Microbial assisted multifaceted amelioration processes of heavy-metal remediation: a clean perspective toward sustainable and greener future.

Rapidly increasing heavy metal waste has adversely affected the environment and the Earth's health. The lack of appropriate remediation technologies has worsened the issue globally, especially in developing countries. Heavy-metals contaminants have severely impacted the environment and led to devastating conditions owing to their abundance and reactivity. As they are nondegradable, the potential risk increases even at a low concentration. However, heavy-metal remediation has increased with the up-gradation of technologies and integration of new approaches. Also, of all the treatment methodologies, microbial-assisted multifaceted approach for ameliorating heavy metals is a promising strategy for propagating the idea of a green and sustainable environment with minimal waste aggregation. Microbial remediation combined with different biotechniques could aid in unraveling new methods for eradicating heavy metals. Thus, the present review focuses on various microbial remediation approaches and their affecting factors, enabling recapitulation of the interplay between heavy-metals ions and microorganisms. Additionally, heavy-metals remediation mechanisms adapted by microorganisms, the role of genetically modified (GM) microorganisms, life cycle assessment (LCA), techno-economic assessment (TEA) limitations, and prospects of microbial-assisted amelioration of heavy-metals have been elaborated in the current review with focus toward "sustainable and greener future."

Allergic sensitization impairs lung resident memory CD8 T-cell response and virus clearance.

BACKGROUND: Patients with asthma often suffer from frequent respiratory viral infections and reduced virus clearance. Lung resident memory T cells provide rapid protection against viral reinfections. OBJECTIVE: Because the development of resident memory T cells relies on the lung microenvironment, we investigated the impact of allergen sensitization on the development of virus-specific lung resident memory T cells and viral clearance. METHODS: Mice were sensitized with house dust mite extract followed by priming with X47 and a subsequent secondary influenza infection. Antiviral memory T-cell response and protection to viral infection was assessed before and after secondary influenza infection, respectively. Gene set variation analysis was performed on data sets from the U-BIOPRED asthma cohort using an IFN-γ-induced epithelial cell signature and a tissue resident memory T-cell signature. RESULTS: Viral loads were higher in lungs of sensitized compared with nonsensitized mice after secondary infection, indicating reduced virus clearance. X47 priming induced fewer antiviral lung resident memory CD8 T cells and resulted in lower pulmonary IFN-γ levels in the lungs of sensitized as compared with nonsensitized mice. Using data from the U-BIOPRED cohort, we found that patients with enrichment of epithelial IFN-γ-induced genes in nasal brushings and bronchial biopsies were also enriched in resident memory T-cell-associated genes, had more epithelial CD8 T cells, and reported significantly fewer exacerbations. CONCLUSIONS: The allergen-sensitized lung microenvironment interferes with the formation of antiviral resident memory CD8 T cells in lungs and virus clearance. Defective antiviral memory response might contribute to increased susceptibility of patients with asthma to viral exacerbations.

Microbial cell factories a new dimension in bio-nanotechnology: exploring the robustness of nature.

Bio-based nanotechnology has its existence in biological dimensions e.g. microbial cell factories (bacteria, fungi. algae, yeast, cyanobacteria) plants, and biopolymers. They provide multipurpose biological platforms to supply well-designed materials for diverse nano-biotechnological applications. The "green or bio-based synthesis of nanoparticles (NPs)" has witnessed a research outburst in the past decade. The bio-based synthesis of NPs using microbial cell factories is a benign process and requires mild conditions for the synthesis with end products being less/non-toxic. As a result, its application has extended in multitudinous industries including environment, cosmetics, and pharmaceutical. Thus, the present review summarizes all the significant aspects of nanotechnology and the reason to switch towards the bio-based synthesis of NPs using microbial cell factories. It consists of a detailed description of the bio-based methods employed for the synthesis and classification of NPs. Also, a comprehensive study on the application of bio-based NPs in the various industrial and biotechnological domains has been discussed. The limitation and its solution would help identify the applicability of NPs to "identified and unidentified" sectors.

Phycoremediation of nitrogen and phosphate from wastewater using Picochlorum sp.: A tenable approach.

