Search results for: yeast enzymes

Authors: A. K. R. Gobinath, He Jianzhong, Kun-Lin Yang

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Fruit waste was used as a feedstock to produce biohydrogen in this study. Fruit waste used in this study was collected from several fruit juice stalls in Singapore. Based on our observation, the fruit waste contained 35-40% orange, 10-20% watermelon, 10-15% apple, 10-15% pineapple, 1-5% mango. They were mixed with water (1:1 ratio based on wet biomass) and blended to attain homogenous mixtures. Later, fruit waste was subjected to one of the following pretreatments: autoclave (121 °C for 20min), microwave (20min) or both. After pretreatment, the total sugar concentration in the hydrolysate was high (>12g/l) when both autoclave and microwave were applied. In contrast, samples without pretreatment measured only less than 2g/l of sugar. While using these hydrolysates as carbon sources, Clostridium strain BOH3 produces 2526-3126 ml/l of hydrogen after 72h of anaerobic fermentation. The hydrogen yield was 295-300 ml/g of sugar which is close to the hydrogen yields from glucose (338 ml/gm) and xylose (330 ml/gm). Our HPLC analysis showed that fruit waste hydrolysate contained oligosugars (25-27%), sucrose (18-23%), fructose (25-30%), glucose (10-15%) and mannose (2-5%). Additionally, pretreatment led to the release of free amino acids (160-512 mg/l), calcium (7.8-12.9 ppm), magnesium (4.32-6.55 ppm), potassium (5.4-65.1 ppm) and sodium (0.4-0.5 ppm) into the hydrolysate. These nutrients were able to support strain-BOH3 to grow and produce high level of hydrogen. Notably, unlike other pretreatment methods (with strong acids and bases), these pretreatment techniques did not generate any inhibitors (e.g. furfural and phenolic acids) to suppress the hydrogen production. Interestingly, strain BOH3 can also ferment pretreated fruit waste slurry and produce hydrogen with a high yield (156-343 ml/gm fruit waste). While fermenting pretreated fruit waste slurry, strain-BOH3 excreted several saccharolytic enzymes majorly xylanase (1.84U/ml), amylase (1.10U/ml), pectinase (0.36U/ml) and cellulase (0.43U/ml). Due to expressions of these enzymes, strain BOH3 was able to directly utilize pretreated fruit waste hydrolysate and produces high-level of hydrogen.

Keywords: autoclave pretreatment, biohydrogen production, clostridial fermentation, fruit waste, and microwave pretreatment

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Authors: C. Müller, T. Ortmann, S. Scholl, H. J. Jördening

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Multienzymatic catalysis can be used as an alternative to chemical synthesis or hydrolysis of polysaccharides for the production of high value oligosaccharides from cheap resources such as sucrose. However, development of multienzymatic processes is complex, especially with respect to suitable conditions for enzymes originating from different organisms. Furthermore, an optimal configuration of the catalysts in a reaction cascade has to be found. These challenges can be approached by design of experiments. The system investigated in this study is a trienzymatic catalyzed reaction which results in laminaribiose production from sucrose and comprises covalently immobilized sucrose phosphorylase (SP), glucose isomerase (GI) and laminaribiose phosphorylase (LP). Operational windows determined with design of experiments and kinetic data of the enzymes were used to optimize the enzyme ratio for maximum product formation and minimal production of byproducts. After adjustment of the enzyme activity ratio to 1: 1.74: 2.23 (SP: LP: GI), different process options were investigated in silico. The considered options included substrate dependency, the use of glucose as co-substrate and substitution of glucose isomerase by glucose addition. Modeling of batch operation in a stirred tank reactor led to yields of 44.4% whereas operation in a continuous stirred tank reactor resulted in product yields of 22.5%. The maximum yield in a bienzymatic system comprised of sucrose phosphorylase and laminaribiose phosphorylase was 67.7% with sucrose and different amounts of glucose as substrate. The experimental data was in good compliance with the process model for batch operation. The continuous operation will be investigated in further studies. Simulation of operational process possibilities enabled us to compare various operational modes regarding different aspects such as cost efficiency, with the minimum amount of expensive and time-consuming practical experiments. This gives us more flexibility in process implementation and allows us, for example, to change the production goal from laminaribiose to higher oligosaccharides.

Keywords: design of experiments, enzyme kinetics, multi-enzymatic system, in silico process development

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Authors: Manuel A. Alonso-Tarajano, Roberto Mosca, Patrick Aloy

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Protein kinases participate in a myriad of cellular processes of major biomedical interest. The in vivo substrate specificity of these enzymes is a process determined by several factors, and despite several years of research on the topic, is still far from being totally understood. In the present work, we have quantified the contributions to the kinase substrate specificity of i) the phosphorylation sites and their surrounding residues in the sequence and of ii) the association of kinases to adaptor or scaffold proteins. We have used position-specific scoring matrices (PSSMs), to represent the stretches of sequences phosphorylated by 93 families of kinases. We have found negative correlations between the number of sequences from which a PSSM is generated and the statistical significance and the performance of that PSSM. Using a subset of 22 statistically significant PSSMs, we have identified specificity determinant residues (SDRs) for 86% of the corresponding kinase families. Our results suggest that different SDRs can function as positive or negative elements of substrate recognition by the different families of kinases. Additionally, we have found that human proteins with known function as adaptors or scaffolds (kAS) tend to interact with a significantly large fraction of the substrates of the kinases to which they associate. Based on this characteristic we have identified a set of 279 potential adaptors/scaffolds (pAS) for human kinases, which is enriched in Pfam domains and functional terms tightly related to the proposed function. Moreover, our results show that for 74.6% of the kinase– pAS association found, the pAS colocalize with the substrates of the kinases they are associated to. Finally, we have found evidence suggesting that the association of kinases to adaptors and scaffolds, may contribute significantly to diminish the in vivo substrate crossed- specificity of protein kinases. In general, our results indicate the relevance of several SDRs for both the positive and negative selection of phosphorylation sites by kinase families and also suggest that the association of kinases to pAS proteins may be an important factor for the localization of the enzymes with their set of substrates.

Keywords: kinase, phosphorylation, substrate specificity, adaptors, scaffolds, cellular colocalization

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Authors: Saule Mussabekova

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Recent advances in genetics have allowed increasing acutely the capacities of the formation of reliable evidence in conducting forensic examinations. Thus, traces of biological origin are important sources of information about a crime. Currently, around the world, sexual offenses have increased, and among them are those in which the criminals use various detergents to remove traces of their crime. A feature of modern synthetic detergents is the presence of biological additives - enzymes. Enzymes purposefully destroy stains of biological origin. To study the nature and extent of the impact of modern washing powders on saliva stains on the physical evidence, specially prepared test specimens of different types of tissues to which saliva was applied have been examined. Materials and Methods: Washing machines of famous manufacturers of household appliances have been used with different production characteristics and advertised brands of washing powder for test washing. Over 3,500 experimental samples were tested. After washing, the traces of saliva were identified using modern research methods of forensic medicine. Results: The influence was tested and the dependence of the use of different washing programs, types of washing machines and washing powders in the process of establishing saliva trace and identify of the stains on the physical evidence while washing was revealed. The results of experimental and practical expert studies have shown that in most cases it is not possible to draw the conclusions in the identification of saliva traces on physical evidence after washing. This is a consequence of the effect of biological additives and other additional factors on traces of saliva during washing. Conclusions: On the basis of the results of the study, the feasibility of saliva traces of the stains on physical evidence after washing is established. The use of modern molecular genetic methods makes it possible to partially solve the problems arising in the study of unlaundered evidence. Additional study of physical evidence after washing facilitates detection and investigation of sexual offenses against women and children.

