Search results for: enzyme pre-treatment

Authors: R. Charoen, S. Tipkanon, W. Savedboworn, N. Phonsatta, A. Panya

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Rice bran protein hydrolysates (RBPH) were prepared from defatted rice bran of two different Thai rice cultivars (Plai-Ngahm-Prachinburi; PNP and Khao Dok Mali 105; KDM105) using an enzymatic method. This research aimed to optimize enzyme-assisted protein extraction. In addition, the functional properties of RBPH and their stabilities to environmental stresses including pH (3 to 8), ionic strength (0 mM to 500 mM) and the thermal treatment (30 °C to 90 °C) were investigated. Results showed that enzymatic process for protein extraction of defatted rice bran was as follows: enzyme concentration 0.075 g/ 5 g of protein, extraction temperature 50 °C and extraction time 4 h. The obtained protein hydrolysate powders had a degree of hydrolysis (%) of 21.05% in PNP and 19.92% in KDM105. The solubility of protein hydrolysates at pH 4-6 was ranged from 27.28-38.57% and 27.60-43.00% in PNP and KDM105, respectively. In general, antioxidant activities indicated by total phenolic content, FRAP, ferrous ion-chelating (FIC), and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) of KDM105 had higher than PNP. In terms of functional properties, the emulsifying activity index (EAI) was was 8.78 m²/g protein in KDM105, whereas PNP was 5.05 m²/g protein. The foaming capacity at 5 minutes (%) was 47.33 and 52.98 in PNP and KDM105, respectively. Glutamine, Alanine, Valine, and Leucine are the major amino acid in protein hydrolysates where the total amino acid of KDM105 gave higher than PNP. Furthermore, we investigated environmental stresses on the stability of 5% oil in water emulsion (5% oil, 10 mM citrate buffer) stabilized by RBPH (3.5%). The droplet diameter of emulsion stabilized by KDM105 was smaller (d < 250 nm) than produced by PNP. For environmental stresses, RBPH stabilized emulsions were stable at pH around 3 and 5-6, at high salt (< 400 mM, pH 7) and at temperatures range between 30-50°C.

Keywords: functional properties, oil in water emulsion, protein hydrolysates, rice bran protein

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Authors: Vadim Makarov, Stewart T.Cole

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PBTZ169 is novel drug candidate with high efficacy in animals models, and its combination treatment of PBTZ169 with BDQ and pyrazinamide was shown to be more efficacious than the standard treatment for tuberculosis in a mouse model. The target of PBTZ169 is famous DprE1, an essential enzyme in cell wall biosynthesis. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Furthermore, this drug candidate demonstrated during preclinical research ‘drug like’ properties what made it an attractive drug candidate to treat tuberculosis in humans. During first clinical trials several cohorts of the healthy volunteers were treated by the single doses of PBTZ169 as well as two weeks repeated treatment was chosen for two maximal doses. As expected PBTZ169 was well tolerated, and no significant toxicity effects were observed during the trials. The study of the metabolism shown that human metabolism of PBTZ169 is very different from microbial or animals compound transformation. So main pathway of microbial, mice and less rats metabolism connected with reduction processes, but human metabolism mainly connected with oxidation processes. Due to this difference we observed several metabolites of PBTZ169 in humans with antitubercular activity, and now we can conclude that animal antituberculosis activity of PBTZ169 is a result not only activity of the drug itself, but it is a result of the sum activity of the drug and its metabolites. Direct antimicrobial plasma activity was studied, and such activity was observed for 24 hours after human treatment for some doses. This data gets high chance for good efficacy of PBTZ169 in human for treatment TB infection. Second phase of clinical trials was started summer of 2017 and continues to the present day. Available data will be presented.

Keywords: clinical trials, DprE1, PBTZ169, metabolism

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Authors: Zoi Konsoula

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The increasing prevalence of Alzheimer’s disease (AD) and diabetes mellitus (DM) constitutes them major global health problems. Recently, the inhibition of key enzyme activity is considered a potential treatment of both diseases. Specifically, inhibition of acetylcholinesterase (AChE), the key enzyme involved in the breakdown of the neurotransmitter acetylcholine, is a promising approach for the treatment of AD, while inhibition of α-amylase retards the hydrolysis of carbohydrates and, thus, reduces hyperglycemia. Unfortunately, commercially available AChE and α-amylase inhibitors are reported to possess side effects. Consequently, there is a need to develop safe and effective treatments for both diseases. In the present study, pomegranate peel (PP) was extracted using various solvents of increasing polarity, while two extraction methods were employed, the conventional maceration and the ultrasound assisted extraction (UAE). The concentration of bioactive phytoconstituents, such as total phenolics (TPC) and total flavonoids (TFC) in the prepared extracts was evaluated by the Folin-Ciocalteu and the aluminum-flavonoid complex method, respectively. Furthermore, the anti-neurodegenerative and anti-hyperglycemic activity of all extracts was determined using AChE and α-amylase inhibitory activity assays, respectively. The inhibitory activity of the extracts against AChE and α-amylase was characterized by estimating their IC₅₀ value using a dose-response curve, while galanthamine and acarbose were used as positive controls, respectively. Finally, the kinetics of AChE and α-amylase in the presence of the most inhibitory potent extracts was determined by the Lineweaver-Burk plot. The methanolic extract prepared using the UAE contained the highest amount of phytoconstituents, followed by the respective ethanolic extract. All extracts inhibited acetylcholinesterase in a dose-dependent manner, while the increased anticholinesterase activity of the methanolic (IC₅₀ = 32 μg/mL) and ethanolic (IC₅₀ = 42 μg/mL) extract was positively correlated with their TPC content. Furthermore, the activity of the aforementioned extracts was comparable to galanthamine. Similar results were obtained in the case of α-amylase, however, all extracts showed lower inhibitory effect on the carbohydrate hydrolyzing enzyme than on AChE, since the IC₅₀ value ranged from 84 to 100 μg/mL. Also, the α-amylase inhibitory effect of the extracts was lower than acarbose. Finally, the methanolic and ethanolic extracts prepared by UAE inhibited both enzymes in a mixed (competitive/noncompetitive) manner since the Kₘ value of both enzymes increased in the presence of extracts, while the Vmax value decreased. The results of the present study indicate that PP may be a useful source of active compounds for the management of AD and DM. Moreover, taking into consideration that PP is an agro-industrial waste product, its valorization could not only result in economic efficiency but also reduce the environmental pollution.