The wastewater originates from different industrial, municipal, and agriculture processes and contains different nitrogen sources, for example, nitrate, ammonium, nitrite, and phosphate such as inorganic and organic sources. The discharge of high nitrate and phosphate to the ecosystem or nearby water bodies can cause eutrophication which disbalances the aquatic ecosystem. Furthermore, ingestion of these pollutants can cause severe toxicity and disease to humans and animals. Thus, from an environmental and social perspective, its treatment is essential with no negative impact on the ecosystem. Microalgae are fundamental, mixotrophic microorganisms that treat different wastewater and utilize nitrate and phosphate in the medium as a source of nutrients. Among them, Picochlorum sp., have the potential to remove nitrogen and phosphate from wastewater. The biomass produced by Picochlorum sp. can be a promising candidate as a sustainable feedstock for biofuel and bioproducts formation. Thus, the present review provides a brief knowledge and understanding about the concentration of nitrogen and phosphate in different wastewater, their negative impacts, and the uptake mechanism of microalgae. Furthermore, the review also provides an insight into Picochlorum sp., and the effects of different physiological and nutritional factors on their growth, wastewater treatment efficacy, and biomass for value-added products and biorefinery applications. In addition, the review is useful to understand the potential of Picochlorum sp. for a tenable wastewater treatment process.

Insight into multicopper oxidase laccase from Myrothecium verrucaria ITCC-8447: a case study using in silico and experimental analysis.

The oxidation activity of multicopper-oxidases overlaps with different substrates of laccases and bilirubin oxidases, thus in the present study an integrated approach of bioinformatics using homology modeling, docking, and experimental validation was used to confirm the type of multicopper-oxidase in Myrothecium verrucaria ITCC-8447. The result of peptide sequence of M. verrucaria ITCC-8447 enabled to predict the 3 D-structure of multicopper-oxidase. It was overlapped with the structure of laccase and root mean square deviation (RMSD) was 1.53 Å for 533 and, 171 residues. The low binding energy with azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (-5.64) as compared to bilirubin (-4.39) suggested that M. verrucaria ITCC-8447 have laccase-like activity. The experimental analysis confirmed high activity with laccase specific substrates, phenol (18.3 U/L), ampyrone (172.4 U/L) and, ampyrone phenol coupling (50 U/L) as compared to bilirubin oxidase substrate bilirubin (16.6 U/L). In addition, lowest binding energy with ABTS (-5.64), syringaldazine SYZ (-4.83), guaiacol GCL (-4.42), and 2,6-dimethoxyphenol DMP (-4.41) confirmed the presence of laccase. Further, complete remediation of two hazardous model pollutants i.e., phenol and resorcinol (1.5 mM) after 12 h of incubation and low binding energy of -4.32 and, -4.85 respectively confirmed its removal by laccase. The results confirmed the presence of laccase in M. verrucaria ITCC-8447 and its effective bioremediation potential.

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL-23 and OX40L.

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL-17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL-12p70 secretion as compared to heat inactivated Per a 10. IL-23, TNF-α, and IL-6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC-T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL-4 and IL-13 that were reduced on blocking of either IL-23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL-23 secretion and OX40L expression on dendritic cells.

Differential proteomic expression of himalayan psychrotrophic diazotroph Pseudomonas palleroniana N26 under low temperature diazotrophic conditions.

BACKGROUND: In low temperature nitrogen-deficient ecosystems, native microorganisms must possess adaptive mechanisms to cope with environmental stress as well as nitrogen (N) starvation-like conditions. However, moderate information is available about the cold adapted diazotrophs and diazotrophy. OBJECTIVE: The aim of this study was to examine the proteomic response(s) of Himalayan psychrotrophic diazotroph under low temperature nitrogen fixing conditions. MATERIALS AND METHODS: Proteomic analysis of Pseudomonas palleroniana N26 was carried out using two dimensional electrophoresis technique. RESULTS: Altogether, fifty three protein spots were found to be differentially expressed revealing several mechanisms thought to be involved in low temperature adaptation and nitrogen fixation, including general stress adaptation, protein synthesis and modifications, and energy metabolism. Expression profiling of the spots revealed the up-regulation of low molecular weight acidic proteins; a majority of which were stress proteins. The largest group of down-regulated proteins were related to biosynthetic processes; thereby, providing the evidence for stress-associated metabolic adaptations. CONCLUSION: The present study, which provides an overview of the cold diazotrophy of a Himalayan psychrotrophic bacterium and its adaptive responses, can facilitate further studies of low temperature nitrogen fixing mechanisms, psychrophilic diazotrophic markers, and transgenic microorganism(s)/crop(s) development.

Nonextraction Treatment of Maxillary Canine and Lateral Incisor Transposition: A Case Report.

The transposition of teeth is an infrequent dental abnormality characterized by the exchange of position between two adjacent teeth. This report presents a unique case of transposition involving the maxillary right canine and lateral incisor in a 20-year-old female patient. Clinical assessment and radiographic evaluation revealed the unconventional positioning of the affected teeth. Treatment planning necessitated collaborative efforts between orthodontic specialists to achieve optimal esthetic and functional outcomes. The patient underwent a comprehensive treatment regimen, including orthodontic alignment by distal drifting of the canine and mesial shifting of the lateral incisor. Subsequent long-term follow-ups confirmed stable occlusion and satisfactory esthetics in a time period of 18 months. This case underscores the significance of precise diagnosis and a multidisciplinary approach in managing intricate dental anomalies such as tooth transposition.