Keywords: saliva research, modern synthetic detergents, laundry detergents, forensic medicine

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Authors: Loren S. Bernal., Catalina Castillo, Carel E. Carvajal, José F. Ibla

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Plastic pollution, specifically microplastics, has become a significant issue in aquatic ecosystems worldwide. The large amount of plastic waste carried by water tributaries has resulted in the accumulation of microplastics in water bodies. The polymer aging process caused by environmental influences such as photodegradation and chemical degradation of additives leads to polymer embrittlement and properties change that require degradation or reduction procedures in rivers. However, there is a lack of such procedures for freshwater entities that develop over extended periods. The aim of this study is evaluate the potential of Pleurotus ostreatus a fungus, in reducing lowdensity polyethylene microplastics present in freshwater samples collected from the middle basin of the Magdalena River in Colombia. The study aims to evaluate this process both in vitro and in silico by identifying the growth capacity of Pleurotus ostreatus in the presence of microplastics and identifying the most likely interactions of Pleurotus ostreatus enzymes and their affinity energies. The study follows an engineering development methodology applied on an experimental basis. The in vitro evaluation protocol applied in this study focused on the growth capacity of Pleurotus ostreatus on microplastics using enzymatic inducers. In terms of in silico evaluation, molecular simulations were conducted using the Autodock 1.5.7 program to calculate interaction energies. The molecular dynamics were evaluated by using the myPresto Portal and GROMACS program to calculate radius of gyration and Energies.The results of the study showed that Pleurotus ostreatus has the potential to degrade low-density polyethylene microplastics. The in vitro evaluation revealed the adherence of Pleurotus ostreatus to LDPE using scanning electron microscopy. The best results were obtained with enzymatic inducers as a MnSO4 generating the activation of laccase or manganese peroxidase enzymes in the degradation process. The in silico modelling demonstrated that Pleurotus ostreatus was able to interact with the microplastics present in LDPE, showing affinity energies in molecular docking and molecular dynamics shown a minimum energy and the representative radius of gyration between each enzyme and its substract. The study contributes to the development of bioremediation processes for the removal of microplastics from freshwater sources using the fungus Pleurotus ostreatus. The in silico study provides insights into the affinity energies of Pleurotus ostreatus microplastic degrading enzymes and their interaction with low-density polyethylene. The study demonstrated that Pleurotus ostreatus can interact with LDPE microplastics, making it a good agent for the development of bioremediation processes that aid in the recovery of freshwater sources. The results of the study suggested that bioremediation could be a promising approach to reduce microplastics in freshwater systems.

Keywords: bioremediation, in silico modelling, microplastics, Pleurotus ostreatus

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Authors: Razieh Karimi Aghcheh, Christian Kubicek, Joseph Strauss, Gerhard Braus

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Filamentous fungi are of considerable economic and social significance for human health, nutrition and in white biotechnology. These organisms are dominant producers of a range of primary metabolites such as citric acid, microbial lipids (biodiesel) and higher unsaturated fatty acids (HUFAs). In particular, they produce also important but structurally complex secondary metabolites with enormous therapeutic applications in pharmaceutical industry, for example: cephalosporin, penicillin, taxol, zeranol and ergot alkaloids. Several fungal secondary metabolites, which are significantly relevant to human health do not only include antibiotics, but also e.g. lovastatin, a well-known antihypercholesterolemic agent produced by Aspergillus. terreus, or aflatoxin, a carcinogen produced by A. flavus. In addition to their roles for human health and agriculture, some fungi are industrially and commercially important: Species of the ascomycete genus Hypocrea spp. (teleomorph of Trichoderma) have been demonstrated as efficient producer of highly active cellulolytic enzymes. This trait makes them effective in disrupting and depolymerization of lignocellulosic materials and thus applicable tools in number of biotechnological areas as diverse as clothes-washing detergent, animal feed, and pulp and fuel productions. Fungal LaeA/LAE1 (Loss of aflR Expression A) homologs their gene products act at the interphase between secondary metabolisms, cellulase production and development. Lack of the corresponding genes results in significant physiological changes including loss of secondary metabolite and lignocellulose degrading enzymes production. At the molecular level, the encoded proteins are presumably methyltransferases or demethylases which act directly or indirectly at heterochromatin and interact with velvet domain proteins. Velvet proteins bind to DNA and affect expression of secondary metabolites (SMs) genes and cellulases. The dynamic interplay between LaeA/LAE1, velvet proteins and additional interaction partners is the key for an understanding of the coordination of metabolic and morphological functions of fungi and is required for a biotechnological control of the formation of desired bioactive products. Aspergilli and Trichoderma represent different biotechnologically significant species with significant differences in the LaeA/LAE1-Velvet protein machinery and their target proteins. We, therefore, performed a comparative study of the interaction partners of this machinery and the dynamics of the various protein-protein interactions using our robust proteomic and mass spectrometry techniques. This enhances our knowledge about the fungal coordination of secondary metabolism, cellulase production and development and thereby will certainly improve recombinant fungal strain construction for the production of industrial secondary metabolite or lignocellulose hydrolytic enzymes.

Keywords: cellulases, LaeA/1, proteomics, secondary metabolites

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Authors: Carolina Gutierrez-Cortes, Hector Suarez, Gustavo Buitrago

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Bacteriocins are antimicrobial peptides produced by bacteria as a competitive strategy for substrate and habitat. Those peptides have a potential use as food biopreservatives due to their antimicrobial activity against foodborne pathogens, avoiding the use of additives that can be harmful to consumers. The industrial production of bacteriocins is currently expensive; one of the options to be competitive is the development of economic culture media, for example, with the use of agro-industrial wastes such as whey. This study evaluated the growth and production of bacteriocins from four strains: Pediococcus pentosaceus 63, Pediococcus pentosaceus 145, Pediococcus pentosaceus 146 and Pediococcus pentosaceus 147 isolated from ‘minas cheese’ (artisanal cheese made from raw milk in the state of Minas Gerais, Brazil) in order to select a strain with growth at high rates and higher antimicrobial activity against Listeria monocytogenes 104 after incubation on the culture medium designed with whey and other components. The media used were: MRS broth, modified MRS broth (using different sources of carbon and nitrogen and different amounts of micronutrients) and a culture medium designed by a factorial design using whey and other components. The final biomass concentrations of the four strains in MRS broth after 24 hours of incubation were very similar 9.25, 9.33, 9.25 and 9.22 (log CFU/mL) for P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146 and P. pentosaceus 147 respectively. In the same assays, antimicrobial activity of 3200 AU/mL for the first three and of 12800 AU/mL for P. pentosaceus 147 were obtained. Culture of P. pentosaceus 63 on modified MRS broth, showed the effect of some sources of carbon on the activity of bacteriocin, obtaining 12800 AU/mL with dextrose and 25600 AU/mL with maltose. Cultures of P. pentosaceus 145, 146 and 147 with these same sugars presented activity of 12800 AU/mL. It was observed that the modified MRS medium using whey increased the antimicrobial activity of the strains at 16000, 6400, 16000 and 19200 AU/mL for each strain respectively, keeping the biomass at values close to 9 log units. About nitrogen sources, it was observed that the combination of peptone (10 g /L), meat extract (10 g/L) and yeast extract (5 g/L) promoted the highest activity (12800 AU/mL), and in all cases MgSO4, MnSO4, K2HPO4 and ammonium citrate at low concentrations adversely affected bacteriocin production. Because P. pentosaceus 147 showed the highest antimicrobial activity in the presence of whey, it was used to evaluate the culture medium (peptone (10 g/L), meat extract (8 g/L), yeast extract (2 g/L), Tween® 80 (1 g/L), ammonium citrate (2 g/L), sodium acetate (5 g/L), MgSO4 (0.2 g/L), MnSO4 (0.04 g/L)). With the designed medium added with whey, 9.34 log units of biomass concentration and 19200 AU/mL were achieved for P. pentosaceus 147. The above suggest that the new medium promotes the antimicrobial activity of P. pentosaceus 147 allowing the use of an economic medium using whey.

