Pharma Applications Research Articles

Single stage bi-substrate transglycosylation reaction for the synthesis of ascorbic acid 2 glucoside using immobilized α-glucosidase

Alpha glucosidases are multifunctional glycoside hydrolases with hydrolysis and transglycosylation ability. It can be utilized for the glycosidic bond synthesis or glycosylation of Ascorbic acid/Vitamin C to its stable analogue, Ascorbic acid 2 glucoside (AA2G), a compound with wide applications in cosmetics and pharma. The application of α-glucosidases for industrial scale transglycosylation is limited due to the low transglycosylation yield of free enzymes. Enzyme immobilization techniques could enable the development of efficient, reusable catalysts. Only a few glycoside hydrolases have been studied in immobilized form for transglycosylation reactions, and α-glucosidases are probably the least explored in this form. Transglycosylation activity of immobilized α-glucosidase from Aspergillus carbonarius BTCF 5 was studied for AA2G synthesis, where different immobilization techniques like calcium alginate encapsulation, adsorption on chitosan beads, covalent cross-linking on magnetic nanoparticles, and cross-linked enzyme aggregates (CLEA) were employed for the immobilization. The immobilization yield of calcium alginate encapsulated enzyme, enzyme immobilized on Fe-MNP support, enzyme immobilized on chitosan beads and as CLEA were 107%, 99%, 46% and 486%, respectively. CLEA was identified as the best immobilization technique for this bi-substrate reaction due to the high immobilization yield and activity retention (30% activity retained after 5 consecutive cycles). Enzyme immobilization increased the transglycosylation activity by 38%, yielding 118 mM AA2G against 72 mM by the free enzyme. This indicates the potential of immobilized α-glucosidase as a catalyst for synthesizing AA2G at an industrial scale.

The emulsifying ability of oleosomes and their interfacial molecules

Oleosomes are natural oil droplets, present in all organisms and abundant in oilseeds. After their aqueous extraction from oilseeds, they can be directly utilized as oil droplets in food, cosmetics and all types of oil-in-water emulsion systems. However, to expand the potential uses of oleosomes as green ingredients and to valorize oilseeds as efficient as possible, we explored their emulsifying ability. Oleosomes were extracted from rapeseeds, and 10.0 wt% oil-in-water emulsions were created after homogenization with 0.5–6.0 wt% oleosomes, and the droplet size of the emulsions and their structure was measured by laser diffraction and confocal laser scanning microscopy (CLSM), respectively. The emulsion with an oleosome concentration lower than 1.0 wt% gave unstable emulsions with visible free oil. At oleosome concentrations at 1.5 wt% or higher, we obtained stable emulsions with droplet sizes between 2.0 and 12.0 µm. To investigate the role of the oleosome interfacial molecules in stabilizing emulsions we also studied their emulsifying and interfacial properties (using drop tensiometry) after isolating them from the oleosome structure. Both oleosomes and their isolated interfacial molecules exhibited a similar behavior on the oil-water interfaces, forming predominantly elastic interfacial films, and also showed a similar emulsifying ability. Our results show that oleosomes are not stabilizing the oil-in-water emulsions as intact particles, but they provide their interfacial molecules, which are enough to stabilize an oil-water surface up to about 2 times bigger than the initial oleosome surface. The understanding of the behavior of oleosomes as emulsifiers, opens many possibilities to use oleosomes as alternative to synthetic emulsifiers in food and pharma applications.

Acute and subacute in vivo safety assessment of developed chitosan derivatives for food applications

The acute and subacute studies of two chitosan derivatives was conducted by oral administration on Swiss albino mice. Chitosan soluble derivatives were prepared by depolymerization method using potassium persulfate. 13C NMR data have revealed that Low Molecular Weight Chitosan (LMWC) has higher (∼70%) ‘acetylated’ residues and Self Assembled Chitosan Microparticles (SAMC) consists of ‘deacetylated’ residues. For acute toxicity study, a single dose of two concentrations 2000 and 5000 mg/kg bw of the chitosan derivatives (LMWC, or SAMC) was given orally to healthy mice. Results indicated that the LD50 value for chitosan derivatives was greater than 5000 mg/kg bw. In the sub-acute toxicity studies, administration of concentrations 1000 and 2000 mg/kg bw dose of chitosan derivatives (repeated daily dose for 28 days) also did not reveal any toxicological changes in clinical observations, viz. relative organs, body weight, food consumption, biochemical parameters studied, antioxidant markers, and histopathological studies has been compared with control group. Together, present systematic study supports the oral administration of non-viscous and soluble chitosan derivatives (LMWC & SAMC) will have no toxicity may be safe to use in food and pharma applications.