Misdiagnosis of an Odontogenic Infection as a Skin Lesion: The Diagnostic Dilemma.

Extraoral sinus tracts of endodontic origin might be confused for a variety of dermatological conditions. Differential diagnosis of this clinical condition plays an essential role in providing appropriate clinical care because misdiagnosis is the most prevalent cause of prolonged therapy and healing failure. As a result, every cutaneous sinus structure affecting the face or neck should be investigated for dental issues. Its diagnosis can sometimes be difficult until the treating clinician examines the potential of a dental cause. Once an appropriate diagnosis has been established, definitive treatment, consisting of root canal therapy or tooth extraction, to remove the primary source of infection is a straightforward and successful operation.

Acute liver failure histopathology: A 5-year retrospective study.

BACKGROUND: Acute liver failure (ALF) is a rare, life-threatening disease of diverse etiology. It is defined as severe acute liver injury for fewer than 26 weeks' duration with encephalopathy and impaired synthetic function (international normalized ratio [INR] of 1.5 or higher) in a patient without cirrhosis or pre-existing liver disease. The diagnosis rests mainly on the clinical ground with wide range of pathological features. The present study seeks to explore the diverse histological patterns observed in cases for ALF and assess their usefulness in determining the underlying causes for the condition. METHODOLOGY: A retrospective cross-sectional study was conducted among patients of ALF who underwent liver transplant and transjugular liver biopsy over a five-year period. From 1082 explant liver and 2446 liver biopsies, 22 cases of ALF (10 explants and 12 liver biopsies) were included in the study. Clinical and laboratory details were retrieved and histological findings were reviewed. RESULT: Age ranged from 10 to 72 years (mean age, 40 years). There was a female predominance with a male:female ratio of 1:1.7. The commonest cause for ALF was virus-induced hepatocellular damage in 36.3% (eight patients), followed by autoimmune hepatitis in 22.7% (five patients), drug-induced liver injury (DILI) in 18.1% (four patients), cryptogenic in 13.6% (three patients) and ischemic injury secondary to large vein thrombosis in 9.0% (two) patients. The histological patterns identified were categorized into six categories. A more comprehensive morphological evaluation was conducted specifically for cases of ALF associated with autoimmune hepatitis (AIH) and compared with other cases of ALF. CONCLUSION: In summary, our present study illustrates a morphological overlap in various patterns for the purpose of etiological assessment. In cases of AIH ALF, the presence of portal plasma cell infiltrate and central perivenulitis were identified as significant histological features to guide diagnosis.

Treatment of Vertical Maxillary Excess and Skeletal Class II Malocclusion: A Case Report.

Class II malocclusion is a recurrent problem that may occur at a young age. If treated initially, the malocclusion can be corrected by redirecting the growth without invasive modalities and avoiding orthognathic surgeries. A female patient aged 10 years three months came to the department of orthodontics having a complaint of upper front teeth placed forwardly, diagnosed with skeletal class II due to retrusive mandible and vertical maxillary excess with hyper divergent growth pattern with increased anterior facial height, with Angle's molar class II division 1 malocclusion, increased overjet of 13 mm and overbite of 7 mm, acute nasolabial angle, deep mentolabial sulcus, and hyperactive mentalis. It was treated using an activator with medium-high-pull headgear (modified Herren activator) passing through the maxillary center of resistance. A fixed mechanotherapy with high-pull headgear was given using the anterior inclined plane acrylic plate in the maxilla and McLaughlin, Bennett, and Trevisi (MBT). Begg's wrap was used for the retention plan. This case report shows the significance of proper treatment results obtained due to correct identification and planning in treating malocclusion. This case report shows the significance of proper treatment results obtained due to correct identification and planning in treating malocclusion.

A comparative study of blood cell count in four automated hematology analyzers: An evaluation of the impact of preanalytical factors.

Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.

Organosolv pretreatment: an in-depth purview of mechanics of the system.