Keywords: antimicrobial activity, bacteriocins, pediococcus, whey

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Authors: Adamu Muhammed Tukur

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Pulp and paper industry is one of the fastest growing industries due to an increased demand in paper products. For it to satisfy this ever increasing demand, it adopts new technological innovations some of which are proved to affect our environment negatively. Global consumption of paper has increased by 400% in the last four decades and this suggests that more research is required to assess the impact of industrial effluents to our environment and public health. Paper products are generally biodegradable, however, the processes involved in its production which involve the use of mainly bleaching agents and other non-biodegradable substances pose serious problem to the environment. There are more than 250 chemicals released in paper mill waste and some are xenobiotics. Different methods such as physical and chemical methods can be adopted for the remediation of the effluents but are proved to be costly and not safe to the environment. On the other hand, biological method is shown to be less costly and environmentally friendly. Microorganisms and their enzymes have shown a promising future for bioremediation of effluents related to paper mill. Many studies prove that one of the major pollutants in the paper mill effluent is phenol especially its chlorinated derivatives. Pentachlorophenol is extremely hazardous to living cells and therefore need to be removed from the environment. Microorganisms including bacteria and fungi have the potential to degrade phenolic compounds e.g. Bacillus stearothermiphilus, Pseudomonas putida, Coricus versicolor, Sphingomonas chlorophenolica, Fusarium sp, Bacillus subtilis and P. aeroginosa. Enzymes used for the degradation include phenol hydrooxylase, polyphenoloxylase, laccase, peroxidase among others. Lignin is another important pollutant and is resistant to microbial degradation but it has been proved that certain bacteria and fungi like can degrade it. Among the fungi white-rot fungi like Fomes lividus and Trametes vesicolor are the most important bioremediators. This review focused on use of microorganism to reduce or eradicate pollutants released from the paper industry. It can serve as a review for further research to be conducted especially in the field of Biotechnology.

Keywords: bioremediation, pulp and paper, pentachlorophenol, environment

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Authors: M. Gowri, E. K. Girija, V. Ganesh

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Background and Significance: Among the dental infections, inflammation and infection of the root canal are common among all age groups. Currently, the management of root canal infections involves cleaning the canal with powerful irrigants followed by intracanal medicament application. Though these treatments have been in vogue for a long time, root canal failures do occur. Treatment for root canal infections is limited due to the anatomical complexity in terms of small micrometer volumes and poor penetration of drugs. Thus, infections of the root canal seem to be a challenge that demands development of new agents that can eradicate C. albicans. Methodology: In the present study, we synthesized and screened silver-curcumin nanoparticle against Candida albicans. Detailed molecular studies were carried out with silver-curcumin nanoparticle on C. albicans pathogenicity. Morphological cell damage and antibiofilm activity of silver-curcumin nanoparticle on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model. Results: Screening data showed that silver-curcumin nanoparticle was active against C. albicans. Silver-curcumin nanoparticle exerted time kill effect and post antifungal effect. When used in combination with fluconazole or nystatin, silver-curcumin nanoparticle revealed a minimum inhibitory concentration (MIC) decrease for both drugs used. In-depth molecular studies with silver-curcumin nanoparticle on C. albicans showed that silver-curcumin nanoparticle inhibited yeast to hyphae (Y-H) conversion. Further, SEM images of C. albicans showed that silver-curcumin nanoparticle caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed eradication of C. albicans and reduction in colony forming unit (CFU) after 24 h treatment in the infected tooth samples in this model. Conclusion: The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.

Keywords: C. albicans, ex vivo dentine model, inhibition of biofilm formation, root canal infection, yeast to hyphae conversion inhibition

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Authors: Boudjelal Amel

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Arisarum vulgare, a member of the Araceae family, is an herbaceous perennial widely distributed in the Mediterranean region. A. vulgare is recognized for its medicinal properties and holds significant traditional importance in Algeria for the treatment of various human ailments, including pain, infections, inflammation, digestive disorders, skin problems, eczema, cancer, wounds, burns and gynecological diseases. Despite its extensive traditional use, scientific exploration of A. vulgare remains limited. The study aims to investigate for the first time the therapeutic potential of A. vulgare ethanolic extract obtained by ultrasound-assisted extraction. The chemical composition of the extract was determined by LC-MS/MS analysis. For in vitro phytopharmacological evaluation, several assays, including DPPH, ABTS, FRAP and reducing power, were employed to evaluate the antioxidant activity. The antibacterial activity was assessed againt Escherichia coli, Salmonella typhimurium, Staphylococus aureus, Enterococcus feacium by disk diffusion and microdilution methods. The possible inhibitory activity of ethanolic extract was analyzed against the cholinesterases enzymes (AChE and BChE). The DNA protection activity of A. vulgare ethanolic extract was estimated using the agarose gel electrophoresis method. The capacities of the extract to protect plasmid DNA (pBR322) from the oxidizing effects of H2O2 and UV treatment were evaluated by their DNA-breaking forms. The in vivo wound healing potential of a traditional ointment containing 5% of A. vulgare ethanolic extract was also investigated. The LC-MS/MS profiling of the extract revealed the presence of various bioactive compounds, including naringenin, chlorogenic, vanillic, cafeic, coumaric acids, trans-cinnamic and trans ferrulic acids. The plant extract presented considerable antioxidant potential, being the most active for Reducing power (0,07326±0.001 mg/ml) and DPPH (0.14±0.004 mg/ml). The extract showed the highest inhibition zone diameter against Enterococcus feacium (36±0.1 mm). The ethanolic extract of A. vulgare suppressed the growth of Staphylococus aureus, Escherichia coli and Salmonella typhimurium according to the MIC values. The extract of the plant significantly inhibited both AChE and BChE enzymes. DNA protection activity of the A. vulgare extract was determined as 90.41% for form I and 51.92% for form II. The in vivo experiments showed that 5% ethanolic extract ointment accelerated the wound healing process. The topical application of the traditional formulation enhanced wound closure (95,36±0,6 %) and improved histological parameters in the treated group compared to the control groups. The promising biological properties of Arisarum vulgare revealed that the plant could be appraised as a potential origin of bioactive molecules having multifunctional medicinal uses.