Keywords: acetylcholinesterase, Alzheimer’s disease, α-amylase, diabetes mellitus, pomegranate

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Authors: Mei Han, Jinkou Zhao, Yuan Yao, Liangui Feng, Xianbin Ding, Guohui Wu, Chao Zhou, Lin Ouyang, Rongrong Lu, Bo Zhang

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To compare the HIV incidence estimated using BED capture enzyme linked immunosorbent assay (BED-CEIA) and observed in a cohort against the HIV incidence among men who have sex with men (MSM) measured by pooling polymerase chain reaction (pooling-PCR). A total of 617 MSM subjects were included in a respondent driven sampling survey in Chongqing in 2008. Among the 129 that were tested HIV antibody positive, 102 were defined with long-term infection, 27 were assessed for recent HIV infection (RHI) using BED-CEIA. The remaining 488 HIV negative subjects were enrolled to the prospective cohort and followed-up every 6 months to monitor HIV seroconversion. All of the 488 HIV negative specimens were assessed for acute HIV infection (AHI) using pooling-PCR. Among the 488 negative subjects in the open cohort, 214 (43.9%) were followed-up for six months, with 107 person-years of observation and 14 subjects seroconverted. The observed HIV incidence was 12.5 per 100 person-years (95% CI=9.1-15.7). Among the 488 HIV negative specimens, 5 were identified with acute HIV infection using pooling-PCR at an annual rate of 14.02% (95% CI=1.73-26.30). The estimated HIV-1 incidence was 12.02% (95% CI=7.49-16.56) based on BED-CEIA. The HIV incidence estimated with three different approaches was different among subgroups. In the highly HIV prevalent MSM, it costs US$ 1724 to detect one AHI case, while detection of one case of RHI with BED assay costs only US$ 42. Three approaches generated comparable and high HIV incidences, pooling PCR and prospective cohort are more close to the true level of incidence, while BED-CEIA seemed to be the most convenient and economical approach for at-risk population’s HIV incidence evaluation at the beginning of HIV pandemic. HIV-1 incidences were alarmingly high among MSM population in Chongqing, particularly within the subgroup under 25 years of age and those migrants aged between 25 to 34 years.

Keywords: BED-CEIA, HIV, incidence, pooled PCR, prospective cohort

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Authors: Ahmed F. Azmy, Amal E. Saafan, Tamer M. Essam, Magdy A. Amin, Shaban H. Ahmed

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Bacterial strains capable of degradation of malathion from the domestic sewage were isolated by an enrichment culture technique. Three bacterial strains were screened and identified as Acinetobacter baumannii (AFA), Pseudomonas aeruginosae (PS1),andPseudomonas mendocina (PS2) based on morphological, biochemical identification and 16S rRNA sequence analysis. Acinetobacter baumannii AFA was the most efficient malathion degrading bacterium, so used for further biodegradation study. AFA was able to grow in mineral salt medium (MSM) supplemented with malathion (100 mg/l) as a sole carbon source, and within 14 days, 84% of the initial dose was degraded by the isolate measured by high performance liquid chromatography. Strain AFA could also degrade other organophosphorus compounds including diazenon, chlorpyrifos and fenitrothion. The effect of different culture conditions on the degradation of malathion like inoculum density, other carbon or nitrogen sources, temperature and shaking were examined. Degradation of malathion and bacterial cell growth were accelerated when culture media were supplemented with yeast extract, glucose and citrate. The optimum conditions for malathion degradation by strain AFA were; an inoculum density of 1.5x 1012CFU/ml at 30°C with shaking. A specific polymerase chain reaction primers were designed manually using multiple sequence alignment of the corresponding carboxylesterase enzymes of Acinetobacter species. Sequencing result of amplified PCR product and phylogenetic analysis showed low degree of homology with the other carboxylesterase enzymes of Acinetobacter strains, so we suggested that this enzyme is a novel esterase enzyme. Isolated bacterial strains may have potential role for use in bioremediation of malathion contaminated.

Keywords: Acinetobacter baumannii, biodegradation, malathion, organophosphate pesticides

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Authors: Marivane Turim Koschevic, Maycon dos Santos, Marcello Lima Bertuci, Farayde Matta Fakhouri, Silvia Maria Martelli

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Natural fibers are rich raw materials in cellulose and abundant in the world, its use for the cellulose nanocrystals extraction is promising as an example cited is the cattail, macrophyte native weed in South America. This study deals with the pre-treatment cattail of crushed fibers, at six different methods of mercerization, followed by the use of bleaching. As a result, have found The positive effects of treating fibers by means of optical microscopy and spectroscopy, Fourier transform (FTIR). The sample selected for future testing of cellulose nanocrystals extraction was treated in 2.5% NaOH for 2 h, 60 °C in the first stage and 30vol H2O2, NaOH 5% in the proportion 30/70% (v/v) for 1 hour 60 °C, followed by treatment at 50/50% (v/v) 15 minutes, 50°C, with the same constituents of the solution.

Keywords: cellulose nanocrystal, chemical treatment, mercerization, natural fibers

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Authors: António Fiúza, Aurora Futuro, Joana Monteiro, Joaquim Góis

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Lepidolite is an important lithium mineral that, to the author’s best knowledge, has not been used to produce lithium hydroxide, which is necessary for energy conversion to electric vehicles. Alkaline leaching of lithium concentrates allows the establishment of a production diagram avoiding most of the environmental drawbacks that are associated with the usage of acid reagents. The tested processes involve a pretreatment by digestion at high temperatures with additives, followed by leaching at hot atmospheric pressure. The solutions obtained must be compatible with solutions from the leaching of spodumene concentrates, allowing the development of a common treatment diagram, an important accomplishment for the feasible exploitation of Portuguese resources. Statistical programming and interpretation techniques minimize the laboratory effort required by conventional approaches and allow phenomenological comprehension.