The concept of biorefinery has been advancing globally and organosolv pretreatment strategy has seen an upsurge in research due to its efficiency in removing the recalcitrant lignin and dissolution of cellulose. The high-performance organosolv system uses green solvents and its reusability contributes concurrently to the biorefinery sector and sustainability. The major advantage of the current system involves the continuous removal of lignin to enhance cellulose accessibility, thereby easing the later biorefinery steps, which were immensely restricted due to the recalcitrant lignin. The current system process can be further explored and enhanced via the amalgamation of new technologies, which is still a work in progress. Thus, the current review summarizes organosolv pretreatment and the range of solvents used, along with a detailed mechanistic approach that results in efficient pretreatment of LCB. The latest developments for designing high-performance pretreatment systems, their pitfalls, and advanced assessments such as Life Cycle Assessment along with Techno-Economic Assessment have also been deliberated to allow an insight into its diverse potential applicability towards a sustainable future.

Development of economical and sustainable cultivation system for biomass production and simultaneous treatment of municipal wastewater using Tetraselmis indica BDUG001.

Microalgal-based bioprocess offers several advantages including wastewater reclamations, therefore present study assessed the usability of the combination of untreated municipal sewage wastewater (UTMSWW) and secondary treated municipal sewage wastewater (STSWW) for nutrient removal and recovery by Tetraselmis indica (T. indica) BDUG001. The present study optimized the additional nutrient supplementations (e.g. ASN-III) percentage and day-night cycle, pH and pH with aeration for monitoring high-rate biomass production and nutrient recovery. The study results showed that the combination of 75% UTMSWW + 25% ASN-III supported maximum biomass production (2.65 ± 0.07 g/L). In the optimized day-night cycle (12:12 h), T. indica BDUG001 showed improved biomass production (2.75 ± 0.07 g/L), biomass productivity (165.63 ± 4.42 mg/L/d), and photosynthetic pigments production. Under optimized pH∼ 7.0 with aeration, maximum total nitrate (TN) removal efficiency (87.67 ± 3.08-91.55 ± 1.92%) was observed, while COD and TP removal was maximum at pH ∼ 9.0. The maximum biomass production (2.35 ± 0.07-2.77 ± 0.04 g/L) with biomass productivity (93.75 ± 167.19 ± 2.21 mg/L/d) and lipid content (42.98 ± 1.86-47.85 ± 0.21% DCW) were also at pH 7.0. with aeration. The present study verified the utilization of UTMSWW with the combination of conventional medium, optimized day-night cycle, pH with aeration along with designing low-cost PBR. It was the ideal system for the cultivation of T. indica BDUG001 for the recovery of nutrients from wastewater, production of biofuels and value-added feedstock.

Characteristics of posterior zone I retinopathy of prematurity.

OBJECTIVE: To report the demographic profile and clinical characteristics of retinopathy of prematurity (ROP) in posterior Zone I. METHODS: In a partly retrospective (ten years) and partly prospective (one year) study, we analysed the demographic profile and clinical characteristics of babies with ROP in posterior Zone I. RESULTS: The study included 130 eyes of 67 infants with a mean gestational age and birth weight of 29.3 (±2.2) weeks and 1217.3 (±381.9) grams, respectively. All babies had received unblended oxygen. In 47 of 51 (91.1%) babies, the weekly weight gain was <100 g (details were not available in 16 babies). The ROP subtypes included aggressive, threshold, hybrid, stage 4, and atypical types in 78 (60%), 20 (15.4%), 11 (8.5%), 15 (11.5%), and 6 (4.6%) eyes, respectively. Fibrovascular proliferation, when present, was prominent nasally, occasionally overriding the disc margin. Extensive arteriovenous tortuosity was more prominent than vascular dilatation. Atypical observations included bleb-like detachment (6 eyes; 4.6%) and candle wax-like preretinal deposits (23 eyes; 17.7%). CONCLUSIONS: Retinopathy of Prematurity in posterior Zone I in this cohort was strongly associated with 100% unblended oxygen supplementation, poor weight gain, and multiple systemic co-morbidities. ROP in posterior zone 1 has a distinct profile with several atypical characteristics different from ROP in other zones.

Current perspective on wastewater treatment using photobioreactor for Tetraselmis sp.: an emerging and foreseeable sustainable approach.