Keywords: arisarum vulgare, LC-MS/MS, antioxidant activity, antimicrobial activity, cholinesterases enzymes inhibition, dna-damage activity, in vivo wound healing

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Authors: Md. Alauddin, Md. Ruhul Amin, Md. Omar Faruque, Muhammad Ali Siddiquee, Zakir Hossain Howlader, Mohammad Asaduzzaman

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Diabetes and hypertension are the major lifestyle related diseases. The α-amylase and angiotensin converting enzymes (ACE) are the key enzymes that regulate diabetes and hypertension. The aim was to develop a drug for the treatment of diabetes and hypertension. The Rice Bran (RB) sample (Oryza sativa; BRRI-Dhan-84) was collected from the Bangladesh Rice Research Institute (BRRI), and rice bran proteins were isolated and hydrolyzed by hydrolyzing enzyme alcalase and trypsin. In vivo experiment suggested that rice bran bioactives has an effect on regulating the expression of several key gluconeogenesis and lipogenesis-regulating genes, such as glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and fatty acid synthase. The above genes have a connection of regulating the glucose level, lipids profile as well as act as an anti-inflammatory agent. A molecular docking, bioinformatics and in vitro experiments were performed. We found rice bran protein hydrolysates significantly (<0.05) influence the peptide concentration in the case of trypsin, alcalase, and (trypsin + alcalase) digestion. The in vitro analysis found that protein hydrolysate significantly (<0.05) reduced diabetic and hypertension as well as oxidative stress. A molecular docking study showed that the YY and IP peptide have a significantly strong binding affinity to the active site of the ACE enzyme and α-amylase with -7.8Kcal/mol and -6.2Kcal/mol, respectively. The Molecular dynamics (MD) simulation and Swiss ADME data analysis showed that less toxicity risk, good physicochemical properties, pharmacokinetics, and drug-likeness with drug scores 0.45 and 0.55 of YY and IP peptides, respectively. Thus, rice bran bioactive could be a good candidate for the treatment of diabetes and hypertension.

Keywords: anti-hypertensive and anti-hyperglycemic, anti-oxidative, bioinformatics, in vitro study, rice bran proteins and peptides

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Authors: F. Saleh, F. Lyall, A. Abdulsid, L. Marks

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Background: Labour in human is a complex biological process that involves interactions of neurological, hormonal and inflammatory pathways, with the placenta being a key regulator of these pathways. It is known that uterine contractions and labour pain cause physiological changes in gene expression in maternal and fetal blood, and in placenta during labour. Oxidative and inflammatory stress pathways are implicated in labour and they may cause alteration of placental gene expression. Additionally, in placental tissues, labour increases the expression of genes involved in placental oxidative stress, inflammatory cytokines, angiogenic regulators and apoptosis. Recently, Hydrogen Sulphide (H2S) has been considered as an endogenous gaseous mediator which promotes vasodilation and exhibits cytoprotective anti-inflammatory properties. The endogenous H2S is synthesised predominantly by two enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). As the H2S pathway has anti-oxidative and anti-inflammatory characteristics thus, we hypothesised that the expression of CBS and CSE in placental tissues would alter during labour. Methods: CBS and CSE expressions were examined in placentas using western blotting and RT-PCR in inner, middle and outer placental zones in placentas obtained from healthy non labouring women who delivered by caesarian section. These were compared with the equivalent zone of placentas obtained from women who had uncomplicated labour and delivered vaginally. Results: No differences in CBS and CSE mRNA or protein levels were found between the different sites within placentas in either the labour or non-labour group. There were no significant differences in either CBS or CSE expression between the two groups at the inner site and middle site. However, at the outer site there was a highly significant decrease in CBS protein expression in the labour group when compared to the non-labour group (p = 0.002). Conclusion: To the best of author’s knowledge, this is the first report to suggest that, CBS is expressed in a spatial manner within the human placenta. Further work is needed to clarify the precise function and mechanism of this spatial regulation although it is likely that inflammatory pathways regulation is a complex process in which this plays a role.

Keywords: anti-inflammatory, hydrogen sulphide, labour, oxidative stress

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Authors: P. S. Percin, O. Inanli, S. Karakaya

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Bitter melon (Momordica charantia) is a medicinal vegetable, which is used traditionally to remedy diabetes. Bitter melon contains several classes of primary and secondary metabolites. In traditional Turkish medicine bitter melon is used for wound healing and treatment of peptic ulcers. Nowadays, bitter melon is used for the treatment of diabetes and ulcerative colitis in many countries. The main constituents of bitter melon, which are responsible for the anti-diabetic effects, are triterpene, protein, steroid, alkaloid and phenolic compounds. In this study total phenolics, total carotenoids and β-carotene contents of mature and immature bitter melons were determined. In addition, in vitro α-amylase and α-glucosidase activities of mature and immature bitter melons were studied. Total phenolic contents of immature and mature bitter melon were 74 and 123 mg CE/g bitter melon respectively. Although total phenolics of mature bitter melon was higher than that of immature bitter melon, this difference was not found statistically significant (p > 0.05). Carotenoids, a diverse group of more than 600 naturally occurring red, orange and yellow pigments, play important roles in many physiological processes both in plants and humans. The total carotenoid content of mature bitter melon was 4.36 fold higher than the total carotenoid content of immature bitter melon. The compounds that have hypoglycaemic effect of bitter melon are steroidal saponins known as charantin, insulin-like peptides and alkaloids. α-Amylase is one of the main enzymes in human that is responsible for the breakdown of starch to more simple sugars. Therefore, the inhibitors of this enzyme can delay the carbohydrate digestion and reduce the rate of glucose absorption. The immature bitter melon extract showed α-amylase and α-glucosidase inhibitory activities in vitro. α-Amylase inhibitory activity was higher than that of α-glucosidase inhibitory activity when IC50 values were compared. In conclusion, the present results provide evidence that aqueous extract of bitter melon may have an inhibitory effect on carbohydrate breakdown enzymes.

