The way forward to produce nutraceuticals from agri-food processing residues: obstacle, solution, and possibility.

Food matrices contain bioactive compounds that have health benefits beyond nutritional value. The bulk of bioactive chemicals are still present in agro-industrial by-products as food matrices. Throughout the food production chain, there is a lot of agro-industrial waste that, if not managed effectively, could harm the environment, company, and how nutritiously and adequately people eat. It's important to establish processes that maximise the use of agro-industrial by-products, such as biological technologies that improve the extraction and acquisition of bioactive compounds for the food and pharmaceutical industries. As opposed to nonbiological processes, biological procedures provide high-quality, bioactive extracts with minimum toxicity and environmental impact. Fermentation and enzymatic treatment are biological processes for obtaining bioactive compounds from agro-industrial waste. In this context, this article summarises the principal bioactive components in agro-industrial byproducts and the biological methods employed to extract them. In this review efficient utilization of bioactive compounds from agro-industrial waste more effectively in food and pharmaceutical industries has been described.

Development of Mupirocin-Impregnated Bacterial Cellulosic Transdermal Patches for the Management of Skin Infection.

The present study reports the production of bacterial cellulose (BC) membranes using Komagataeibacter hansenii for the development of transdermal wound healing patches. BC-based transdermal patches were developed by impregnating varied concentrations of antibiotic mupirocin and characterized by SEM, FTIR, TGA, and DSC to study the interaction of BC with antibiotic. Developed patches were evaluated for antimicrobial activity, in vitro drug release study, in vivo efficacy, and acute dermal toxicity studies. The antibacterial activity of mupirocin-impregnated patches (mup-BC) showed an inhibition zone from 26.16 ± 0.76 to 35.86 ± 0.61 mm. The in vivo efficacy of mup-BC patches on the superficial abrasion mouse model infected with MRSA 15187 was determined. A single application of the mup-BC (Batch-3) showed a significant decrease up to 2.5 log10 colony-forming units (CFUs) in the infected skin. Acute dermal toxicity study showed no notable sign of toxicity. Pharmacokinetic study indicated that an application of mup-BC (Batch-3) showed a peak plasma concentration of around 1.5 µg/mL mupirocin. The overall preparation, ease of application, and efficacy results clearly indicate that the patches developed in the present study find immense application in the healthcare sector, especially for the treatment of burn or dermal wound infections.

Exploring the Antibacterial Potential of Semisynthetic Phytocannabinoid: Tetrahydrocannabidiol (THCBD) as a Potential Antibacterial Agent against Sensitive and Resistant Strains of Staphylococcus aureus.

Antimicrobial resistance (AMR) is one of the most challenging problems and is responsible for millions of deaths every year. We therefore urgently require new chemical entities with novel mechanisms of action. Phytocannabinoids have been adequately reported for the antimicrobial effect but not seriously pursued because of either stringent regulatory issues or poor drug-like properties. In this regard, the current work demonstrated the antibacterial potential of tetrahydrocannabidiol (THCBD, 4), a semisynthetic phytocannabinoid, against Staphylococcus aureus, the second-most widespread bug recognized by the WHO. THCBD (4) was generated from cannabidiol and subjected to extensive antibacterial screening. In in vitro studies, THCBD (4) demonstrated a potent MIC of 0.25 µg/mL against Gram-positive bacteria, S. aureus ATCC-29213. It is interesting to note that THCBD (4) has demonstrated strong effectiveness against efflux pump-overexpressing (SA-1199B, SA-K2191, SA-K2192, and Mupr-1) and multidrug-resistant (MRSA-15187) S. aureus strains. THCBD (4) has also shown a good effect in kill kinetic assays against ATCC-29213 and MRSA-15187. In the checkerboard assay, THCBD (4) has shown additive/indifference effects with several well-known clinically used antibiotics, tetracycline, mupirocin, penicillin G, and ciprofloxacin. THCBD (4) also exhibited good permeability in the artificial skin model. Most importantly, THCBD (4) has significantly reduced CFU in mice's in vivo skin infection models and also demonstrated decent plasma exposure with 16-17% oral bioavailability. Acute dermal toxicity of THCBD (4) suggests no marked treatment-related impact on gross pathophysiology. This attractive in vitro and in vivo profile of plant-based compounds opens a new direction for new-generation antibiotics and warrants further detailed investigation.