Keywords: alkaline leaching, lithium, research design, statistical interpretation

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Authors: Vagolu S. Krishna, Shan Zheng, Estharla M. Rekha, Luke W. Guddat, Dharmarajan Sriram

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Tuberculosis (TB) remains a major threat to human health. This due to the fact that current drug treatments are less than optimal as well as the rising occurrence of multi drug-resistant and extensively drug-resistant strains of the etiological agent, Mycobacterium tuberculosis (Mt). Given the wide-spread significance of this disease, we have undertaken a design and evaluation program to discover new anti-TB drug leads. Here, our attention is focused on ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway. Importantly, this enzyme is present in bacteria but not in humans, making it an attractive proposition for drug discovery. In the present work, we used high-throughput virtual screening to identify seventeen potential inhibitors of KARI using the Birla Institute of Technology and Science in-house database. Compounds were selected based on high docking scores, which were assigned as the result of favourable interactions between the compound and the active site of KARI. The Ki values for two leads, compounds 14 and 16 are 3.71 and 3.06 µM, respectively for Mt KARI. To assess the mode of binding, 100 ns molecular dynamics simulations for these two compounds in association with Mt KARI were performed and showed that the complex was stable with an average RMSD of less than 2.5 Å for all atoms. Compound 16 showed an MIC of 2.06 ± 0.91 µM and a 1.9 fold logarithmic reduction in the growth of Mt in an infected macrophage model. The two compounds exhibited low toxicity against murine macrophage RAW 264.7 cell lines. Thus, both compounds are promising candidates for development as an anti-TB drug leads.

Keywords: ketol-acid reductoisomerase, macrophage, molecular docking and dynamics, tuberculosis

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Authors: Anju Joshi, C. N. Tharamani

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Diabetes is one of the leading cause of death at present and remains an important concern as the prevalence of the disease is increasing at an alarming rate. Therefore, it is crucial to diagnose the accurate levels of glucose for developing an efficient therapeutic for diabetes. Due to the availability of convenient and compact self-testing, continuous monitoring of glucose is feasible nowadays. Enzyme based electrochemical sensing of glucose is quite popular because of its high selectivity but suffers from drawbacks like complicated purification and immobilization procedures, denaturation, high cost, and low sensitivity due to indirect electron transfer. Hence, designing a robust enzyme free platform using transition metal oxides remains crucial for the efficient and sensitive determination of glucose. In the present work, manganese doped nickel oxide nanoparticles (Mn-NiO) has been synthesized onto the surface of multiwalled carbon nanotubes using a simple microwave assisted approach for non-enzymatic electrochemical sensing of glucose. The morphology and structure of the synthesized nanostructures were characterized using scanning electron microscopy (SEM) and X-Ray diffraction (XRD). We demonstrate that the synthesized nanostructures show enormous potential for electrocatalytic oxidation of glucose with high sensitivity and selectivity. Cyclic voltammetry and square wave voltammetry studies suggest superior sensitivity and selectivity of Mn-NiO decorated carbon nanotubes towards the non-enzymatic determination of glucose. A linear response between the peak current and the concentration of glucose has been found to be in the concentration range of 0.01 μM- 10000 μM which suggests the potential efficacy of Mn-NiO decorated carbon nanotubes for sensitive determination of glucose.

Keywords: diabetes, glucose, Mn-NiO decorated carbon nanotubes, non-enzymatic

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Authors: Thanapat Chaireongsirikul, Apichit Svang-Ariyaskul

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An analysis of mass balance, energy performance, and environmental impact assessment were performed to evaluate bioethanol production in Thailand. Thailand is an agricultural country. Thai government plans to increase the use of alternative energy to 20 percent by 2022. One of the primary campaigns is to promote a bioethanol production from abundant biomass resources such as bitter cassava, molasses and sugarcane. The bioethanol production is composed of three stages: cultivation, pretreatment, and bioethanol conversion. All of mass, material, fuel, and energy were calculated to determine the environmental impact of three types of bioethanol production: bioethanol production from cassava (CBP), bioethanol production from molasses (MBP), and bioethanol production from rice straw (RBP). The results showed that bioethanol production from cassava has the best environmental performance. CBP contributes less impact when compared to the other processes.

Keywords: bioethanol production, biofuel, LCA, chemical engineering

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Authors: Reginald Anyanwu

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Enzymes are biological molecules that significantly regulate the rate of almost all of the chemical reactions that take place within cells, and have been widely used for products’ innovations. They are vital for life and serve a wide range of important functions in the body, such as aiding in digestion and metabolism. The present study was aimed at finding out the extent to which biological molecules have been utilized by pharmaceutical, food and beverage, and biofuel industries in commercial and scale up applications. Taking into account the escalating business opportunities in this vertical, biotech firms have also been penetrating enzymes industry especially that of food. The aim of the study therefore was to find out how biocatalysis can be successfully deployed; how enzyme application can improve industrial processes. To achieve the purpose of the study, the researcher focused on the analytical tools that are critical for the scale up implementation of enzyme immobilization to ascertain the extent of increased product yield at minimum logistical burden and maximum market profitability on the environment and user. The researcher collected data from four pharmaceutical companies located at Anambra state and Imo state of Nigeria. Questionnaire items were distributed to these companies. The researcher equally made a personal observation on the applicability of these biological molecules on innovative Products since there is now shifting trends toward the consumption of healthy and quality food. In conclusion, it was discovered that enzymes have been widely used for products’ innovations but there are however variations on their applications. It was also found out that pivotal contenders of enzymes market have lately been making heavy investments in the development of innovative product solutions. It was recommended that the applications of enzymes on innovative products should be widely practiced.

Keywords: enzymes, pharmaceuticals, process development, quality food consumption, scale-up applications

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Authors: Zoltan Sebestyen, Eszter Barta-Rajnai, Emma Jakab, Zsuzsanna Czegeny