Keywords: bitter melon, in vitro antidiabetic activity, total carotenoids, total phenols

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Authors: Serap Gedikli, Hakan Çakmak, M. Buğra Güldiken, Duygu Yalnızoğlu

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Air, which is necessary for living things to survive; while it carries some useful substances in it, it can also carry foreign particles of different sizes that may be harmful to the health. All airborne organic substances of biological origin, including bacteria, fungi, fungal spores, viruses, pollen, and their components, are called "bioaerosols". Nowadays, everyone spends most of their time in closed areas such as home, workplace, school, etc. Although it is known that outdoor air pollution affects health, it is not known that indoor air pollution has harmful effects in terms of health. In this study, indoor air microbial load and SARS-CoV-2 virus cleaning efficiency of Froumann brand air cleaners were studied. This work in 300 m³, 600 m³, and 1000 m³ completely closed areas without any air circulation with Froumann N80, N90, and N100 air-cleaning devices. Analyzes were performed for both areas at 60 minutes before and after the device was operated using a particle measuring device (Particles Plus 7302) and an air sampler (Mas-100 ECO). The measurements were taken by placing the test equipment 1.5-2 m away from the air cleaner. At the same time, the efficiency of the HEPA filter was evaluated by taking samples from the air outlet point of the HEPA filter using the air sampling device (Mas-100 ECO) after the device was started. Nutrient agar and malt agar are used as total mesophilic bacteria and total fungi. The number of colony-forming units per m³ (cfu/m³) was calculated by counting colonies in Petri dishes after incubation for 48 hours at 37°C for bacteria and 72 hours at 30°C for fungi. The change in the number of colonies and the decrease in the microbial load was calculated as a percentage value. SARS-CoV-2 activity analysis studies were carried out by İnönü University Microbiology Department in accordance with the World Health Organization regulations. Finally, the HEPA filter in the devices used was taken and kept under a certain temperature and humidity, and the change in the microbial load on it was monitored over a 6-month period. At the end of the studies, a 91%-94% reduction was determined in the total mesophilic bacteria count of Frouman brand N80, N90, and N100 model air cleaners. A decrease of 94%-96% was detected in the total number of yeast/molds. HEPA filter efficiency was evaluated, and at the end of the analysis, 98% of the bacterial load and approximately 100% of yeast/mold load at the HEPA filter air outlet point were decreased. According to the SARS- CoV-2 analysis results, when the device is operating at the medium airflow level 3, it can filter virus-carrying aerosols by 99%. As a result, it was determined that the Froumann model air cleaner was effective in controlling and reducing the microbial load in the indoor air.

Keywords: HEPA filter, indoor air quality, microbial load, SARS-CoV-2

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Authors: Achiraya Jiraprasertwong, Erdogan Gulari, Sumaeth Chavadej

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Among agricultural residues, sugarcane bagasse is one of the most convincing raw materials for the production of bioethanol due to its availability, and low cost through enzymatic hydrolysis and yeast fermentation. A pretreatment step is needed to enhance the enzymatic step. In this study, sugarcane bagasse (SCB), one of the most abundant agricultural residues in Thailand, was pretreated biologically with various microorganisms of white-rot fungus—Phanerochaete sordid (SK 7), Cellulomonas sp. (TISTR 784), and strain A 002 (Bacillus subtilis isolated from Thai higher termites). All samples with various microbial pretreatments were further hydrolyzed enzymatically by a commercial enzyme obtained from Aspergillus niger. The results showed that the pretreatment with the white-rot fungus gave the highest glucose concentration around two-fold higher when compared with the others.

Keywords: sugarcane bagasse, microorganisms, pretreatment, enzymatic hydrolysis

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Authors: Tanju Teker, Yener Tekeli̇, Esra Karpuz

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Salvia species are known as medicinal plant and often used in public. The antimicrobial effects and essential oil composition of Salvia verticillata L. subsp. amasiaca were determined. The antimicrobial activity is determined by using disk diffusion method against two Gram-positive bacteria, two Gram-negative bacteria and one kind of yeast and essential oil composition was determined by GC - MS. As a result of antimicrobial analysis while sample has shown very strong antimicrobial activity against Staphylococcus aureus, moderately effective against Pseudomonas aeruginosa and low effective against Enterococcus faecalis, it has not shown antimicrobial activity against Escherichia coli and C. albicans. Trans-caryophyllene (% 35.07), germacrene-d (% 10.98) and caryopyllene oxide (% 5.81) are the main components of essential oil composition.

Keywords: salvia, medicinal plant, antimicrobial activity, essential oil

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Authors: Bassma A. Abdel Haleem, Ehab K. Emam, George E. Yacoub, Ashraf M. Salem

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Background: Obesity is an increasingly common problem in childhood. Childhood obesity is considered the main risk factor for the development of metabolic syndrome (MetS) (diabetes type 2, dyslipidemia, and hypertension). Recent studies estimated that among children with obesity 30-60% will develop MetS. Visceral fat thickness is a valuable predictor of the development of MetS. Computed tomography and dual-energy X-ray absorptiometry are the main techniques to assess visceral fat. However, they carry the risk of radiation exposure and are expensive procedures. Consequently, they are seldom used in the assessment of visceral fat in children. Some studies explored the potential of ultrasound as a substitute to assess visceral fat in the elderly and found promising results. Given the vulnerability of children to radiation exposure, we sought to evaluate ultrasound as a safer and more cost-efficient alternative for measuring visceral fat in obese children. Additionally, we assessed the correlation between visceral fat and obesity indicators such as insulin resistance. Methods: A cross-sectional study was conducted on 46 children with obesity (aged 6–16 years). Their visceral fat was evaluated by ultrasound. Subcutaneous fat thickness (SFT), i.e., the measurement from the skin-fat interface to the linea alba, and visceral fat thickness (VFT), i.e., the thickness from the linea alba to the aorta, were measured and correlated with anthropometric measures, fasting lipid profile, homeostatic model assessment for insulin resistance (HOMA-IR) and liver enzymes (ALT). Results: VFT assessed via ultrasound was found to strongly correlate with the BMI, HOMA-IR with AUC for VFT as a predictor of insulin resistance of 0.858 and cut off point of >2.98. VFT also correlates positively with serum triglycerides and serum ALT. VFT correlates negatively with HDL. Conclusions: Ultrasound, a safe and cost-efficient technique, could be a useful tool for measuring the abdominal fat thickness in children with obesity. Ultrasound-measured VFT could be an appropriate prognostic factor for insulin resistance, hypertriglyceridemia, and elevated liver enzymes in obese children.

Keywords: metabolic syndrome, pediatric obesity, sonography, visceral fat

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Authors: N. A. P. Camaforte, P. M. P. Vareda, L. L. Saldanha, A. L. Dokkedal, J. M. Rezende-Neto, M. R. Senger, F. P. Silva-Jr, J. R. Bosqueiro

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Diabetes mellitus is a disease characterized by deficiency of insulin secretion and/or action which results in hyperglycemia. Nowadays, acarbose is a medicine used by diabetic people to inhibit alpha-glucosidases leading to the decreasing of post-feeding glycaemia, but with low effectiveness and many side effects. Medicinal plants have been used for the treatment of many diseases including diabetes and their action occurs through the modulation of insulin-depending processes, pancreas regeneration or inhibiting glucose absorption by the intestine. Previous studies in our laboratory showed that the treatment using two crude extracts of plants from Brazilian cerrado was able to decrease fasting blood glucose and improve glucose tolerance in streptozotocin-diabetic mice. Because of this and the importance of the search for new alternatives to decrease the hyperglycemia, we decided to evaluate the inhibitory action of two plants from Brazilian cerrado - B.H. and Myrcia bella. The enzymatic assay was performed in 50 µL of final volume using pancreatic α-amylase and maltase together with theirs commercial substrates. The inhibition potency (IC50) was determined by the incubation of eight different concentrations of both extracts and the enzymes for 5 minutes at 37ºC. After, the substrate was added to start the reaction. Glucosidases assay was evaluated measuring the quantity of p-nitrophenol in 405 nmin 384 wells automatic reader. The in vitro assay with the extracts of B.H. and M. bella showed an IC50 of 28,04µg/mL and 16,93 µg/mL for α-amilase, and 43,01µg/mL and 17 µg/mL for maltase, respectively. M. bella extract showed a higher inhibitory activity for those enzymes than B.H. extract. The crude extracts tested showed a higher inhibition rate to α-amylase, but were less effective against maltase in comparison to acarbose (IC50 36µg/mL and 9 µg/mL, respectively). In conclusion, the crude extract of B.H. and M. bella showed a potent inhibitory effect against α-amylase and showed promising results to the possible development of new medicines to treat diabetes with less or even without side effects.

