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© 2018 Elsevier Ltd. All rights reserved. In the pharmaceutical industry, organic-based small molecules have been the most widely used therapeutics, but peptides are increasingly becoming an important class of therapeutic agent for biopharmaceutical applications. This trend is enhanced by research advances that have provided cutting-edge techniques and strategies to overcome challenges often encountered with the use of peptides. By their nature, peptide pharmaceutics are plagued with high cost of production, poor solubility, and difficulty in administration. However, the use of techniques such as peptide engineering, peptide conjugation, and formulation strategies to improve parenteral delivery are paving the way for an increased use of peptides as biologics. With these drivers, peptides are anticipated to occupy an increased share of use in the treatment of health conditions, as well as in performance on the therapeutics market. This chapter gives an overview of the drivers, restrains, and advances in the application of peptides as biopharmaceutical agents.

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d ) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed.

© 2017 Wiley Periodicals, Inc. Peptides with biological properties, that is, bioactive peptides, are a class of biomolecules whose health-promoting properties are increasingly being exploited in food and health products. However, research on targeted techniques for the detection and quantification of these peptides is still in its infancy. Such information is needed in order to enhance the biological and chemometric characterization of peptides and their subsequent application in the functional food and pharmaceutical industries. In this review, the role of classic techniques such as electrophoretic, chromatographic, and peptide mass spectrometry in the structure-informed detection and quantitation of bioactive peptides are discussed. Prospects for the use of aptamers in the characterization of bioactive peptides are also discussed. Practical applications: Although bioactive peptides have huge potential applications in the functional foods and health area, there are limited techniques in enhancing throughput detection, quantification, and characterization of these peptides. This review discusses state-of-the-art techniques relevant in complementing bioactive detection and profiling irrespective of the small number of amino acid units. Insights into challenges, possible remedies and prevailing areas requiring thorough research in the extant literature for food chemists and biotechnologists are also presented.

© 2017 Bentham Science Publishers. Bioactive proteins and peptides are recognised as novel therapeutic molecules with varying biological properties for potential medical applications. Development of protein and peptidebased therapeutic products for human use is growing steadily as they continue to receive an increasing rate of approval by the United States Food and Drugs Administration (US FDA). In this short review, we describe the current status and methodologies involved in the synthesis of protein and peptide biopharmaceuticals with an emphasis on the drivers and restrains to their exploitation in the therapeutic products sector.

Bioactive peptides (BPs) are protein hydrolysates able to induce positive physiological responses when introduced into the body, making them useful ingredients in food, cosmetic and pharmaceutical products. However, the full potential of BPs has not been fully explored because research is still lacking on economical and scalable production methods. This study is therefore aimed at the development of a bioprocess for the production of novel bioactive peptides from food proteins by exploiting fermentative and the proteolytic activities of Lactobacillus delbrueckii subsp. lactis 313 (LDL 313). The optimum parameters and conditions for the production of antihypertensive peptides using LDL 313 were obtained from the literature and covered the upstream, midstream and downstream stages. The feasibility of manufacturing BPs in a large scale was also projected by conducting an economic assessment and a quantitative analysis based on the medical needs of hypertensive patients in Malaysia. Results indicated that to meet the needs of the hypertensive population, a production level of 1.267 kg milk protein/kg peptide had to be obtained at an enzyme requirement of 0.2 kg enzyme/kg milk protein. At this production level and at a peptide market value of USD 88.5/g peptide, the annual expected value of peptide will be USD 41,858,307,460. It will take about 19.5 h to produce one batch of the peptides at a total annual cost (equipment and utilities) of USD 15,081,885. An annual revenue of USD 42 billion is therefore expected from the entire bioprocess implying that the process is economically feasible.