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Extraction of the energy content of lignocellulosic biomass is one of the possible pathways to reduce the greenhouse gas emission derived from the burning of the fossil fuels. The application of the bioenergy can mitigate the energy dependency of a country from the foreign natural gas and the petroleum. The diversity of the plant materials makes difficult the utilization of the raw biomass in power plants. This problem can be overcome by the application of thermochemical techniques. Pyrolysis is the thermal decomposition of the raw materials under inert atmosphere at high temperatures, which produces pyrolysis gas, biooil and charcoal. The energy content of these products can be exploited by further utilization. The differences in the chemical and physical properties of the raw biomass materials can be reduced by the use of torrefaction. Torrefaction is a promising mild thermal pretreatment method performed at temperatures between 200 and 300 °C in an inert atmosphere. The goal of the pretreatment from a chemical point of view is the removal of water and the acidic groups of hemicelluloses or the whole hemicellulose fraction with minor degradation of cellulose and lignin in the biomass. Thus, the stability of biomass against biodegradation increases, while its energy density increases. The volume of the raw materials decreases so the expenses of the transportation and the storage are reduced as well. Biooil is the major product during pyrolysis and an important by-product during torrefaction of biomass. The composition of biooil mostly depends on the quality of the raw materials and the applied temperature. In this work, thermoanalytical techniques have been used to study the qualitative and quantitative composition of the pyrolysis and torrefaction oils of a woody (black locust) and two herbaceous samples (rape straw and wheat straw). The biooil contains C5 and C6 anhydrosugar molecules, as well as aromatic compounds originating from hemicellulose, cellulose, and lignin, respectively. In this study, special emphasis was placed on the formation of the lignin monomeric products. The structure of the lignin fraction is different in the wood and in the herbaceous plants. According to the thermoanalytical studies the decomposition of lignin starts above 200 °C and ends at about 500 °C. The lignin monomers are present among the components of the torrefaction oil even at relatively low temperatures. We established that the concentration and the composition of the lignin products vary significantly with the applied temperature indicating that different decomposition mechanisms dominate at low and high temperatures. The evolutions of decomposition products as well as the thermal stability of the samples were measured by thermogravimetry/mass spectrometry (TG/MS). The differences in the structure of the lignin products of woody and herbaceous samples were characterized by the method of pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). As a statistical method, principal component analysis (PCA) has been used to find correlation between the composition of lignin products of the biooil and the applied temperatures.

Keywords: pyrolysis, torrefaction, biooil, lignin

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Authors: Lopamudra Ray, Malla Padma, Dibya Bhol, Samir Ranjan Mishra, A. N. Panda, Gurdeep Rastogi, T. K. Adhya, Ajit Kumar Pattnaik, Mrutyunjay Suar, Vishakha Raina

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Chilika Lake is the largest coastal estuarine brackish water lagoon in Asia situated on the east coast of India and is a designated Ramsar site. In the current study, several chitinolytic microorganisms were isolated and screened by appearance of clearance zone on 0.5% colloidal chitin agar plate. A strain designated as RC 1830 displayed maximum colloidal chitin degradation by release of 112 μmol/ml/min of N-acetyl D-glucosamine (GlcNAc) in 48h. The strain was taxonomically identified by polyphasic approach based on a range of phenotypic and genotypic properties and was found to be a novel species named Streptomyces chilikensis RC1830. The organism was halophilic (12% NaCl w/v), alkalophilic (pH10) and was capable of hydrolyzing chitin, starch, cellulose, gelatin, casein, tributyrin and tween 80. The partial purification of chitinase enzymes from RC1830 was performed by DEAE Sephacel anion exchange chromatography which revealed the presence of a very low molecular weight chitinase(10.5kD) which may be a probable chitobiosidase enzyme. The study reports the presence of a low MW chitinase (10.5kD) and a chitin decaetylase from a novel Streptomyces strain RC1830 isolated from Chilika Lake. Previously chitinases less than 20.5kD have not been reported from any other Streptomyces species. The enzymes was characterized with respect to optimum pH, temperature, and substrate specificity and temperature stability.

Keywords: chitinases, chitobiosidase, Chilika Lake, India

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Authors: Idan Chiyanzu, Maruping Mangena

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Crude glycerol, a major by-product from the transesterification of triacylglycerides with alcohol to biodiesel, is known to have a broad range of applications. For example, its bioconversion can afford a wide range of chemicals including alcohols, organic acids, hydrogen, solvents and intermediate compounds. In bacteria, the 2-oxoglutarate dioxygenase (2-OGD) enzymes are widely found among the Pseudomonas syringae species and have been recognized with an emerging importance in ethylene formation. However, the use of optimized enzyme function in recombinant systems for crude glycerol conversion to ethylene is still not been reported. The present study investigated the production of ethylene from crude glycerol using engineered E. coli MG1655 and JM109 strains. Ethylene production with an optimized expression system for 2-OGD in E. coli using a codon optimized construct of the ethylene-forming gene was studied. The codon-optimization resulted in a 20-fold increase of protein production and thus an enhanced production of the ethylene gas. For a reliable bioreactor performance, the effect of temperature, fermentation time, pH, substrate concentration, the concentration of methanol, concentration of potassium hydroxide and media supplements on ethylene yield was investigated. The results demonstrate that the recombinant enzyme can be used for future studies to exploit the conversion of low-priced crude glycerol into advanced value products like light olefins, and tools including recombineering techniques for DNA, molecular biology, and bioengineering can be used to allowing unlimited the production of ethylene directly from the fermentation of crude glycerol. It can be concluded that recombinant E.coli production systems represent significantly secure, renewable and environmentally safe alternative to thermochemical approach to ethylene production.

Keywords: crude glycerol, bioethylene, recombinant E. coli, optimization

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Authors: Fiúza A., Góis J. Leite M., Braga H., Lima A., Jorge P., Moutela P., Martins L., Futuro A.

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Lepidolite is an important lithium mineral that, to the author’s best knowledge, has not been used to produce lithium hydroxide, necessary for energy conversion to electric vehicles. Alkaline leaching of lithium concentrates allows the establishment of a production diagram avoiding most of the environmental drawbacks that are associated with the usage of acid reagents. The tested processes involve a pretreatment by digestion at high temperatures with additives, followed by leaching at hot atmospheric pressure. The solutions obtained must be compatible with solutions from the leaching of spodumene concentrates, allowing the development of a common treatment diagram, an important accomplishment for the feasible exploitation of Portuguese resources. Statistical programming and interpretation techniques are used to minimize the laboratory effort required by conventional approaches and also allow phenomenological comprehension.