Yeast cell wall extracts from Saccharomyces cerevisiae varying in structure and composition differentially shape the innate immunity and mucosal tissue responses of the intestine of zebrafish (Danio rerio).

With the rising awareness of antimicrobial resistance, the development and use of functional feed additives (FFAs) as an alternative prophylactic approach to improve animal health and performance is increasing. Although the FFAs from yeasts are widely used in animal and human pharma applications already, the success of future candidates resides in linking their structural functional properties to their efficacy in vivo. Herein, this study aimed to characterise the biochemical and molecular properties of four proprietary yeast cell wall extracts from S. cerevisiae in relation to their potential effect on the intestinal immune responses when given orally. Dietary supplementation of the YCW fractions identified that the α-mannan content was a potent driver of mucus cell and intraepithelial lymphocyte hyperplasia within the intestinal mucosal tissue. Furthermore, the differences in α-mannan and β-1,3-glucans chain lengths of each YCW fraction affected their capacity to be recognised by different PRRs. As a result, this affected the downstream signalling and shaping of the innate cytokine milieu to elicit the preferential mobilisation of effector T-helper cell subsets namely Th17, Th1, Tr1 and FoxP3+-Tregs. Together these findings demonstrate the importance of characterising the molecular and biochemical properties of YCW fractions when assessing and concluding their immune potential. Additionally, this study offers novel perspectives in the development specific YCW fractions derived from S. cerievisae for use in precision animal feeds.

Production in Nicotiana benthamiana of a thermotolerant glucose oxidase that shows enzybiotic activity against Escherichia coli and Staphylococcus aureus

Glucose oxidase (GOX) catalyzes the FAD-dependent oxidation of α-D-glucose to D-gluconolactone with production of hydrogen peroxide. This enzyme encounters many biotechnological applications from glucose sensors to applications in food, pharma and textile industries. For this purpose, recombinant GOX versions, usually derived from Aspergillus niger, are produced in fermentation systems, frequently in filamentous fungi because other production platforms such as bacteria or yeast have rendered meager results. We wondered whether A. niger GOX, more specifically a mutant version with superior thermotolerant properties, could be efficiently produced in Nicotiana benthamiana plants. To this aim, we used a tobacco mosaic virus-derived vector that is inoculated into plant tissues using Agrobacterium tumefaciens. Results exhibited the efficient production of the recombinant GOX in plants and the facile downstream purification when the recombinant protein is targeted to the apoplast, the space between plasma membranes and cell walls. The plant-made recombinant GOX displayed excellent catalytic properties in broad pH and temperature conditions. In addition to establishing a new strategy to produce recombinant GOX in plants as a green alternative to traditional fungal fermentation, we further investigated the potential application of this protein as an ezybiotic. Results exhibited a remarkable bacteriocide activity against Escherichia coli and Staphylococcus aureus.

Extrusion for pharma applications: An update

AbstractThis paper aims to give an overview of our current research and development on hot‐melt extrusion (HME) for pharmaceutical formulation and drug delivery. Unfortunately, a large percentage of marketed drugs (40%) and those in the development pipeline (90%) are poorly water soluble. We use a few examples to show that solubility of APIs can be drastically improved by using HME to mix them with different biocompatible polymers and playing with the various process parameters (extrusion temperature, screw speed, screw configuration, etc.). The advantages of this technique are then presented as well as the further development needed to make this process fully industrial for pharmaceutical purposes.

Remodelling metabolism for high-level resveratrol production in Yarrowia lipolytica

Resveratrol is a polyphenol with numerous applications in food, pharma, and cosmetics. Lack of precursors and low titer are the main problems hindering industrial scale resveratrol production. Based on previous prescreening, expressing the combination of FjTAL, Pc4CL1 and VvSTS achieved the best resveratrol titer. This was further improved to 235.1 mg/L through engineering the shikimic acid pathway, applying a modular enzyme assembly of Pc4CL1 and VvSTS, enhancing p-coumaric acid supply and diverting glycolytic flux toward erythrose-4-phosphate. The titer was increased to 819.1 mg/L following two rounds of multicopy integration of resveratrol biosynthesis and malonyl-CoA supply, respectively. The titer reached 22.5 g/L with a yield on glucose of 65.5 mg/g using an optimum fed-batch strategy in a 5 L bioreactor with morphology control. This research is the highest report on the de novo production of resveratrol in Yarrowia lipolytica and the findings lay a solid foundation for other producing polyphenols.

In silico Identification of miRNAs and Their Targets in Cluster Bean for Their Role in Development and Physiological Responses.