Monoliths are continuous adsorbents that can easily be synthesised to possess tuneable meso-/macropores, convective fluid transport, and a plethora of chemistries for ligand immobilisation. They are grouped into three main classes: organic, inorganic, and hybrid, based on their chemical composition. These classes may also be differentiated by their unique morphological and physicochemical properties which are significantly relevant to their specific separation applications. The potential applications of monoliths for molecular separation have created the need to enhance their characteristic properties including mechanical strength, electrical conductivity, and chemical and thermal stability. An effective approach towards monolith enhancement has been the doping and/or hybridization with miniaturized molecular species of desirable functionalities and characteristics. Nanoparticles are usually preferred as dopants due to their high solid phase dispersion features which are associated with improved intermolecular adsorptive interactions. Examples of such nanomaterials include, but are not limited to, carbon-based, silica-based, gold-based, and alumina nanoparticles. The incorporation of these nanoparticles into monoliths via in situ polymerisation and/or post-modification enhances surface adsorption for activation and ligand immobilisation. Herein, insights into the performance enhancement of monoliths as chromatographic supports by nanoparticles doping are presented. In addition, the potential and characteristics of less common nanoparticle materials such as hydroxyapatite, ceria, hafnia, and germania are discussed. The advantages and challenges of nanoparticle doping of monoliths are also discussed.

The genome of virulent strains may possess the ability to mutate by means of antigenic shift and/or antigenic drift as well as being resistant to antibiotics with time. The outbreak and spread of these virulent diseases including avian influenza (H1N1), severe acute respiratory syndrome (SARS-Corona virus), cholera (Vibrio cholera), tuberculosis (Mycobacterium tuberculosis), Ebola hemorrhagic fever (Ebola Virus) and AIDS (HIV-1) necessitate urgent attention to develop diagnostic protocols and assays for rapid detection and screening. Rapid and accurate detection of first cases with certainty will contribute significantly in preventing disease transmission and escalation to pandemic levels. As a result, there is a need to develop technologies that can meet the heavy demand of an all-embedded, inexpensive, specific and fast biosensing for the detection and screening of pathogens in active or latent forms to offer quick diagnosis and early treatments in order to avoid disease aggravation and unnecessary late treatment costs. Nucleic acid aptamers are short, single-stranded RNA or DNA sequences that can selectively bind to specific cellular and biomolecular targets. Aptamers, as new-age bioaffinity probes, have the necessary biophysical characteristics for improved pathogen detection. This article seeks to review global pandemic situations in relation to advances in pathogen detection systems. It particularly discusses aptameric biosensing and establishes application opportunities for effective pandemic monitoring. Insights into the application of continuous polymeric supports as the synthetic base for aptamer coupling to provide the needed convective mass transport for rapid screening is also presented.

Published by Elsevier B.V. All rights reserved. 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.

© 2015 by John Wiley & Sons, Ltd. All rights reserved. Multifunctional proteins and peptides from food proteins have been studied over the past few decades to elucidate their biological potency and the beneficial roles they play in human health. Owing to their multiple biological activities, these peptides have a wider role in modulating physiological functions such as antioxidative, antimicrobial, antihypertensive, cytomodulatory, anxiolytic, anorexic, and immunomodulatory activities in living body systems. Highlighted in this chapter is the biological role of some multifunctional peptides as well as the food proteins and enzyme(s) that are responsible for their release. Other challenges to the bioprocessing of multifunctional peptides and the need for research to address them are also discussed.

© 2015 by John Wiley & Sons, Ltd. All rights reserved. Owing to their biological roles, multifunctional peptides constitute a new generation of biologically active molecules whose potential can be exploited in several industrial applications such as functional food, pharmaceutical, and cosmeceutical industries. With the required combination and balance of research and commercial operations, major corporations can effectively harness the diverse functionalities of these peptides for enhanced nutrition and the treatment and mitigation of ill health. However, further insightful research in vivo and clinical studies are needed to unravel the mechanism and fate of these peptides en route to the body systems. This is needed to firmly establish the therapeutic potency of these peptides.