Utilization of xylose enriched extract from spent lemongrass hydrolysate for clavulanic acid production using Streptomyces clavuligerus (MTCC 1142).

The main goal of this study was to provide possible alternative production medium containing xylose enriched spent lemongrass hydrolysate with glycerol as a feedstock and corn gluten meal as a nitrogen source for their ability to support the cell growth of the Streptomyces clavuligerus MTCC 1142 for the production of clavulanic acid. The xylose was extracted from spent lemongrass by using 0.25% dilute nitric acid and further partial purification of acid spent hydrolysate was performed using ion exchange resin. The method was optimized using xylose enriched hydrolysate as feed stock combined with glycerol at ratio 1:1 and growing the selected strain aerobically in media at neutral pH containing 5 mM phosphate ion concentration and using corn gluten meal as a nitrogen source, fermenting at a temperature of 28-30 °C for 96 h and 0.59 g/L clavulanic acid was effectively produced. These results demonstrate the feasibility of using spent lemongrass as feedstock for the cultivation of S. clavuligerus to produce clavulanic acid.

Efficient production of bacterial cellulose based composites using zein protein extracted from corn gluten meal.

Corn gluten meal (CGM) which is a byproduct of corn wet milling is mainly used in animal and poultry feed. Due to its high protein content in CGM, it has been utilized for the extraction of zein protein which is the main hydrophobic protein present in the corn. The extracted zein protein was used along with bacterial cellulose that is highly pure, biocompatible, biodegradable, and generally regarded as safe for the preparation of composites that have better surface properties and applications. SEM analysis of the synthesized composite showed layering, incorporation of zein protein onto the surface of bacterial cellulose. XRD results showed there were no significant changes in the peak intensity due to the surface modification of BC membranes composites in comparison to pristine BC and TGA showed the thermostable characteristic of bacterial cellulose and are more capable of withstanding high temperature. Maximum production of bacterial cellulose was observed when corn gluten meal and zein protein were used as a cheap nitrogen sources for the production of bacterial cellulose along with other medium components. An increase of approximately 4.0 g/l of bacterial cellulose from 13.561 g/l to 17.83 g/l was observed when corn gluten meal and zein protein were used in the production medium. The prepared BC-based zein protein composites can be utilized for food packaging and storage applications.

Fungal production of kojic acid and its industrial applications.

Kojic acid has gained its importance after it was known worldwide that the substance functions primarily as skin-lightening agent. Kojic acid plays a vital role in skin care products, as it enhances the ability to prevent exposure to UV radiation. It inhibits the tyrosinase formation which suppresses hyperpigmentation in human skin. Besides cosmetics, kojic acid is also greatly used in food, agriculture, and pharmaceuticals industries. Conversely, according to Global Industry Analysts, the Middle East, Asia, and in Africa especially, the demand of whitening cream is very high, and probably the market will reach to $31.2 billion by 2024 from $17.9 billion of 2017. The important kojic acid-producing strains were mainly belongs to the genus Aspergillus and Penicillium. Due to its commercial potential, it continues to attract the attention for its green synthesis, and the studies are still widely conducted to improve kojic acid production. Thus, the present review is focused on the current production processes, gene regulation, and limitation of its commercial production, probable reasons, and possible solutions. For the first time, detailed information on the metabolic pathway and the genes involved in kojic acid production, along with illustrations of genes, are highlighted in the present review. Demand and market applications of kojic acid and its regulatory approvals for its safer use are also discussed. KEY POINTS: • Kojic acid is an organic acid that is primarily produced by Aspergillus species. • It is mainly used in the field of health care and cosmetic industries. • Kojic acid and its derivatives seem to be safe molecules for human use.

Morphologically dissimilar spores of Aspergillus sojae exhibit genomic homogeneity but the differential in Kojic acid accumulation.