Keywords: artificial intelligence, tailings free process, ferroelectric electrolyte battery, life cycle assessment

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Authors: Ozlem Bahadir Acikara, Mert Ilhan, Ekin Kurtul, Karel Smejkal, Esra Kupeli Akkol

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Present study is aimed to investigate in vitro inhibitory effects of the extracts prepared from the aerial parts of Podospermum canum (Asteraceae) on hyaluronidase, collagenase, and elastase enzymes using a bioassay-guided fractionation. Inhibitory effects of the extract, sub-extracts, fractions obtained by column chromatography, and isolated compounds on collagenase, elastase, and hyaluronidase were performed by using in vitro enzyme inhibitory assays based on spectrophotometric evaluation. The ethyl acetate and remaining water extracts prepared from the plant displayed significant inhibitory activities on collagenase and elastase, while petroleum ether and chloroform extracts did not show any inhibitory activity. Eleven known compounds: arbutin, 6'-O-caffeoylarbutin, cichoriin, 3,5-dicaffeoylquinic acid methyl ester, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, apigenin-7-O-β-rutinoside, isoorientin, orientin, vitexin, procatechuic acid, and compound 4-hydroxy-benzoic acid 4-(6-O-α-rhamnopyranosyl-β-glucopyranosyl) benzyl ester have been obtained from ethyl acetate sub-extract of the plant through bioassay-guided fractionation and isolation. Results of the present study have revealed that among the isolated compounds, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, apigenin-7-O-β-rutinoside and isoorientin showed potent enzyme inhibitory activities. However, methanolic extract of P. canum displayed a greater inhibitory activity than fractions and isolated compounds both on collagenase and elastase.

Keywords: Asteraceae, collagenase, elastase, hyaluronidase, Podospermum canum

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Authors: Srestha Chaturabul, Wanchalerm Srirachat, Thanaporn Wannachod, Prakorn Ramakul, Ura Pancharoen, Soorathep Kheawhom

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The separation of mercury(II) from petroleum-produced water from the Gulf of Thailand was carried out using a hollow fiber supported liquid membrane system (HFSLM). Optimum parameters for feed pretreatment were 0.2 M HCl, 4% (v/v) Aliquat 336 for extractant and 0.1 M thiourea for stripping solution. The best percentage obtained for extraction was 99.73% and for recovery 90.11%, respectively. The overall separation efficiency noted was 94.92% taking account of both extraction and recovery prospects. The model for this separation developed along a combined flux principle i.e. convection–diffusion–kinetic. The results showed excellent agreement with theoretical data at an average standard deviation of 1.5% and 1.8%, respectively.

Keywords: separation, mercury(ii), petroleum produced water, hollow fiber, liquid membrane

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Authors: Ragy Raafat Gaber Attaalla

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For cardiovascular illness, oral P2Y12 inhibitors including clopidogrel, prasugrel, and ticagrelor are frequently recommended. Each of these medications has advantages and disadvantages. In the absence of genotyping, it has been demonstrated that the stronger platelet aggregation inhibitors prasugrel and ticagrelor are superior than clopidogrel at preventing significant adverse cardiovascular events following an acute coronary syndrome and percutaneous coronary intervention (PCI). Both, nevertheless, come with a higher risk of bleeding unrelated to a coronary artery bypass. As a prodrug, clopidogrel needs to be bioactivated, principally by the CYP2C19 enzyme. A CYP2C19 no function allele and diminished or absent CYP2C19 enzyme activity are present in about 30% of people. The reduced exposure to the active metabolite of clopidogrel and reduced inhibition of platelet aggregation among clopidogrel-treated carriers of a CYP2C19 no function allele likely contributed to the reduced efficacy of clopidogrel in clinical trials. Clopidogrel's pharmacogenetic results are strongest when used in conjunction with PCI, but evidence for other indications is growing. One of the most typical examples of clinical pharmacogenetic application is CYP2C19 genotype-guided antiplatelet medication following PCI. Guidance is available from expert consensus groups and regulatory bodies to assist with incorporating genetic information into P2Y12 inhibitor prescribing decisions. Here, we examine the data supporting genotype-guided P2Y12 inhibitor selection's effects on clopidogrel response and outcomes and discuss tips for pharmacogenetic implementation. We also discuss procedures for using genotype data to choose P2Y12 inhibitor therapies as well as any unmet research needs. Finally, choosing a P2Y12 inhibitor medication that optimally balances the atherothrombotic and bleeding risks may be influenced by both clinical and genetic factors.

Keywords: inhibitors, cardiovascular events, coronary intervention, pharmacogenetic implementation

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Authors: F. Ambreen, A.Naheed

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Osteoarthritis (OA) is a degenerative disorder affecting millions of people worldwide. Nitric oxide (NO) was found to play a catabolic role in the development of osteoarthritis. It is a toxic free radical gas generated during the metabolism of L-arginine by the enzyme Nitric oxide synthase (NOS). Inducible Nitric Oxide Synthase (iNOS) is one of the isoform of NOS, and its overexpression leads to the excessive formation of NO that results in pathophysiological joint conditions. Several synthetic anti-inflammatory drugs and inhibitors are present to date, but all showed side effects and complications. Therefore, the pursuit of natural disease-modifying drugs remains a top priority. Curcumin is an active component of turmeric, and the past few decades have witnessed intense research devoted to the antioxidant and anti-inflammatory properties of curcumin. The present study focused on curcumin and its derivatives in the search for new iNOS inhibitors for the treatment of osteoarthritis. We conducted a molecular docking study on curcumin and its four derivatives; cyclocurcumin, tetrahydrocurcumin, demethoxycurcumin and curcumin monoglucoside with iNOS using CLC Drug discovery work bench 3.02. We selected two co-crystallized ligands for this study; tetrahydrobiopterin and N-omega-propyl-L-arginine present in complex with the enzyme iNOS. Results showed the best binding affinity of N-omega-propyl-L-arginine with cyclocurcumin and curcumin monoglucoside that exhibit binding energies of -65.2 kcal/mol and -68 kcal/mol respectively. Whereas with tetrahydrobiopterin, best binding scores of -64.7 kcal/mol and -62.2 kcal/mol were found with tetrahydrocurcumin and demethoxycurcumin respectively. This information could open doors of research for the designing of novel drugs using herbs such as curcumin for the treatment of inflammatory joint diseases.