© Springer India 2015. The search for natural antioxidants is an ongoing endeavour as an aid to combat the harmful effects of free radicals. Research advances in the past few decades have shown that, by controlled enzymatic hydrolysis, natural antioxidants can be produced from food proteins. In this chapter, the role of certain antioxidative peptides derived from food proteins is discussed in relation to their prospect in the prevention of oxidative stress. The molecular diversity of these food peptides is described together with their pharmacological effects and mechanisms of action in relation to antioxidation. The production of these peptides and the elucidation of their antioxidative peptides are also presented. Owing to their therapeutic potential, antioxidative peptides derived from food proteins can be incorporated as ingredients in functional foods, nutraceuticals and pharmaceuticals, where their biological activities may inhibit product oxidation or assist in the control and prevention of diseases induced by free radicals. However, further insightful research is needed to overcome certain scientific challenges and thereby increase and promote consumer acceptance of these natural antioxidants.

Background: Effective bimolecular adsorption of proteins onto solid matrices is characterized by in-depth understanding of the biophysical features essential to optimize the adsorption performance, Results: The adsorption of bovine serum albumin (BSA) onto anion-exchange Q-sepharose solid particulate support was investigated in batch adsorption experiments, Adsorption kinetics and isotherms were developed as a function of key industrially relevant parameters such as polymer loading, stirring speed, buffer pH, protein concentration and the state of protein dispersion (solid/aqueous) in order to optimize binding performance and adsorption capacity, Experimental results showed that the first order rate constant is higher at higher stirring speed, higher polymer loading, and under alkaline conditions, with a corresponding increase in equilibrium adsorption capacity, Increasing the stirring speed and using aqueous dispersion protein system increased the adsorption rate, but the maximum protein adsorption was unaffected, Regardless of the stirring speed, the adsorption capacity of the polymer was 2.8 mg/ml, However, doubling the polymer loading increased the adsorption capacity to 9.4 mg/ml, Conclusions: The result demonstrates that there exists a minimum amount of polymer loading required to achieve maximum protein adsorption capacity under specific process conditions.

A microchip pressure-driven liquid chromatographic system with a packed column has been designed and fabricated by using poly(dimethylsiloxane) (PDMS). The liquid chromatographic column was packed with mesoporous silica beads of Ia3d space group. Separation of dyes and biopolymers was carried out to verify the performance of the chip. A mixture of dyes (fluorescein and rhodamine B) and a biopolymer mixture (10 kDa Dextran and 66 kDa BSA) were separated and the fluorescence technique was employed to detect the movement of the molecules. Fluorescein molecule was a nonretained species and rhodamine B was attached onto silica surface when dye mixture in deionized water was injected into the microchannel. The retention times for dextran molecule and BSA molecule in biopolymer separation experiment were 45 s and 120 s, respectively. Retention factor was estimated to be 3.3 for dextran and 10.4 for BSA. The selectivity was 3.2 and resolution was 10.7. Good separation of dyes and biopolymers was achieved and the chip design was verified.

A microfabricated poly(dimethylsiloxane) (PDMS) chip containing channel filled with polymer monolith has been developed for on-chip biomolecule separation. Methacrylate monolithic polymers were prepared by photo-initiated polymerization within the channel to serve as a continuous stationary phase. The monolithic polymer was functionalized with a weak anion-exchange ligand, and key parameters affecting the binding characteristics of the system were investigated. The total binding capacity was unaffected by the flow rate of the mobile phase but varied significantly with changes in ionic strength and pH of the binding buffer. The binding capacity decreased with increasing buffer ionic strength, and this is due to the limited available binding sites for protein adsorption resulting from cationic shielding effect. Similarly, the binding capacity decreased with decreasing buffer pH towards the isoelectric point of the protein. A protein mixture, BSA and ovalbumin, was used to illustrate the capacity of the methacrylate-based microfluidic chip for rapid biomolecule separation.