Keywords: alfa-glucosidases, diabetes mellitus, glycaemia, medicinal plants

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Authors: Govindan Sudha, Mathumitha Subramaniam, Alamelu Govindasamy, Sasikala Gunasekaran

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The aim of this study was to investigate the protective effect of Hypsizygus ulmarius polysaccharides (HUP) on reducing oxidative stress, cognitive impairment and neurotoxicity in D-galactose induced aging mice. Mice were subcutaneously injected with D-galactose (150 mg/kg per day) for 6 weeks and were administered HUP simultaneously. Aged mice receiving vitamin E (100 mg/kg) served as positive control. Chronic administration of D-galactose significantly impaired cognitive performance oxidative defence and mitochondrial enzymes activities as compared to control group. The results showed that HUP (200 and 400 mg/kg) treatment significantly improved the learning and memory ability in Morris water maze test. Biochemical examination revealed that HUP significantly increased the decreased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), mitochondrial enzymes-NADH dehydrogenase, malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH), Na+K+, Ca2+, Mg2+ATPase activities, elevated the lowered total anti-oxidation capability (TAOC), glutathione (GSH), vitamin C and decreased the raised acetylcholinesterase (AChE) activities, malondialdehyde (MDA), hydroperoxide (HPO), protein carbonyls (PCO), advanced oxidation protein products (AOPP) levels in brain of aging mice induced by D-gal in a dose-dependent manner. In conclusion, present study highlights the potential role of HUP against D-galactose induced cognitive impairment, biochemical and mitochondrial dysfunction in mice. In vitro studies on the effect of HUP on scavenging DPPH, ABTS, DMPD, OH radicals, reducing power, B-carotene bleaching and lipid peroxidation inhibition confirmed the free radical scavenging and antioxidant activity of HUP. The results suggest that HUP possesses anti-aging efficacy and may have potential in treatment of neurodegenerative diseases.

Keywords: aging, antioxidants, mushroom, neurotoxicity

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Authors: Ihcen Khacheba, Amar Djeridane, Abdelkarim Kamli, Mohamed Yousfi

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Plant materials constitute an important source of natural bioactive molecules. Thus plants have been used from antiquity as sources of medicament against various diseases. These properties are usually attributed to secondary metabolites that are the subject of a lot of research in this field. This is particularly the case of phenolic compounds plants that are widely renowned in therapeutics as anti-inflammatories, enzyme inhibitors, and antioxidants, particularly flavonoïds. With the aim of acquiring a better knowledge of the secondary metabolism of the vegetable kingdom in the region of Laghouat and of the discovering of new natural therapeutics, 10 extracts from 5 Saharan plant species were submitted to chemical screening.The analysis of the preceding biological targets led to the evaluation of the biological activity of the extracts of the species Genista Corsica. The first step, consists in extracting and quantifying phenolic compounds. The second step has been devoted to stugying the effects of phenolic compounds on the kinetics catalyzed by two enzymes belonging to the class of hydrolase (the α-amylase and α-glucosidase) responsible for the digestion of sugars and finally we evaluate the antiantioxidant potential. The analysis results of phenolic extracts show clearly a low content of phenolic compounds in investigated plants. Average total phenolics ranged from 0.0017 to 11.35 mg equivalent gallic acid/g of the crude extract. Whereas the total flavonoids content lie between 0.0015 and 10.,96 mg/g equivalent of rutin. The results of the kinetic study of enzymatic reactions show that the extracts have inhibitory effects on both enzymes, with IC50 values ranging from 95.03 µg/ml to 1033.53 µg/ml for the α-amylase and 279.99 µg/ml to 1215.43 µg/ml for α-glucosidase whose greatest inhibition was found for the acetone extract of June (IC50 = 95.03 µg/ml). The results the antioxidant activity determined by ABTS, DPPH, and phosphomolybdenum tests clearly showed a good antioxidant capacity comparatively to antioxidants taken as reference the biological potential of these plants and could find their use in medicine to replace synthetic products.

Keywords: phenolic extracts, inhibition effect, α-amylase, α-glucosidase, antioxidant activity

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Authors: L. García, N. Cobas, M. López

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Acrylamide, a probable carcinogen, is formed in high-temperature processed food (>120ºC) when the free amino acid asparagine reacts with reducing sugars, mainly glucose and fructose. Cane juices' repeated heating would potentially form acrylamide during brown sugar production. This study aims to determine if using panela in yogurt cake preparation increases acrylamide formation. A secondary aim is to analyze the acrylamide concentration in four cake confections with different caloric sweetener ingredients: beet sugar (BS), cane sugar (CS), panela (P), and a panela and chocolate mix (PC). The doughs were obtained by combining ingredients in a planetary mixer. A model system made up of flour (25%), caloric sweeteners (25 %), eggs (23%), yogurt (15.7%), sunflower oil (9.4%), and brewer's yeast (2 %) was applied to BS, CS and P cakes. The ingredients of PC cakes varied: flour (21.5 %), panela chocolate (21.5 %), eggs (25.9 %), yogurt (18 %), sunflower oil (10.8 %), and brewer’s yeast (2.3 %). The preparations were baked for 45' at 180 ºC. Moisture was estimated by AOAC. Protein was determined by the Kjeldahl method. Ash percentage was calculated by weight loss after pyrolysis (≈ 600 °C). Fat content was measured using liquid-solid extraction in hydrolyzed raw ingredients and final confections. Carbohydrates were determined by difference and total sugars by the Luff-Schoorl method, based on the iodometric determination of copper ions. Finally, acrylamide content was determined by LC-MS by the isocratic system (phase A: 97.5 % water with 0.1% formic acid; phase B: 2.5 % methanol), using a standard internal procedure. Statistical analysis was performed using SPSS v.23. One-way variance analysis determined differences between acrylamide content and compositional analysis, with caloric sweeteners as fixed effect. Significance levels were determined by applying Duncan's t-test (p<0.05). P cakes showed a lower energy value than the other baked products; sugar content was similar to BS and CS, with 6.1 % mean crude protein. Acrylamide content in caloric sweeteners was similar to previously reported values. However, P and PC showed significantly higher concentrations, probably explained by the applied procedure. Acrylamide formation depends on both reducing sugars and asparagine concentration and availability. Beet sugar samples did not present acrylamide concentrations within the detection and quantification limit. However, the highest acrylamide content was measured in the BS. This may be due to the higher concentration of reducing sugars and asparagine in other raw ingredients. The cakes made with panela, cane sugar, or panela with chocolate did not differ in acrylamide content. The lack of asparagine measures constitutes a limitation. Cakes made with panela showed lower acrylamide formation than products elaborated with beet or cane sugar.