Keywords: curcumin, iNOS, molecular docking, osteoarthritis

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Authors: Jyoti Kumari, Pavuluri Srinivasa Rao

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Jackfruit (ArtocarpusheterophyllusL.) is an underutilized yet highly nutritious fruit with unique flavour, known for its therapeutic and culinary properties. Fresh jackfruit bulb has a very short shelf life due to high moisture and sugar content leading to microbial and enzymatic browning, hindering its consumer acceptability and marketability. An attempt has been made for the preservation of the ripe jackfruit bulbs, by the application of high pressure (HP) over a range of 200-500 MPa at ambient temperature for dwell times ranging from 5 to 20 min. The physicochemical properties of jackfruit bulbs such as the pH, TSS, and titrable acidity were not affected by the pressurization process. The ripening index of the fruit bulb also decreased following HP treatment. While the ascorbic acid and antioxidant activity of jackfruit bulb were well retained by high pressure processing (HPP), the total phenols and carotenoids showed a slight increase. The HPP significantly affected the colour and textural properties of jackfruit bulb. High pressure processing was highly effective in reducing the browning index of jackfruit bulbs in comparison to untreated bulbs. The firmness of the bulbs improved upon the pressure treatment with longer dwelling time. The polyphenol oxidase has been identified as the most prominent oxidative enzyme in the jackfruit bulb. The enzymatic activity of polyphenol oxidase and peroxidase were significantly reduced by up to 40% following treatment at 400 MPa/15 min. HPP of jackfruit bulbs at ambient temperatures is shown to be highly beneficial in improving the shelf stability, retaining its nutrient profile, color, and appearance while ensuring the maximum inactivation of the spoilage enzymes.

Keywords: antioxidant capacity, ascorbic acid, carotenoids, color, HPP-high pressure processing, jackfruit bulbs, polyphenol oxidase, peroxidase, total phenolic content

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Authors: K. S. Zaytsev, Y. R. Nartsissov

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Glycine is one of the key inhibitory neurotransmitters in Central nervous system (CNS) meanwhile glycinergic transmission is highly dependable on its appropriate reuptake from synaptic cleft. Glycine transporters (GlyT) of types 1 and 2 are the enzymes providing glycine transport back to neuronal and glial cells along with Na⁺ and Cl⁻ co-transport. The distribution and stoichiometry of GlyT1 and GlyT2 differ in details, and GlyT2 is more interesting for the research as it reuptakes glycine to neuron cells, whereas GlyT1 is located in glial cells. In the process of GlyT2 activity, the translocation of the amino acid is accompanied with binding of both one chloride and three sodium ions consequently (two sodium ions for GlyT1). In the present study, we developed a computer simulator of GlyT2 and GlyT1 activity based on known experimental data for quantitative estimation of membrane glycine transport. The trait of a single protein functioning was described using the probability approach where each enzyme state was considered separately. Created scheme of transporter functioning realized as a consequence of elemental steps allowed to take into account each event of substrate association and dissociation. Computer experiments using up-to-date kinetic parameters allowed receiving the number of translocated glycine molecules, Na⁺ and Cl⁻ ions per time period. Flexibility of developed software makes it possible to evaluate glycine reuptake pattern in time under different internal characteristics of enzyme conformational transitions. We investigated the behavior of the system in a wide range of equilibrium constant (from 0.2 to 100), which is not determined experimentally. The significant influence of equilibrium constant in the range from 0.2 to 10 on the glycine transfer process is shown. The environmental conditions such as ion and glycine concentrations are decisive if the values of the constant are outside the specified range.

Keywords: glycine, inhibitory neurotransmitters, probability approach, single protein functioning

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Authors: Sarah Gaßmeyer, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist

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Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find frequent applications in deracemization and dynamic kinetic resolutions. Arylmalonate decarboxylase (AMDase) from Bordetella Bronchiseptica has high structural similarity to amino acid racemases. These cofactor-free racemases are able to break chemically strong CH-bonds under mild conditions. The racemase-like catalytic machinery of mutant G74C conveys it a unique activity in the racemisation of pharmacologically relevant derivates of 2-phenylpropionic acid (profenes), which makes AMDase G74C an interesting object for the mechanistic investigation of cofactor-independent racemases. Structure-guided protein engineering achieved a variant of this unique racemase with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. By saturation–transfer–difference NMR spectroscopy (STD-NMR), substrate binding during catalysis was investigated. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose variation increased the activity of G74C. While single-amino acid exchanges increased the activity moderately, structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

Keywords: racemase, rational protein design, STD-NMR, structure guided saturation mutagenesis

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Authors: Carlos A. C. G. Neto, Natan C. G. e Silva , Thaís de O. Costa, Luciana R. B. Gonçalves, Maria V. P. Rocha

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β-galactosidases (E.C. 3.2.1.23) are enzymes that have attracted by catalyzing the hydrolysis of lactose and in producing galacto-oligosaccharides by favoring transgalactosylation reactions. These enzymes, when immobilized, can have some enzymatic characteristics substantially improved, and the coating of supports with multifunctional polymers is a promising alternative to enhance the stability of the biocatalysts, among which polyethylenimine (PEI) stands out. PEI has certain properties, such as being a flexible polymer that suits the structure of the enzyme, giving greater stability, especially for multimeric enzymes such as β-galactosidases. Besides that, protects them from environmental variations. The use of chitosan support coated with PEI could improve the catalytic efficiency of β-galactosidase from Kluyveromyces lactis in the transgalactosylation reaction for the production of prebiotics, such as lactulose since this strain is more effective in the hydrolysis reaction. In this context, the aim of the present work was first to develop biocatalysts of β-galactosidase from K. lactis immobilized on chitosan-coated with PEI, determining the immobilization parameters, its operational and thermal stability, and then to apply it in hydrolysis and transgalactolisation reactions to produce lactulose using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% (v/v) glutaraldehyde and then coated with 10% (w/v) PEI solution was evaluated using an enzymatic load of 10 mg protein per gram support. Subsequently, the hydrolysis and transgalactosylation reactions were conducted at 50 °C, 120 RPM for 20 minutes, using whey supplemented with fructose at a ratio of 1:2 lactose/fructose, totaling 200 g/L. Operational stability studies were performed in the same conditions for 10 cycles. Thermal stabilities of biocatalysts were conducted at 50 ºC in 50 mM phosphate buffer, pH 6.6 with 0.1 mM MnCl2. The biocatalyst whose support was coated was named CHI_GLU_PEI_GAL, and the one that was not coated was named CHI_GLU_GAL. The coating of the support with PEI considerably improved the parameters of immobilization. The immobilization yield increased from 56.53% to 97.45%, biocatalyst activity from 38.93 U/g to 95.26 U/g and the efficiency from 3.51% to 6.0% for uncoated and coated support, respectively. The biocatalyst CHI_GLU_PEI_GAL was better than CHI_GLU_GAL in the hydrolysis of lactose and production of lactulose, converting 97.05% of lactose at 5 min of reaction and producing 7.60 g/L lactulose in the same time interval. QUI_GLU_PEI_GAL biocatalyst was stable in the hydrolysis reactions of lactose during the 10 cycles evaluated, converting 73.45% lactose even after the tenth cycle, and in the lactulose production was stable until the fifth cycle evaluated, producing 10.95 g/L lactulose. However, the thermal stability of CHI_GLU_GAL biocatalyst was superior, with a half-life time 6 times higher, probably because the enzyme was immobilized by covalent bonding, which is stronger than adsorption (CHI_GLU_PEI_GAL). Therefore, the strategy of coating the supports with PEI has proven to be effective for the immobilization of β-galactosidase from K. lactis, considerably improving the immobilization parameters, as well as, the catalytic action of the enzyme. Besides that, this process can be economically viable due to the use of an industrial residue as a substrate.