Cluster bean popularly known as “guar” is a drought-tolerant, annual legume that has recently emerged as an economically important crop, owing to its high protein and gum content. The guar gum has wide range of applications in food, pharma, and mining industries. India is the leading exporter of various cluster bean-based products all across the globe. Non-coding RNAs (miRNAs) are involved in regulating the expression of the target genes leading to variations in the associated pathways or final protein concentrations. The understanding of miRNAs and their associated targets in cluster bean is yet to be used to its full potential. In the present study, cluster bean EST (Expressed Sequence Tags) database was exploited to identify the miRNA and their predicted targets associated with metabolic and biological processes especially response to diverse biotic and abiotic stimuli using in silico approach. Computational analysis based on cluster bean ESTs led to the identification of 57 miRNAs along with their targets. To the best of our knowledge, this is the first report on identification of miRNAs and their targets using ESTs in cluster bean. The miRNA related to gum metabolism was also identified. Most abundant miRNA families predicted in our study were miR156, miR172, and miR2606. The length of most of the mature miRNAs was found to be 21nt long and the range of minimal folding energy (MFE) was 5.8–177.3 (−kcal/mol) with an average value of 25.4 (−kcal/mol). The identification of cluster bean miRNAs and their targets is predicted to hasten the miRNA discovery, resulting in better knowledge of the role of miRNAs in cluster bean development, physiology, and stress responses.

Chemoenzymatic Asymmetric Synthesis of Pyridine-Based α-Fluorinated Secondary Alcohols.

Fluoro‐substituted and heteroaromatic compounds are valuable intermediates for a variety of applications in pharma‐ and agrochemistry and synthetic chemistry. This study investigates the chemoenzymatic preparation of chiral alcohols bearing a heteroaromatic ring with an increasing degree of fluorination in α‐position. Starting from readily available picoline derivatives prochiral α‐halogenated acyl moieties were introduced with excellent selectivity and 64–95 % yield. The formed carbonyl group was subsequently reduced to the corresponding alcohols using the alcohol dehydrogenase from Lactobacillus kefir, yielding an enantiomeric excess of 95–>99 % and up to 98 % yield.

Profiling of bioactives and in vitro evaluation of antioxidant and antidiabetic property of polyphenols of marine algae Padina tetrastromatica

This study aimed to characterize and quantify the phenolics, hydrocolloids, lipids and carotenoids and to study antioxidant and antidiabetic property of phenolics of marine brown algae Padina tetrastromatica. Extraction using 1% acidic methanol yielded 3.6mg/g total polyphenol, 2.86mg/g flavonoid and 21.8μg/g tannin. Ultra high performance liquid chromatography-high resolution mass spectrometry/mass spectrometry (UPLC-HRMS/MS) analysis resulted in free phenolics: danshensu, quercetin derivative, luteolin, genistein, and hydroxyl-ferulic acid derivative; bound phenolics: luteolin, acacetin derivative, dioxinodehydroeckol derivative, and rosmarinic acid. The radical scavenging activities of the phenolic extracts were 10.83±2.81 and 58.85±2.28μg/mL by the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) methods, respectively. The total antioxidant activity of seaweed phenolics was 29.13±2.31μg of ascorbic acid equivalent, and the reductive capability was 32.39±3.11μg of quercetin equivalent. In addition, the extract exhibited a half maximal inhibitory concentration (IC50) of α-amylase (47.2±2.9μg) and α-glucosidase (28.8±2.3μg) inhibitory activities. Fatty acids methyl-palmitate and methyl-palmitoleate were present in neutral-, glyco-, and phospholipid fractions of total lipids (0.82%). The seaweed is rich in bioactives fucoxanthin (96.66%), polysaccharides (mg/g dry wt.) laminarin (14.78), fucoidan (1.38) and alginate (70.16) respectively. Based on the nutraceutical density, there will be potential food and pharma applications for P. tetrastromatica.

Failure analysis of AISI 440C strand dies used in twin screw extruders during polymer compounding