A microchip pressure-driven liquid chromatographic system with a packed column has been designed and fabricated by using poly(dimethylsiloxane) (PDMS). The liquid chromatographic column was packed with mesoporous silica beads of Ia3d space group. Separation of dyes and biopolymers was carried out to verify the performance of the chip. A mixture of dyes (fluoresceinand rhodamine B) and a biopolymer mixture (10 kDa Dextran and 66 kDa BSA) were separated and the fluorescence technique was employed to detect the movement of the molecules. Fluorescein molecule was a nonretained species and rhodamine B was attached onto silica surface when dye mixture in deionized water was injected into the microchannel. The retention times for dextran molecule and BSA molecule in biopolymer separation experiment were 45 s and 120 s, respectively. Retention factor was estimated to be 3.3 for dextran and 10.4 for BSA. The selectivity was 3.2 and resolution was 10.7. Good separation of dyes and biopolymers was achieved and the chip design was verified.

This chapter deals with the downstream processes of lactobacilli proteinases recovery, isolation and purification strategies. One of the most pressing bioprocess challenges in the area of production of pharmaceutical/biological materials is that of isolation and purification of the finished products. Although several isolation and purification methods exist for proteins, they must all be weighed out based on their merits and demerits. From the already established molecular and particle separations using membranes, centrifugation and phase-partitioning techniques, combinatoric techniques can be developed to ensure proteinase purification at the minimum allowed cost and high product purity. The industrial-scale use and commercialisation of lactobacilli proteinases will require the inputs of scientists, engineers, controls specialists, quality experts and economists. Further, with the important role and prospect of lactobacilli proteinases in industry, more targeted research is needed to aid in the development, purification and characterisation of robust enzymes with desired properties for wider industrial purposes.

Lactobacilli cell-envelope proteinases (CEPs) have demonstrated numerous biopharmaceutical applications in the development of new streams of blockbuster nutraceuticals; thus, the development of efficient and commercially viable methods for CEP extraction will promote their full-scale application. In this study, the sub-cellular location of CEPs in Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was identified and the effects of different extraction methods were investigated for their ability to efficiently release CEPs from LDL 313. Significantly high relative proteinase activity of~95% was detected in cell-wall fractions and ~5% activity was observed for osmotic fluids, implying that proteinases in LDL 313 are cell-wall bound. CEPs were released from cell-wall via incubation in calcium-free buffer, indicating the enzyme is liable to self-digestion and ionic misfolding. Of the different extraction methods investigated, the use of 5. M LiCl was the most suitable, under the conditions of experimentation, for releasing high levels of CEPs from LDL 313.

This chapter focuses on the metabolism of genus Lactobacillus, with emphasis on the proteolytic potential of the well-known species in order to highlight their potential use in the production of bioactive peptides. Fermentation activities for most of the literature references used in this chapter were conducted in batch mode but the focus of the studies was not on comparing fermentation modes and their effects on proteinase yields. The vast diversity of lactobacilli proteinases represents an industrially feasible avenue for exploitation in biotechnological applications. Continued improvement of proteinase yields depends on a number of factors: identification of highly proteolytic strains, improvement of proteinase yields via optimisation of mediacomponent and process parameters and efficient product recovery. Successfully addressing these issues will require a multidisciplinary approach, encompassing fields such as microbiology, statistics and mathematical modelling and bioprocess engineering.

There is a mounting interest in the therapeutic potential of bioactive peptides which collectively present a cornucopia of bioactivities for exploitation in vivo. Bioactive peptides trigger certain functionalities such as antioxidative, antimicrobial, antihypertensive, cytomodulatory and immunomodulatory activities in the living body system. With research and development, there exists an opportunity to effectively harness these functionalities for the treatment, prevention and mitigation of different medical conditions. This critical review discusses some potential therapeutic applications of bioactive peptides in the light of advances in general biopharmaceutical production based on proteomics and genomics.