Keywords: β-galactosidase, immobilization, kluyveromyces lactis, lactulose, polyethylenimine, transgalactosylation reaction, whey

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Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi

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Seed aging during storage is a complex biochemical and physiological processes that can lead to reduce seed germination. This phenomenon associated with increasing of total antioxidant activity during aging. To study the effects of hormones on seed aging, aged wheat seeds (control, 90 and 80% viabilities) were treated with GA3, Salicylic Acid, and paclobutrazol and antioxidant system were investigated as molecular biomarkers for seed vigor. The results showed that, seed priming treatment significantly affected germination percentage, normality seedling percentage, H2O2, MDA, CAT, APX, and GPX activates. Maximum germination percentage achieve in GA3 priming in control treatment. Germination percentage and normal seedling percentage increased in other GA3 priming treatment compared with other hormones. Also aging increased MDA, H2O2 content. MDA is considered sensitive marker commonly used for assessing membrane lipid peroxidation and H2O2result in toxicity to cellular membrane system and damages to plant cells. Amount of H2O2 and MDA declined in GA3 treatment. CAT, GPX and APX activities were reduced by increasing the aging time and at different levels of priming. The highest APX activity was observed in Salicylic Acid control treatment and the highest GPX and CAT activity was obtained in GA3 control treatment. The lowest MDA and H2O2 showed in GA3 control treatment, too. Hormone priming increased Antioxidant enzyme activity and decreased amount of reactive oxygen space and malondialdehyde (MDA) under aging treatment. Also, GA3 priming treatments have a significant effect on germination percentage and number of normal seedling. Generally aging seed, increase ROS and lipid peroxidation. Antioxidant enzymes activity of aged seeds increased after hormone priming.

Keywords: hormones priming, wheat, aging seed, antioxidant, lipid peroxidation

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Authors: Sánchez Acuña, Juan Camilo, Granados Gómez, Mildred Magaly, Navarrete Rodríguez, Luisa Fernanda

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Nowadays, the main biofuels source production as bioethanol is food crops. This means a high competition between foods and energy production. For this reason, it is necessary to take into account the use of new raw materials friendly to the environment. The main objective of this paper is to evaluate the potential of the agro-industrial banana crop residues in the production of bioethanol. A factorial design of 2⁴ was used, the design has variables such as pH, time and concentration of hydrolysis, another variable is the time of fermentation that is of 7 or 15 days. In the hydrolysis phase, the pH is acidic (H₂SO₄) or basic (NaOH), the time is 30 or 15 minutes and the concentration is 0.1 or 0.5 M. It was observed that basic media, low concentrations, fermentation, and higher pretreatment times produced better performance in terms of biofuel obtained.

Keywords: bioethanol, biofuels, banana waste, hydrolysis

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Authors: V. Karuppaiah, J. C. Padaria, C. Srivastava

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The tobacco caterpillar, Spodoptera litura Fab (Lepidoptera: Noctuidae) is a polyphagous pest various field and horticulture crops in India. Pest had virtually developed resistance to all commonly used insecticides. Enhanced detoxification is the prime mechanism that is dictated by detoxification different enzymes and carboxylesterase is one of the major enzyme responsible development of resistance. In India, insecticide resistance studies on S. litura are mainly deployed on detoxification enzymes activity and investigation at gene level alteration i.e. at nucleotide level is very merger. In the present study, we collected the S. litura larvae from three different cauliflower growing belt viz., IARI, New Delhi (Delhi), Palari, Sonepat (Haryana) and Varanasi (Uttar Pradesh) to study the role of carboxylesterase activity and its gene level variation The CarE activity was measured using UV-VIS spectrophotometer with 3rd instar larvae of S. litura. The elevated activity of CarE was observed in Sonepat strain (28.09 ± 0.09 µmol/min/mg of protein) followed by Delhi (26.72 ± 0.04 µmol/min/mg of protein) and Varanasi strain (10.00 ± 0.44 µmol/min/mg of protein) of S. litura. The genomic DNA was isolated from 3rd instar larvae and CarE gene was amplified using a primer sequence, F:5’tccagagttccttgtcaggcac3’; R:5’ctgcatcaagcatgtctc3. CarE gene, about 500bp was partially amplified, sequenced and submitted to NCBI (Accession No. KF835886, KF835887 and KF835888). The sequence data revealed polymorphism at nucleotide level in all the three strains and gene found to have 88 to 97% similarity with previous available nucleotide sequences of S. litura, S. littoralis and S. exiqua. The polymorphism at the nucleotide level could be a reason for differential activity of carboxylesterase enzymes among the strains. However, investigation at gene expression level would be useful to analyze the overproduction of carboxylesterase enzyme.