In view of excellent wear and corrosion resistance, AISI 440C Martensitic Stainless steel finds wide use as Dies in Twin Screw Extruders in Polymer, Food and Pharma applications, especially when either additives, which could be corrosive, are added to aid compounding of polymers or moisture and/or free radicals are present in the processing material. Two AISI 440C Die Plates part of Strand Die Assembly in a φ40 mm size Twin Screw Extruder abruptly cracked during operation in succession disrupting regular production with the second failure occurring after about two years of the Extruder in service. While visual examination of the failed Die Plates showed presence of surface / sub-surface cracks and marks of corrosion, the sectioned Die Plate revealed multiple internal cracks. The hardness survey on the failed Die Plates confirmed values between 30 and 36 HRC, which are about 20–25 HRC lower than the hardness measured in as-supplied condition. Macro-examination of the sectioned Die Plate revealed multiple micro-crack origin locations together with strong evidence of corrosion. The microstructural examination showed carbide network along grain boundaries in a matrix consisting of tempered martensite and revealed intergranular fracture. Fractographic observations using Scanning Electron Microscopy on the cracked Die Plate clearly revealed segregation of fine carbides along crack boundaries. Based on detailed analyses, which included site data collection and investigation, the failure of Die Plates was attributed to Stress Corrosion Cracking. Finally, recommendations were proposed to prevent recurrence of such failures in service.

Aerogels are not regulated as nanomaterials, but can be assessed by tiered testing and grouping strategies for nanomaterials.

Aerogels contribute to an increasing number of novel applications due to many unique properties, such as high porosity and low density. They outperform most other insulation materials, and some are also useful as carriers in food or pharma applications. Aerogels are not nanomaterials by the REACH definition but retain properties of nanoscale structures. Here we applied a testing strategy in three tiers. In Tier 1, we examined a panel of 19 aerogels (functionalized chitosan, alginate, pyrolyzed carbon, silicate, cellulose, polyurethane) for their biosolubility, and oxidative potential. Biosolubility was very limited except for some alginate and silicate aerogels. Oxidative potential, as by the ferric reduction ability of human serum (FRAS), was very low except for one chitosan and pyrolyzed carbon, both of which were <10% of the positive control Mn2O3. Five aerogels were further subjected to the Tier 2 alveolar macrophage assay, which revealed no in vitro cytotoxicity, except for silicate and polyurethane that induced increases in tumor necrosis factor α. Insufficiently similar aerogels were excluded from a candidate group, and a worst case identified. In the Tier 3 in vivo instillation, polyurethane (0.3 to 2.4 mg) elicited dose-dependent but reversible enzyme changes in lung lavage fluid on day 3, but no significant inflammatory effects. Overall, the results show a very low inherent toxicity of aerogels and support a categorization based on similarities in Tier 1 and Tier 2. This exemplifies how nanosafety concepts and methods developed on particles can be applied to specific concerns on advanced materials that contain or release nanostructures.

National conference on Biotech and Pharma Applications (NCBA)

Warm and Happy greetings to all. I am immensely happy that Department of Biotechnology of our K L University is organizing a National Conference on Biotech and Pharma Applications (NCBA-2020) during 12 March, 2020 and is going to discuss on a collection of technical papers in the proceedings. Department of Biotechnology, K L University continues to march on the way of success with confidence. On this occasion, I wish all the very best. I congratulate HOD, staff members, students of Department of Biotechnology, Delegates and Participants from different parts of the country and nations for their efforts in participating in this conference and wish the conference all the success.

Deep convolutional neural networks: Outperforming established algorithms in the evaluation of industrial optical coherence tomography (OCT) images of pharmaceutical coatings

This paper presents a novel evaluation approach for optical coherence tomography (OCT) image analysis of pharmaceutical solid dosage forms based on deep convolutional neural networks (CNNs). As a proof of concept, CNNs were applied to image data from both, in- and at-line OCT implementations, monitoring film-coated tablets as well as single- and multi-layered pellets. CNN results were compared against results from established algorithms based on ellipse-fitting, as well as to human-annotated ground truth data. Performance benchmarks used include, efficiency (computation speed), sensitivity (number of detections from a defined test set) and accuracy (deviation from the reference method). The results were validated by comparing the output of several algorithms to data manually annotated by human experts and microscopy images of cross-sectional cuts of the same dosage forms as a reference method. In order to guarantee comparability for all results, the algorithms were executed on the same hardware. Since modern OCT systems must operate under real-time conditions in order to be implemented in-line into manufacturing lines, the necessary steps are discussed on how to achieve this goal without sacrificing the algorithmic performance and how to tailor a deep CNN to cope with the high amount of image noise and alterations in object appearance. The developed deep learning approach outperforms static algorithms currently available in pharma applications with respect to performance benchmarks, and represents the next level in real time evaluation of challenging industrial OCT image data.