Keywords: beet sugar, cane sugar, panela, yogurt cake

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Authors: Parikshit Gogo, N. N. Dutta

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The oxidative enzymes specially laccase (benzenediol: oxygen oxidoreductase, E.C.1.10.3.2) from bacteria, fungi and plants have been playing an important role in green technologies due to their specific advantageous properties. Laccase from different sources and in different forms was used as a biocatalyst in many oxidation and conjugation reactions starting from phenol to hydrocarbons. Tea polyphenols and its derivatives attract the scientific community because of their potential use as antioxidants in food, pharmaceutical and cosmetic industries. Conjugate of polyphenols emerged as a novel materials which shows better stability and antioxidant properties in applied fields. The conjugation reaction of catechin with poly (allylamine) has been studied using free, immobilized and cross-linked enzyme crystals (CLEC) of laccase from Trametes versicolor with particular emphasis on the effect of pertinent variables and kinetic aspects of the reaction. The stability and antioxidant property of the conjugated product was improved as compared to the unconjugated tea polyphenols. The reaction was studied in 11 different solvents in order to deduce the solvent effect through an attempt to correlate the initial reaction rate with solvent properties such as hydrophobicity (logP), water solubility (logSw), electron pair acceptance (ETN) and donation abilities (DNN), polarisibility and dielectric constant which exhibit reasonable correlations. The study revealed, in general that polar solvents favour the initial reaction rate. The kinetics of the conjugation reaction conformed to the so-called Ping-Pong-Bi-Bi mechanism with catechin inhibition. The stability as well as activity of the CLEC was better than the free enzymes and immobilized laccase for practical application. In case of immobilized laccase system marginal diffusional limitation could be inferred from the experimental data. The kinetic parameters estimated by non-linear regression analysis were found to be KmPAA(mM) = 0.75, 1.8967 and Kmcat (mM) = 11.769, 15.1816 for free and immobilized laccase respectively. An attempt has been made to assess the activity of the laccase for the conjugation reaction in relation to other reactions such as dimerisation of ferulic acids and develop a protocol to enhance polyphenol antioxidant activity.

Keywords: laccase, catechin, conjugation reaction, antioxidant properties

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Authors: Anil Kumar, Diwakar Aggarwal, M. Sudhakara Reddy

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The wood of Eucalyptus, Populus and bamboos are some important species used as raw material for the manufacture of pulp. However, higher levels of lignin pose a problem during Kraft pulping and yield of pulp is also lower. In order to increase the yield of pulp per unit wood and reduce the use of chemicals during kraft pulping it is important to reduce the lignin content and/or increase cellulose content in wood. Cellulose biosynthesis in wood takes place by the coordinated action of many enzymes. The two important enzymes are KORRIGAN and SUCROSE SYNTHASE. KORRIGAN (Endo-1,4--glucanase) is implicated in the process of editing growing cellulose chains and improvement of the crystallinity of produced cellulose, whereas SUCROSE SYNTHASE is involved in providing substrate (UDP-glucose) for growing cellulose chains. The present study was aimed at the cloning, characterization and overexpression of these genes in Eucalyptus and Populus. An efficient shoot organogenesis protocol from leaf explants taken from micro shoots of the species has been developed. Agrobacterium mediated genetic transformation using Agrobacterium tumefaciens strain EHA105 and LBA4404 harboring binary vector pBI121 was achieved. Both the genes were cloned from cDNA library of Populus deltoides. These were subsequently characterized using various bioinformatics tools. The cloned genes were then inserted into pBI121 under the CaMV35S promotors replacing GUS gene. The constructs were then mobilized into above strains of Agrobacterium and used for the transformation work. Subsequently, genetic transformation of these clones with target genes following already developed protocol is in progress. Four transgenic lines of Eucalyptus tereticornis overexpressing Korrigan gene under the strong constitutive promoters CaMV35S have been developed, which are being further evaluated. Work on development of more transgenic lines overexpressing these genes in Populus and Eucalyptus is also in progress. This presentation will focus on important developments in this direction.

Keywords: Eucalyptus tereticornis, genetic transformation, Kraft pulping Populus deltoides

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Authors: Thi Huong Vu, Vijay Jayasena, Zhongxiang Fang, Gary Dykes

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D-pinitol (3-O-methyl ether of D-chiro-inosito) has been known to have health benefits for diabetic patients. Carob kibble has received attention due to the presence of high value D-pinitol and polyphenol antioxidants. D-pinitol was concentrated from carob kibble using submerged fermentation with Saccharomyces cerevisiae. Total carbohydrates and D-pinitol were determined by the phenol-sulphuric acid method and HPLC, respectively. The content of D-pinitol increased from approximately 43 to 70 mg/g dry weight after fermentation. The yeast consumed over 70% of total carbohydrates in carob kibble without any negative effect on D-pinitol content. A range of substrate medium pH’s from 5.0 – 7.0 had no significant effect on the removal of carbohydrates and D-pinitol. This method may provide a practical solution for production of D-pinitol from carob in a cost effective manner.

Keywords: carob kibble, d-pinitol, saccharomyces cerevisiae, submerged fermentation, total carbohydrates

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Authors: Weeraya Samnuknit, Apichat Boontawan

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A vacuum fractionation technique was introduced to remove ethanol from fermentation broth. The effect of initial glucose and ethanol concentrations were investigated for specific productivity. The inhibitory ethanol concentration was observed at 100 g/L. In order to increase the fermentation performance, the ethanol product was removed as soon as it is produced. The broth was boiled at 35°C by reducing the pressure to 65 mBar. The ethanol/water vapor was fractionated for up to 90 wt% before leaving the column. Ethanol concentration in the broth was kept lower than 25 g/L, thus minimized the product inhibition effect to the yeast cells. For batch extractive fermentation, a high substrate utilization rate was obtained at 26.6 g/L.h and most of glucose was consumed within 21 h. For repeated-batch extractive fermentation, addition of glucose was carried out up to 9 times and ethanol was produced more than 8-fold higher than batch fermentation.

Keywords: ethanol, extractive fermentation, product inhibition, vacuum fractionation

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Authors: M. Caramihai, Irina Severin

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This paper deals with the design of an intelligent control structure for a bioprocess of Hansenula polymorpha yeast cultivation. The objective of the process control is to produce biomass in a desired physiological state. The work demonstrates that the designed Hybrid Control Techniques (HCT) are able to recognize specific evolution bioprocess trajectories using neural networks trained specifically for this purpose, in order to estimate the model parameters and to adjust the overall bioprocess evolution through an expert system and a fuzzy structure. The design of the control algorithm as well as its tuning through realistic simulations is presented. Taking into consideration the synergism of different paradigms like fuzzy logic, neural network, and symbolic artificial intelligence (AI), in this paper we present a real and fulfilled intelligent control architecture with application in bioprocess control.