We reported that Aspergillus sojae (SSC-3), an indigenous isolate from rice husk, is a potent kojic acid producer. During optimization, it was observed that under static fermentation conditions, this fungal strain produces two dissimilar morphological green and yellow spores, i.e., SSC-3(Y) and SSC-3(G). Furthermore, these different spore types differ in color, morphology, and in kojic acid metabolite accumulation, with green spores producing 12.87 g/l and yellow spores producing 8.63 g/l of kojic acid on the 12th day of fermentation. To understand if there is a genetic basis for the difference in morphology and metabolite accumulation characteristics, sequencing of internal transcribed spacer regions (ITS) and RAPD analysis from both the spore were carried out. Our study revealed that though the spores are dissimilar with respect to morphology and metabolite accumulation profile, they are genetically homogenous. This suggests that there could be epigenetic differences in these spore types, which may be explored in detail in further studies.

Boswellic acids, as novel inhibitor targeting peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) in Escherichia coli.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an essential cytosolic enzyme in the biosynthesis of peptidoglycan. It becomes a potential bacterial target for screening promising antibacterial compounds as it is associated with the early phases of peptidoglycan production. MurA enzyme is conserved and necessary for bacterial viability with no mammalian homolog, which is a well-proven therapeutic research target. The present study reports the natural compounds from Boswellia serrata targeting the MurA enzyme. The identified inhibitors against MurA Escherichia coli (E. coli): ß-boswellic acid (IC50 33.65 µM), Acetyl-ß-boswellic acid (IC50 30.17 µM), and Acetyl-11-keto-ß-boswellic acid (IC50 37.67 µM). Inhibitors showed a fourfold decrease in IC50 values on pre-incubation with substrate-UDP-N-acetyl-glucosamine (UDP-GlcNAc). Mode-of-inhibition studies revealed their uncompetitive nature with both the substrates. Although these boswellic acids have been explored for their pharmacological potential, this is the first study reporting these compounds' E. coli MurA inhibiting potential.

Active pharmaceutical ingredient (API) chemicals: a critical review of current biotechnological approaches.

The aim of this article was to generate a framework of bio-based economy by an effective utilization of biomass from the perspectives of agriculture for developing potential end bio-based products (e.g. pharmaceuticals, active pharmaceutical ingredients). Our discussion is also extended to the conservatory ways of bioenergy along with development of bio-based products and biofuels. This review article further showcased the fundamental principles for producing these by-products. Thereby, the necessity of creating these products is to be efficaciously utilization by small-scale farmers that can aid the local needs for bio-based materials and energy. Concurrently, the building up of small markets will open up the avenues and linkages for bigger markets. In nutshell, the aim of the review is to explore the pathway of the biotechnological approaches so that less chosen producers and underdeveloped areas can be allied so that pressure on the systems of biomass production can be relaxed.

Potential Inhibitors Targeting Escherichia coli UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): An Overview.

Antibiotic resistance is one of the biggest challenges that is escalating and affecting humanity across the globe. To overcome this increasing burden of resistance, discovering novel hits by targeting the enzymes involved in peptidoglycan (murein) biosynthesis has always been considered better in antimicrobial drug discovery. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme has been identified as essential for Escherichia coli survival and catalyzes the early-stage step in bacterial cell wall synthesis. The present article gives a brief overview of the role of enzymes in peptidoglycan synthesis and MurA enzyme (previously known as MurZ in E. coli), in particular, including its structural and active site features. This review also provides an insight into the current knowledge of the reported MurA inhibitors, their mechanism of action and drawbacks of these hits that hinder their clinical trials, which would be helpful for synthesis and discovering potent molecules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00988-6.

A critical review on exploitation of agro-industrial biomass as substrates for the therapeutic microbial enzymes production and implemented protein purification techniques.