Keywords: carboxylesterase, CarE gene, nucleotide polymorphism, insecticide resistance, spodoptera litura

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Authors: Ali Aydin, Mert Sudagidan, Aysen Coban, Alparslan Kadir Devrim

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Pseudomonas spp. (specifically, P. fluorescens and P. fragi) are considered the principal spoilage microorganisms of refrigerated poultry meats. The higher the level psychrophilic spoilage Pseudomonas spp. on carcasses at the end of processing lead to decrease the shelf life of the refrigerated product. The aim of the study was the identification of psychrophilic Pseudomonas spp. having proteolytic and lipolytic activities from poultry meats by 16S rRNA and rpoB gene sequencing, investigation of protease and lipase related genes and determination of proteolytic activity of Pseudomonas spp. In the of isolation procedure, collected chicken meat samples from local markets and slaughterhouses were homogenized and the lysates were incubated on Standard method agar and Skim Milk agar for selection of proteolytic bacteria and tributyrin agar for selection of lipolytic bacteria at +4 °C for 7 days. After detection of proteolytic and lipolytic colonies, the isolates were firstly analyzed by biochemical tests such as Gram staining, catalase and oxidase tests. DNA gene sequencing analysis and comparison with GenBank revealed that 126 strong enzyme Pseudomonas spp. were identified as predominantly P. fluorescens (n=55), P. fragi (n=42), Pseudomonas spp. (n=24), P. cedrina (n=2), P. poae (n=1), P. koreensis (n=1), and P. gessardi (n=1). Additionally, protease related aprX gene was screened in the strains and it was detected in 69/126 strains, whereas, lipase related lipA gene was found in 9 Pseudomonas strains. Protease activity was determined using commercially available protease assay kit and 5 strains showed high protease activity. The results showed that psychrophilic Pseudomonas strains were present in chicken meat samples and they can produce important levels of proteases and lipases for food spoilage to decrease food quality and safety.

Keywords: Pseudomonas, chicken meat, protease, lipase

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Authors: Ekpekpo Arthur

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Thin films can either be conductive or dielectric (non-conductive). It is formed through atom/molecules state or formed after decomposing the materials into atomic/molecular scale by physical or chemical processes. In this study, thin films of Lead Sulphide were deposited on glass substrate prepared from lead acetate and thiourea solution using chemical bath deposition (CBD). The glass slides were subjected to the pretreatment by soaking them in a solution of 50% sulphuric acid and 50% nitric acid. Lead sulphide was deposited at different parameters such as deposition time and temperature. The optical properties of the thin films were determined from spectroscopy measurements of absorbance and reflectance. Optical studies show that the band gap of lead sulphide ranges between 0.41 eV to 300K.

Keywords: lead sulphide, spectroscopy, absorbance, reflectance

Procedia PDF Downloads 416

Authors: Chukwuma S. Ezeonu, Ikechukwu N. E. Onwurah, Uchechukwu U. Nwodo, Chibuike S. Ubani, Chigozie M. Ejikeme

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Trichophyton soudanense and Trichophyton mentagrophyte were isolated from the rice mill environment, cultured and used singly and as di-culture in the treatment of measure quantities of preheated rice husk. Optimized conditions studied showed that carboxymethylcellulase (CMCellulase) activity of 57.61 µg/ml/min was optimum for Trichophyton mentagrophyte heat pretreated rice husk crude enzymes at 50oC and 80oC respectively. Duration of 120 hours (5 days) gave the highest CMcellulase activity of 75.84 µg/ml/min for crude enzyme of Trichophyton mentagrophyte heat pretreated rice husk. However, 96 hours (4 days) duration gave maximum activity of 58.21 µg/ml/min for crude enzyme of Trichophyton soudanense heat pretreated rice husk. Highest CMCellulase activities of 67.02 µg/ml/min and 69.02 µg/ml/min at pH of 5 were recorded for crude enzymes of monocultures of Trichophyton soudanense (TS) and Trichophyton mentagrophyte (TM) heat pretreated rice husk respectively. Biomass components showed that rice husk cooled after heating followed by treatment with Trichophyton mentagrophyte gave 44.50 ± 10.90 (% ± Standard Error of Mean) cellulose as the highest yield. Maximum total lignin value of 28.90 ± 1.80 (% ± SEM) was obtained from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM). The hemicellulose content of 30.50 ± 2.12 (% ± SEM) from pre-heated rice husk treated with Trichophyton soudanense (TS); lignin value of 28.90 ± 1.80 from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM); also carbohydrate content of 16.79 ± 9.14 (% ± SEM) , reducing and non-reducing sugar values of 2.66 ± 0.45 and 14.13 ± 8.69 (% ± SEM) were all obtained from for pre- heated rice husk treated with Trichophyton mentagrophyte (TM). All the values listed above were the highest values obtained from each rice husk treatment. The pre-heated rice husk treated with Trichophyton mentagrophyte (TM) fermented with palmwine yeast gave bio-ethanol value of 11.11 ± 0.21 (% ± Standard Deviation) as the highest yield.

Keywords: Trichophyton soudanense, Trichophyton mentagrophyte, biomass, bioethanol, rice husk

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Authors: Massimo Mozzon, Nadia Raffaelli

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Proteases from herbs, woody plants, and trees are exploited for cheesemaking in several countries, especially in South Europe and West Africa. Particularly, “thistles” belonging to several genera within the Asteraceae family (Cynara, Silybum, Centaurea, Carlina, Cirsium, Onopordum) are traditionally used in Mediterranean countries for clotting raw ewe’s and goat’s milk. For the first time, the clotting performance of an aqueous extract from flowers of Onopordum tauricum Willd. (Taurian thistle, bull cottonthistle) were tested in milk of different origin (cow, goat, ewe). The vegetable material was collected in the Central Apennines range, between the Marche and Umbria regions. A response surface methodology (RSM) approach was used to study the effect of the curdling variables (temperature, pH, amount of enzymatic extract) on the technological performance of the thistle extract. A three-step procedure for the purification of the enzyme (ammonium sulphate precipitation, gel filtration and ion-exchange chromatography) was also carried out. The milk clotting activity (MCA) of O. tauricum crude extracts was strongly affected by temperature, pH and by the interaction between these two variables, according to a second-order response surface model, while the milk/coagulant ratio did not affect in a significant way the clotting properties. Experimental data showed that the addition of 10 mM CaCl2 reduced the clotting time of ewe’s, goat’s, and cow’s milk by about 3-fold, 8-fold, and 14-fold, respectively, at 35°C and pH 6.7-6.8. After purification, an enzymatic preparation very close to homogeneity was obtained, which showed a major band at about 30 kDa when analyzed by SDS-PAGE. The identity of the enzyme as an aspartic protease was confirmed by inhibition studies. Cheese-making trials were carried out to check the scale-up (1 to 5 L of milk; 37 °C; 10 mM CaCl2 fortification) and set the recipe: 35-45% of curd yields were recorded, according to curd cutting and pressing.

Keywords: milk clotting activity, Onopordum tauricum, plant proteases, vegetable rennet

Procedia PDF Downloads 139