass occurred. In conclusion, the major contributions of the study are the development of reliable SPR quantification assay and the EBA process template that is useful for rhEGF purification from real feedstock

The potential of Mimosa pudica as a biopreservative for food products: a bioprocessing perspective

The plant extracts from various parts of Mimosa pudica exhibits significant antimicrobial activities against a wide range of food borne pathogens. The presence of active constituents can be manipulated for the biopreservative application in food industry. Biotechnological approach for the production of the antimicrobial compounds from M. pudica can be achieved via cell suspension culture and hairy root culture. The bioprocessing aspects of the two systems are similar to microbial biotechnology, except the bioreactor design required. In addition, the downstream processing of the two cell culture systems is also easier compared to the conventional whole plant extraction, as metabolites secretion can be achieved using exudation technique. The selection of solvent during the extraction should be made in regard to the stability of compounds and food safety. This mini review presents an up-to-date picture on the potential use of natural antimicrobial derived from M. pudica from a bioprocessing point of view that could be the key to successful production of plant-based biopreservative in food industry

Recovery of a bacteriocin-like inhibitory substance from Pediococcus acidilactici Kp10 using surfactant precipitation

Bacteriocin is an important peptide which can be used as an anti-microbial agent in food. However, simpler and more cost-effective purification methods need to be developed compared to chromatography to enhance its commercial viability. Surfactant precipitation was employed for the first time to purify bacteriocin-like inhibitory substance (BLIS) from a fermentation broth of Pediococcus acidilactici Kp10, and the amount precipitated was investigated as a function of anionic surfactant (AOT) concentration, and pH. Protein recovery from the precipitate was accomplished using solvent extraction, and solvent type, NaCl concentration, and ionic strength of the final solution were optimised. Optimal conditions were; 1.05 mM of AOT at pH 4 for precipitation, and acetone extraction (with 1 mM NaCl), which resulted in an 86.3% yield, and 53.8 purification factor. This study highlighted the fact that surfactant precipitation can be used as a primary recovery method for BLIS from a complex fermentation broth

Lysozymes from natural rubber latex (Hevea brasiliensis): assay development and recovery using ammonium sulphate and surfactant precipitations

To date, the C-serum and the bottom fraction of natural rubber latex (NRL) are still generally being considered as waste. However, they are rich sources of lysozyme, which is a bacteriolytic enzyme that has huge potential for biotechnological exploitations. So far, there are no reports on lysozyme recovery for the valorisation of NRL by-products. Therefore, this study aimed to (1) develop and validate a lysozyme assay based on the lysoplate method using the cloudy NRL sample and Micrococcus lysodeikticus cells and (2) recover lysozyme from the NRL fractions using ammonium sulphate ([NH4]2SO4) precipitation (direct and fractional modes) and the novel sodium di-(2-ethylhexyl) sulfosuccinate (AOT) surfactant precipitation. The assay developed in this study was high in both accuracy (97.54%) and precision (13.61% relative standard deviation). The detection and quantification limits, as well as the linearity range of the assay were 0.13 U/mL, 0.04 U/mL and 0-10,445.40 U/mL, respectively. As for the lysozyme recovery from the C-serum of NRL, the performance of the AOT precipitation via negative mode (activity recovery [AR] = 67.29%; purification factor [PF] = 2.42) was comparable (p > 0.05) to that of (NH4)2SO4 precipitation (direct mode: AR = 88.44%, PF = 2.18; fractional mode: AR = 95.44%, PF = 3.05). Meanwhile, for the lysozyme recovery from the bottom fraction of NRL, the AOT precipitation (AR = 71.94%; PF = 1.73) was shown to be superior than (NH4)2SO4 precipitation (direct mode: AR = 25.68%, PF = 3.23; fractional mode: AR = 27.88%, PF = 12.87). Hence, considering its effectiveness, simplicity, rapidity, as well as its great potential to be integrated with rubber product manufacturing, the AOT precipitation is an attractive alternative recovery method for lysozyme recovery from NRL. The findings described herein are significant for the development of a downstream processing scheme for lysozyme recovery from NRL

Evaluation of antioxidant and antibacterial activities of fish protein hydrolysate produced from Malaysian fish sausage (Keropok Lekor) by-products by indigenous Lactobacillus casei fermentation

Malaysian fish sausage, Keropok Lekor (KL), is a popular fish snack in Malaysia. The fish by-products (FBPs) from KL processing comprise a significant quantity of proteins with high nutritional value. However, in Malaysia, such FBPs are either disposed of into waterways, which may cause environmental pollution, or are processed into products of low economic value, such as fertiliser and animal feed. To maximise the potential of FBPs, a microbial fermentation approach can be used to convert FBPs into bioactive fish protein hydrolysate (FPH). Bioactive FPH has higher economic value due to its potential as a bio-ingredient in the nutraceutical and functional food industries. This study aimed to evaluate antioxidant and antibacterial activities of FPHs from FBPs of KL processing produced by four indigenous Lactobacillus casei strains (LC216, LC217, LC219, and LC220). The relationship between the level and composition of free amino acids during fermentation and the antioxidant and antibacterial activities of the produced FPH was evaluated. The results showed that the FPHs produced using the four L. casei strains had higher 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities (82.8–88.4%) than that of the unfermented FBPs (78.9%). The obtained half-maximal effective concentrations (EC50) of the FPHs were also significantly lower (p < 0.05) than those of the unfermented FBPs. The ferrous chelating ability of the FPHs produced using strain LC217 improved by 68.43% compared to that of the unfermented FBPs. Furthermore, compared to the unfermented FBPs, the FPHs produced using strains LC216 and LC217 had a significantly higher (p < 0.05) maximum growth inhibition against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli and Listeria innocua. Moreover, there was a positive relationship between the degree of hydrolysis and the bioactivities. A strong positive correlation also existed between the level of antioxidant amino acids content and antioxidant activity. Overall, the L. casei fermentative production of bioactive FPHs from the FBPs from KL processing represents an effective, environmentally sustainable and economical strategy for producing bioactive bioingredients, which can be used in functional food production and food preservation