Keywords: bioprocess, intelligent control, neural nets, fuzzy structure, hybrid techniques

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Authors: Cheng-Kuang Hsu, Chih-Hsiang Chang, Chi-Chih Wang

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Black soybeans contain high amount of antioxidants including polyphenols, anthocyanins and flavones. The protective function of black soybean against CCl4 (a strong oxidant) induced acute and chronic liver damage was investigated in vivo using SD rats or ICR mouse. The evaluation of CCl4 induced oxidative stress in the liver tissues included the measurements of the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the concentration of thiobarbituric acid reactive substances (TBARS), the activities of antioxidant enzymes (superoxide dismutase SOD, catalase, and glutathione peroxidase GPx), as well as the level of histological lesion in the liver tissues. For chronic experiment, a decoction at the concentration of 100 or 1000 mg/kg of body weight, produced by baking black soybean at 130°C for 5 min and followed by immerging in 100°C hot water for 20 min, showed the inhibitory effect against CCl4 induced liver damage in SD rats. Hot-water extract (80 °C for 30 min) from un-preheated black soybean at the concentration of 200 mg/kg of body weight could not reduce ALT and AST levels in CCl4 treated SD rats, but the hot-water extract from preheated black soybean did enhance antioxidant enzymes activities, decline ALT and AST levels. Specially, the hot-water extract from the seed cost of black soybean had the highest liver protective function since it can reduce vacuolization and necrosis in the liver tissues. For acute experiment, the hot-water extracts from black soybean and the seed coat, as well as pure cyanidin-3-glucoside (C3G) could reduce ALT and AST levels of CCl4 induced ICR mouse. The decoction and hot-water extract from the seed coat of black soybean had higher total polyphenols, anthocyanins and flavones contents than those extracts from whole black soybean. Such results agreed with high liver protective function in the decoction and hot-water from the seed coat of black soybean. Black soybean showed protective function only after preheating process (baking at 130°C for 5 to 10 min) because preheating treatment damaged the cell wall and made the extraction of the antioxidants more effectively.

Keywords: black soybean, liver protective function, antioxidant, antioxidative stress

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Authors: Ruolan Zhang, Ryo Imai, Naoko Senda, Tomoyuki Sakai

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Enzymes have attracted increasing attentions in industrial manufacturing for their applicability in catalyzing complex chemical reactions under mild conditions. Directed evolution has become a powerful approach to optimize enzymes and exploit their full potentials under the circumstance of insufficient structure-function knowledge. With the incorporation of cell-free synthetic biotechnology, rapid enzyme synthesis can be realized because no cloning procedure such as transfection is needed. Its open environment also enables direct enzyme measurement. These properties of cell-free biotechnology lead to excellent throughput of enzymes generation. However, the capabilities of current screening methods have limitations. Fluorescence-based assay needs applicable fluorescent label, and the reliability of acquired enzymatic activity is influenced by fluorescent label’s binding affinity and photostability. To acquire the natural activity of an enzyme, another method is to combine pre-screening step and high-performance liquid chromatography (HPLC) measurement. But its throughput is limited by necessary time investment. Hundreds of variants are selected from libraries, and their enzymatic activities are then identified one by one by HPLC. The turn-around-time is 30 minutes for one sample by HPLC, which limits the acquirable enzyme improvement within reasonable time. To achieve the real high-throughput enzyme screening, i.e., obtain reliable enzyme improvement within reasonable time, a widely applicable high-throughput measurement of enzymatic reactions is highly demanded. Here, a high-throughput screening method using broadband coherent anti-Stokes Raman spectroscopy (CARS) was proposed. CARS is one of coherent Raman spectroscopy, which can identify label-free chemical components specifically from their inherent molecular vibration. These characteristic vibrational signals are generated from different vibrational modes of chemical bonds. With the broadband CARS, chemicals in one sample can be identified from their signals in one broadband CARS spectrum. Moreover, it can magnify the signal levels to several orders of magnitude greater than spontaneous Raman systems, and therefore has the potential to evaluate chemical's concentration rapidly. As a demonstration of screening with CARS, alcohol dehydrogenase, which converts ethanol and nicotinamide adenine dinucleotide oxidized form (NAD+) to acetaldehyde and nicotinamide adenine dinucleotide reduced form (NADH), was used. The signal of NADH at 1660 cm⁻¹, which is generated from nicotinamide in NADH, was utilized to measure the concentration of it. The evaluation time for CARS signal of NADH was determined to be as short as 0.33 seconds while having a system sensitivity of 2.5 mM. The time course of alcohol dehydrogenase reaction was successfully measured from increasing signal intensity of NADH. This measurement result of CARS was consistent with the result of a conventional method, UV-Vis. CARS is expected to have application in high-throughput enzyme screening and realize more reliable enzyme improvement within reasonable time.

Keywords: Coherent Anti-Stokes Raman Spectroscopy, CARS, directed evolution, enzyme screening, Raman spectroscopy

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Authors: Bassam Al Johny

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Halophilic bacteria are organisms which thrive in salt-rich environments, such as salt lakes, solar salterns and salt mines which contain large populations of these organisms. In biotechnology, such salt-tolerant bacteria are widely used for the production of valuable enzymes, and more than a thousand years ago humans began using salt to cure and thereby preserve perishable foods and other materials, such as hides; halophiles can be detrimental to the preservation of salt brine cured hides. The aim of this review is to provide an overview of the taxonomy of these organisms including novel isolates from rock salt, and also to discuss their current and future biotechnological and environmental uses.

Keywords: hypersaline environments, halophilic bacteria, environmental application, industrial application

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Authors: Aruma Baduge Kithma De Silva

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Haskap (Lonicera caerulea L.), also known as blue honeysuckle, is a newly commercialized berry crop in Canada. Haskap berries are rich in polyphenols, including, anthocyanins, which are known for potential health-promoting properties. Cyanidin-3-O-glucoside (C3G) is the most abundant anthocyanin of haskap berries. The compound C3G has the ability to reduce the risk of type 2 diabetes (T2D), which has become an increasingly common health issue around the world. The T2D is characterized as a metabolic disorder of hyperglycemia and insulin resistance. It has been demonstrated that C3G has anti-diabetic effects through several ways, including inhibition of dipeptidyl peptidase-4 (DPP-4), reduction of gluconeogenesis, improvement in insulin sensitivity, and inhibition of activities of carbohydrate hydrolyzing enzymes, including α-amylase and α-glucosidase. The goal of this study was to investigate the influence of variety and harvests maturity of haskap on C3G, other fruit quality characteristics and anti-diabetic activities of haskap berries using in vitro studies. The polyphenols present in four commercially grown haskap cultivars, Aurora, Rebecca, Larissa, and Evie harvested at five harvesting dates (H1-H5) apart from 2-3 days, were extracted separately. High-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) analyzes of polyphenols revealed that haskap berries contain predominantly anthocyanins, flavonols, flavan-3-ols, and phenolic acids. The compound C3G was the most prominent anthocyanin, which is available in approximately 79% of total anthocyanin in four cultivars. The Larissa at H5 contained the highest C3G content. The antioxidant capacity of Evie at H5 was greater than other cultivars. Furthermore, Larissa H5 showed the greatest inhibition of carbohydrate hydrolyzing enzymes including alpha-glucosidase and alpha-amylase. In conclusion, the haskap variety and harvesting date influenced the polyphenol composition and biological properties. The variety Larissa, at H5 harvesting date, contained the highest polyphenol content and the ability of inhibition of the carbohydrate hydrolyzing enzyme as well as DPP4 enzyme in order to reduce type 2 diabetes.

Keywords: anthocyanin, Haskap, type 2 diabetes, polyphenol

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