Annually, a huge amount of waste is generated by the industries that use agricultural biomass. Researchers have looked into employing this cheap and renewable agro-biomass as a substrate for enzyme production via fermentation processes to meet the ever-increasing worldwide need. Although there are a number of sources for enzyme extraction, microbial sources have dominated industrial sectors due to their easy availability and rapid growth. Microbial enzymes are currently used in a variety of industries, including pharmaceuticals, food, biofuels, textiles, paper, detergents, and so on, and using these nutritious feedstocks not only reduces production costs but also helps to reduce environmental concerns. The present review focuses on the therapeutic microbial enzymes produced using different agro-industrial biomass as raw materials, with down-streaming techniques for obtaining a final pure product. Additionally, the article also discussed biomass pretreatment processes, including physical, chemical and biological. The type of pretreatment method to be used is mostly governed by the intended use of the major molecular components of biomass (cellulose, hemicelluloses and lignin). Finally, purification challenges are included. All of this information will be useful in the industrial synthesis of high-purity targeted enzymes if the crucial aspects that have been discussed are taken into account.

Optimization and validation of RT-LAMP assay for diagnosis of SARS-CoV2 including the globally dominant Delta variant.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. OBJECTIVE: Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). RESULTS: Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. CONCLUSION: In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.

Recent advances in biodiesel production: Challenges and solutions.

Mono alkyl fatty acid ester or methyl ethyl esters (biodiesel) are the promising alternative for fossil fuel or petroleum derived diesel with similar properties and could reduce the carbon foot print and the greenhouse gas emissions. Biodiesel can be produced from renewable and sustainable feedstocks like plant derived oils, and it is biodegradable and non-toxic to the ecosystem. The process for the biodiesel production is either through traditional chemical catalysts (Acid or Alkali Transesterification) or enzyme mediated transesterification, but as enzymes are natural catalysts with environmentally friendly working conditions, the process with enzymes are proposed to overcome the drawbacks of chemical synthesis. At present 95% of the biodiesel production is contributed by edible oils worldwide whereas recycled oils and animal fats contribute 10% and 6% respectively. Although every process has its own limitations, the enzyme efficiency, resistance to alcohols, and recovery rate are the crucial factors to be addressed. Without any benefit of doubt, production of biodiesel using renewable feedstocks and enzymes as the catalysts could be recommended for the commercial purpose, but further research on improving the efficiency could be an advantage.

Screening of compound library identifies novel inhibitors against the MurA enzyme of Escherichia coli.

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC50 of 14.03 to 32.30 µM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

Recent developments on solid-state fermentation for production of microbial secondary metabolites: Challenges and solutions.

Microbial secondary metabolites (SMs) are the intermediate or the product of metabolism produced during fermentation process. SMs are produced during stationary phase and play a major role in competition, antagonism and self defence mechanisms. These metabolites finds application in the pharmaceuticals, food, cosmetics etc. These are produced besides primary key metabolites (e.g., amino acids, lipids, carbohydrates etc.). Growth condition in solid-state fermentation (SSF) resembles microorganism's own native environment allowing the microorganisms to adapt best. Recent developments in bioprocessing has identified specific SSF practices that have a significant impact on SMs production. The practice of SSF, representing new opportunities to design better bioprocessing with potential genetic development goals for expanding the list of exciting SMs. Current updates cover advanced techniques on SSF to improve microbial SMs production and their ease of operation and cost-effective production strategies. Various factors affecting the SSF have been discussed with respect to sustainable development of novel SSF strategies for SMs production.

Conversion of amino acids to aryl/heteroaryl ethanol metabolites using human CYP2D6-expressing live baker's yeast.

Different natural aromatic/heterocyclic l-amino acids were biotransformed into aryl/heteroaryl ethanol metabolites via oxidative deamination, decarboxylation and reduction cascades using live baker's yeast cells producing intracellular human CYP2D6 enzyme. Among the three yeast strains expressing 3 different CYP2D6 variants, CYP2D6(2) (i.e. CYP2D6 wild-type) provided the best result under neutral pH conditions at RT. We have successfully converted six natural amino acids into their corresponding alcohols, having one carbon atom less, with moderate yields. Some of the downstream products like tryptophol and tyrosol induced the pTrKB (Tropomyosin receptor kinase B) activation pattern similar to that of BDNF (brain-derived neurotrophic factor), thereby depicting potential antidepressant activity. Control experiments and molecular modelling studies revealed that this tandem bio-transformation probably happens via a pyruvate intermediate. This study establishes that CYP2D6-expressing live yeast cells can be a powerful tool for the enzymatic C-N, C-C bond cleavage of amino-acids.