Experimental investigation on extractive separation of vanillic acid

Vanillic acid has received remarkable attention due to its diversified applications in cosmetics, food, flavours, tobacco, pharma, alcohols, beverages, and polymer industries. However, its separation from fermentation broths and wastewater of olive oil mills, or paper and pulp industries is still a problematic process. In this paper, the liquid – liquid extraction has been investigated for the separation of vanillic acid from aqueous streams. The assessment of equilibrium has been done by calculating the partition coefficient, dimerization constant, distribution coefficient, and extraction efficiency. The extraction ability is given in terms of extraction efficiency and distribution coefficient followed a trend:1-octanol (54.07%) > iso-butanol (51.73%) > toluene (45.23%) > xylenes mix (35.47%) > petroleum ether (31.54%) > benzene (30.44%). The obtained results were discussed with help of acid properties, type of solvent used, and their physicochemical properties such as dipole moment ( D m ), dielectric constant ( ε ), solvent polarity parameter ( E T ), solubility in water, octanol-water partition coefficient ( log K ow ), etc. This is the first attempt of study using the extractive separation method for removal or recovery of vanillic acid from aqueous solutions by the above-mentioned solvents.

Effect of Arthrospira platensis microalgae protein purification on emulsification mechanism and efficiency

In light of environmental concerns and changing consumer demands, efforts are increasing to replace frequently used animal-based emulsifiers. We demonstrate the interfacial network formation and emulsifying potential of Arthrospira platensis protein extracts and hypothesize a mechanistic change upon progressing purification. A microalgae suspension of A. platensis powder in phosphate buffer solution (pH 7, 0.1 M) was homogenized and insoluble components separated by centrifugation. Proteins were precipitated at the identified isoelectric point at pH 3.5 and diafiltrated. In interfacial shear rheology measurements, the build-up of an interfacial viscoelastic network was faster and final network strength increased with the degree of purification. It is suggested that isolated A. platensis proteins rapidly form an interconnected protein layer while coextracted surfactants impede protein adsorption for crude and soluble extracts. Emulsions with 20 vol % medium chain triglycerides (MCT) oil could be formed with all extracts of different degrees of purification. Normalized by protein concentration, smaller droplets could be stabilized with the isolated fractions. For potential applications in food, pharma and cosmetic product categories, the enhanced functionality has to be balanced against the loss in biomass while purifying microalgae proteins or other alternative single cell proteins.

De novo production of resveratrol from glycerol by engineering different metabolic pathways in Yarrowia lipolytica

Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct Yarrowia lipolytica strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 ​g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 ​g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 ​g/L was reached. This study suggests that Y. lipolytica is a promising host for resveratrol production from glycerol.

Biodegradable chitosan-sodium alginate-oleic acid nanocarrier promotes bioavailability and target delivery of lutein in rat model with no toxicity.

Efficient delivery of macular carotenoid lutein to target retinal tissue is possible with enhanced intestinal uptake remains a major challenge owing to the polarity, sensitivity to light, heat and solubility. In this study, to overcome such constraints, biodegradable polymers chitosan-sodium alginate-oleic acid based nano-carrier loaded with lutein (LNCs) was prepared and safety efficacy was examined in vivo. Acute-toxicity of LNCs (0.1, 1, 10 and 100mg/kg body weight) revealed that the LD50 of LNCs was higher than 100mg/kg body weight. In subacute-toxicity of LNCs (1 and 10mg/kg body weight) revealed no mortality with no morphological and clinical changes in rats. Histology, haematology and biochemical analysis of urine and plasma confirmed no toxicity of LNCs compared to control. Post-prandial plasma and tissue (retina) levels of lutein from LNCs were higher. Results demonstrate increased bioavailability of lutein from LNCs with no toxicity suggests applications in food and pharma.

Versatile and Valuable Utilization of Amidohydrolase L-glutaminase in Pharma and Food industries: A Review.

L-glutaminase has versatile applications in pharma and food industries. In pharmaceutical industry, L-glutaminase can be used as anti-oxidant and anti-cancer agent to treat Acute Lymphocytic Leukaemia (ALL). Whereas, in the food industry, L-glutaminase is used for acrylamide degradation, theanine production, flavour enhancer, soy sauce and many. The other applications include nitrogen metabolism and its use as biosensor in hybridoma technology. Both intra-cellular and extra-cellular L-glutaminases from wide range of sources were identified. Because of its diverse applications, there is a need to improve the production of L-glutaminase by enzyme engineering technology. Effect of recombination on L-glutaminase production was also reported. Researchers also confirmed the antitumor properties of L-glutaminase by conducting in vitro, in vivo and in silico studies. Bacillus sps. and Aspergillus sps. are the commercial producers of L-glutaminase. In this review, the applications, different sources of Lglutaminase, anti-cancer properties were discussed.

Purification, characterization and specificity of a new GH family 35 galactosidase from Aspergillus awamori

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and β-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50–80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and β-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55–60 °C, 5.0–5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and β-galactosidase activities, but also exhibited β-xylosidase and β-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable β-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.