The effect of different sugars (glucose, glycerol, maltose, galactose and lactose) on cell-membrane-associated proteinase production by Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was investigated. The experimental results showed that aside glycerol and galactose, all the other sugars supported high growth levels of LDL 313, with glucose displaying the maximum biomass concentration of 0.85 mg/mL dry cell weight for cells harvested at the mid-exponential phase of ~12 h after inoculation. The specific proteinase yield, a measure of the rate of proteinase production relative to cell wall biosynthesis, was used to evaluate the preferential degree of proteinase metabolism as induced by the consumption of different sugar substrates by LDL 313. It was found that maltose displayed the highest specific proteinase yield of 12.59 U/mg sugar consumed. Further, molecular differences were observed in the SDS electrophoretic profile of cell surface proteins generated for the different carbon substrates. This is a preliminary study which supports the inference that different sugars stimulate the production of different cell-surface proteins with a significant effect on cell proteinase activity.

Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1 < X < 10 % vol/vol), agitation speed (0 < S < 200 rpm), varying incubation temperature (30 < T < 50 °C), starting pH (4.5 < pH < 7) and carbon/nitrogen ratio of production medium (0.2 < r < 5) had an individual effect on proteinase yield (p < 0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45 °C incubation temperature, 2 % (v/v) inoculum size of OD560 = 1, 150 rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8 h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions.

The effect of process conditions on the growth and production of cell-envelope-associated proteinase (PrtL) by Lactobacillus delbrueckii subsp. lactis ATCC ® 7830™ (LDL 313) was studied. Cell growth was profuse under the temperature conditions of 37°C, 40°C and 45°C, with cell growth rates, µ max increasing with temperatures. Initial culture pH of 6 displayed the highest PrtL yield of 2.33U. For each growth temperature the cell growth rates under anaerobic conditions were markedly higher than microaerophilic conditions, attributable to efficient sugar utilization under anaerobic fermentation. Overall, the proteolytic activities of cell-bound PrtL (bPrtL) were found to be higher than that of released PrtL (rPrtL). Higher rPrtL activities were displayed by anaerobic cultures over the entire fermentation period whereas the converse was true for bPrtL with microaerophilic cultures in the mid-late exponential phase displaying higher bPrtL activities than anaerobic cultures. Further, the proteinases had caseinolytic specificity for ß-casein and k-casein placing them in the cell-envelope-associated proteinases (CEPs) class I (CEP I) of the lactococcal CEPs grouping. This study provides insights into conditions for profuse growth and proteinase production by LDL 313 for subsequent technological applications in fermented foods, the dairy industry and bioactive peptide production.

Bioactive peptides from food proteins have been studied over the past decade to elucidate their biological potency in the major systems of the body such as the digestive, cardiovascular, nervous, and immune systems. Some bioactive peptides have been established for their antimicrobial roles as well as humoral and cell-mediated immune functions and thus have prospects of being incorporated as ingredients in functional foods, nutraceuticals and pharmaceuticals where their biological activities may assist in the control and prevention of diseases.However, further insightful research on the pharmacokinetics of immunomodulatory peptides invivo and clinical studies are needed to firmly establish their therapeutic potency.

Recent studies have shown that most peptide sequences encrypted in food proteins confer bioactive properties after release by enzymatic hydrolysis. Such bioactivities, which include antithrombotic, antihypertensive, immunomodulatory and antioxidant properties, are among the traits that are of biological significance in therapeutic products. Bioactive peptides could therefore serve as potential therapeutic agents. Moreover, research has shown that peptide therapeutics are toxicologically safe, and present less side effects when compared to small molecule drugs. However, the major conventional methods i.e. the synthetic and biotechnological methods used in the production of peptide therapeutics are relatively expensive. The lack of commercially-viable processes for large-scale production of peptide therapeutics has therefore been a major hindrance to the application of peptides as therapeutic aids. This paper therefore discusses the plausibility of manufacturing pharmaceutical-grade bioactive peptides from food proteins; the challenges and some implementable strategies for overcoming those challenges.