Urbanization is a revolutionary and necessary step for the development of nations. However, with development emanates its drawback i.e., generation of a huge amount of wastewater. The existence of diverse types of nutrient loads and toxic compounds in wastewater can reduce the pristine nature of the ecosystem and adversely affects human and animal health. The conventional treatment system reduces most of the chemical contaminants but their removal efficiency is low. Thus, microalgae-based biological wastewater treatment is a sustainable approach for the removal of nutrient loads from wastewater. Among various microalgae, Tetraselmis sp. is a robust strain that can remediate industrial, municipal, and animal-based wastewater and reduce significant amounts of nutrient loads and heavy metals. The produced biomass contains lipids, carbohydrates, and pigments. Among them, carbohydrates and lipids can be used as feedstock for the production of bioenergy products. Moreover, the usage of a photobioreactor (PBR) system improves biomass production and nutrient removal efficiency. Thus, the present review comprehensively discusses the latest studies on Tetraselmis sp. based wastewater treatment processes, focusing on the use of different bioreactor systems to improve pollutant removal efficiency. Moreover, the applications of Tetraselmis sp. biomass, advancement and research gap such as immobilized and co-cultivation have also been discussed. Furthermore, an insight into the harvesting of Tetraselmis biomass, effects of physiological, and nutritional parameters for their growth has also been provided. Thus, the present review will broaden the outlook and help to develop a sustainable and feasible approach for the restoration of the environment.

An insight into the principles of lignocellulosic biomass-based zero-waste biorefineries: a green leap towards imperishable energy-based future.

Lignocellulosic biomass (LCB) is an energy source that has a huge impact in today's world. The depletion of fossil fuels, increased pollution, climatic changes, etc. have led the public and private sectors to move towards sustainability i.e. using LCB for the production of biofuels and value-added compounds. A major bottleneck of the process is the recalcitrant nature of LCB. This can be overcome by using various pretreatment strategies like physical, chemical, biological, physicochemical, etc. Further, the pretreated biomass is made to undergo various steps like hydrolysis, saccharification, etc. for the conversion of value-added products and the remaining waste residues can be further utilized for the synthesis of secondary products thus favouring the zero-waste biorefinery concept. Currently, microorganisms are being explored for their use in biorefinery but the unavailability of commercial strains is a major limitation. Thus, the use of metagenomics can be used to overcome the limitation which is both cost-effective and environmentally friendly. The review deliberates the composition of LCBs, and their recalcitrance nature, followed by the structural changes caused by various pretreatment methods. The further steps in biorefineries, strategies for the development of zero-waste refineries, bottlenecks, and suggestions are also discussed. Special emphasis is given to the use of metagenomics for the discovery of microorganisms efficient for zero-waste biorefineries.

Current perspective on production and applications of microbial cellulases: a review.

The potential of cellulolytic enzymes has been widely studied and explored for bioconversion processes and plays a key role in various industrial applications. Cellulase, a key enzyme for cellulose-rich waste feedstock-based biorefinery, has increasing demand in various industries, e.g., paper and pulp, juice clarification, etc. Also, there has been constant progress in developing new strategies to enhance its production, such as the application of waste feedstock as the substrate for the production of individual or enzyme cocktails, process parameters control, and genetic manipulations for enzyme production with enhanced yield, efficiency, and specificity. Further, an insight into immobilization techniques has also been presented for improved reusability of cellulase, a critical factor that controls the cost of the enzyme at an industrial scale. In addition, the review also gives an insight into the status of the significant application of cellulase in the industrial sector, with its techno-economic analysis for future applications. The present review gives a complete overview of current perspectives on the production of microbial cellulases as a promising tool to develop a sustainable and greener concept for industrial applications.

Multicopper oxidase laccases with distinguished spectral properties: A new outlook.

Multicopper oxidases (MCOs) has a unique feature of having the presence of four Cu atoms arranged into three (Type I, II and III) spectral classification. MCOs laccase due to its broad range of substrate specificity has numerous biotechnological applications. The two types of laccases include the typical blue and the atypical white, yellow laccases which have been isolated from diverse geographical locations globally. In the present study laccases were identified using Liquid Chromatograph Mass Spectrometer Studies (LCMS) study where blue laccase exhibited homology with Trametes villosa Q99044 and Q99046 and white, yellow laccase exhibited homology with Myrothecium verrucaria OX = 1859699; Q12737 and Trametes versicolor Q12717 respectively. The spectral comparison between laccases were determined via spectroscopic analysis where UV-spectra of blue laccase from Trametes versicolor had a peak at 605 nm (Type I Cu atom) whereas in case of white and yellow laccases the peak was absent and in addition had an absorption peak at 400nm. It was followed by X-Ray Diffraction (XRD) analysis of proteins where α-helix (10°) and ß-sheet (22°) structure were observed in case of all the three laccases. However, the intensity of α-helix in white and yellow laccase was stronger as compared to the blue laccase whereas the intensity of ß-sheet was stronger in case of blue laccase as compared to other two laccases. Further, Fourier-transform infrared spectroscopy (FTIR) analysis was performed which enabled the analysis of proteins where α-helix (1650-1658 cm-1), ß-sheets (1620-1640 cm-1), amide I (1700-1600 cm-1) amide II (bands at under 1400 cm-1) and amide A, B (bands above 3000 cm-1).