A Modified, Efficient and Sensitive pH Indicator Dye Method for the Screening of Acid-Producing Acetobacter Strains Having Potential Application in Bio-Cellulose Production.

It is imperative that promising bacterial cellulose-producing bacteria mainly belongs to genera Acetobacter (acid-producing bacteria). In order to screen cellulose-producing Acetobacter, the isolated cultures from vinegar/rotten fruits were inoculated in Hestrin-Schramm (HS) medium containing ethanol and CaCO3. After the desired incubation, the positive cultures form a zone, which is observed around the bacterial growth, resulted from the solubilization of CaCO3 by acetic acid produced from the oxidation of ethanol during fermentation. However, in this method, the clarity of the solubilized zone is not very sharp and distinct. In the present, investigation, an improved method for screening, of the microorganisms producing acetic acid has been developed. In this method, methyl red (MR) is incorporated as a pH indicator in HS medium containing ethanol and CaCO3. Plates containing MR at alkaline pH are yellow and turn dark red at acidic pH. Thus, a distinctive, clear zone is formed around bacterial colonies producing acetic acid and is easy to differentiate between acid producers and non-producers. The present method is more rapid, accurate, and sensitive and can be successfully be used for the detection of acetic acid-producing bacteria particularly for the screening of potent cellulose producer Acetobacter sp.

Consistent production of kojic acid from Aspergillus sojae SSC-3 isolated from rice husk.

A consistent kojic acid producing fungal strain has been isolated from rice husk using glucose-peptone medium. The isolate was identified as Aspergillus sojae SSC-3 on 18S rDNA analysis. A. sojae was capable of producing substantially good amount of kojic acid, however the production was varying from batch to batch. In order to obtain consistent, repeated and high levels of kojic acid, monospore isolation procedures was adopted. The highest production of kojic acid obtained was 12 ± 2 g/L in 120 h with sucrose (10%) and yeast extract (0.5%) as carbon and nitrogen source respectively. The process was scale up to 10 L fermenter size which repeatedly resulted in the production of 18 ± 2 g/L of kojic acid in 96 h. Kojic acid was recovered (> 82%) from the fermentation broth with > 99% purity. Best to our knowledge this is the first report were kojic acid production is reported from Aspergillus sojae strain.

Biotransformation of Chrysin to Baicalein: Selective C6-Hydroxylation of 5,7-Dihydroxyflavone Using Whole Yeast Cells Stably Expressing Human CYP1A1 Enzyme.

Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer, and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multistep chemical approaches. Here, we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7-trihydroxyflavone (baicalein). Molecular modeling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake-flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds.

High production of erythritol from Candida sorbosivorans SSE-24 and its inhibitory effect on biofilm formation of Streptococcus mutans.

Amongst different isolates screened for erythritol production, isolate no. SSE-24 was found to be the best erythritol producer and identified as Candida sorbosivorans SSE-24. Statistical optimization was used to determine the optimum level of the significant variables for maximum erythritol production. The interactive effects of glucose, inoculum level and yeast extract were determined to be significant. The optimum medium composition for erythritol production was 160 g/L glucose, 12 g/L yeast extract, 10% inoculum level and 0.35 g/L FeSO4⋅7H2O. The production of erythritol was successfully scaled up to a 30 L level, where 60.20 g/L of erythritol was produced, with a yield of 0.38 g/g. The fermentation broth was purified by activated charcoal followed by vacuum concentration, ion exchange chromatography and crystallization. Purity of erythritol was further determined by NMR. Significant inhibitory effect of erythritol on growth (>78%) and biofilm formation (40.2%) of Streptococcus mutans enhances the importance of this study.