Search results for: enzymatic biosensor

Authors: Shohei Matsuo, Saki Mikai, Hiroshi Morita

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Objectives: Shochu is a popular Japanese distilled spirits. In the production of shochu, the filamentous fungus Aspergillus kawachii has traditionally been used. A. kawachii produces two types of starch hydrolytic enzymes, α-amylase (enzymatic liquefaction) and glucoamylase (enzymatic saccharification). Liquid culture system is a relatively easy microorganism to ferment with relatively low cost of production compared for solid culture. In liquid culture system, acid-unstable α-amylase (α-A) was produced abundantly, but, acid-stable α-amylase (Aα-A) was not produced. Since there is high enzyme productivity, most in shochu brewing have been adopted by a solid culture method. In this study, therefore, we investigated production of Aα-A in liquid culture system. Materials and methods: Microorganism Aspergillus kawachii NBRC 4308 was used. The mold was cultured at 30 °C for 7~14 d to allow formation of conidiospores on slant agar medium. Liquid Culture System: A. kawachii was cultured in a 100 ml of following altered SLS medium: 1.0 g of rice flour, 0.1 g of K2HPO4, 0.1 g of KCl, 0.6 g of tryptone, 0.05 g of MgSO4・7H2O, 0.001 g of FeSO4・7H2O, 0.0003 g of ZnSO4・7H2O, 0.021 g of CaCl2, 0.33 of citric acid (pH 3.0). The pH of the medium was adjusted to the designated value with 10 % HCl solution. The cultivation was shaking at 30 °C and 200 rpm for 72 h. It was filtered to obtain a crude enzyme solution. Aα-A assay: The crude enzyme solution was analyzed. An acid-stable α-amylase activity was carried out using an α-amylase assay kit (Kikkoman Corporation, Noda, Japan). It was conducted after adding 9 ml of 100 mM acetate buffer (pH 3.0) to 1 ml of the culture product supernatant and acid treatment at 37°C for 1 h. One unit of a-amylase activity was defined as the amount of enzyme that yielded 1 mmol of 2-chloro-4-nitrophenyl 6-azide-6-deoxy-b-maltopentaoside (CNP) per minute. Results and Conclusion: We experimented with co-culture of A. kawachii and lactobacillus in order to get control of pH in altered SLS medium. However, high production of acid-stable α-amylase was not obtained. We experimented with yoghurt or milk made an addition to liquid culture. The result indicated that high production of acid-stable α-amylase (964 U/g-substrate) was obtained when milk made an addition to liquid culture. Phosphate concentration in the liquid medium was a major cause of increased acid-stable α-amylase activity. In liquid culture, acid-stable α-amylase activity was enhanced by milk, but Fats and oils in the milk were oxidized. In addition, Tryptone is not approved as a food additive in Japan. Thus, alter SLS medium added to skim milk excepting for the fats and oils in the milk instead of tryptone. The result indicated that high production of acid-stable α-amylase was obtained with the same effect as milk.

Keywords: acid-stable α-amylase, liquid culture, milk, shochu

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Authors: Bo Ram Choi, Ji Su Kim, Juyeon Cho, Hyukjin Lee

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Food contaminated by bacteria (E.coli), causes food poisoning, which occurs to many patients worldwide annually. We have introduced an application of rolling circle amplification (RCA) as a versatile biosensor and developed a diagnostic platform composed of capillary tube and microbeads for rapid and easy detection of Escherichia coli (E. coli). When specific mRNA of E.coli is extracted from cell lysis, rolling circle amplification (RCA) of DNA template can be achieved and can be visualized by beads aggregation in capillary tube. In contrast, if there is no bacterial pathogen in sample, no beads aggregation can be seen. This assay is possible to detect visually target gene without specific equipment. It is likely to the development of a genetic kit for point of care testing (POCT) that can detect target gene using microbeads.

Keywords: rolling circle amplification (RCA), Escherichia coli (E. coli), point of care testing (POCT), beads aggregation, capillary tube

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Authors: Shahram Naghizadeh Raeisi, Ali Alghooneh

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The loss of curd cohesiveness and syneresis are two common problems in the ultrafiltered cheese industry. In this study, by using membrane technology and protein modification, a modified cheese was developed and its properties were compared with a control sample. In order to decrease the lactose content and adjust the protein, acidity, dry matter and milk minerals, a combination of ultrafiltration, nanofiltration and reverse osmosis technologies was employed. For protein modification, a two-stage chemical and enzymatic reaction was employed before and after ultrafiltration. The physicochemical and microstructural properties of the modified ultrafiltered cheese were compared with the control one. Results showed that the modified protein enhanced the functional properties of the final cheese significantly (pvalue< 0.05), even if the protein content was 50% lower than the control one. The modified cheese showed 21 ± 0.70, 18 ± 1.10 & 25±1.65% higher hardness, cohesiveness and water-holding capacity values, respectively, than the control sample. This behavior could be explained by the developed microstructure of the gel network. Furthermore, chemical-enzymatic modification of milk protein induced a significant change in the network parameter of the final cheese. In this way, the indices of network linkage strength, network linkage density, and time scale of junctions were 10.34 ± 0.52, 68.50 ± 2.10 & 82.21 ± 3.85% higher than the control sample, whereas the distance between adjacent linkages was 16.77 ± 1.10% lower than the control sample. These results were supported by the results of the textural analysis. A non-linear viscoelastic study showed a triangle waveform stress of the modified protein contained cheese, while the control sample showed rectangular waveform stress, which suggested a better sliceability of the modified cheese. Moreover, to study the shelf life of the products, the acidity, as well as molds and yeast population, were determined in 120 days. It’s worth mentioning that the lactose content of modified cheese was adjusted at 2.5% before fermentation, while the lactose of the control one was at 4.5%. The control sample showed 8 weeks shelf life, while the shelf life of the modified cheese was 18 weeks in the refrigerator. During 18 weeks, the acidity of modified and control samples increased from 82 ± 1.50 to 94 ± 2.20 °D and 88 ± 1.64 to 194 ± 5.10 °D, respectively. The mold and yeast populations, with time, followed the semicircular shape model (R2 = 0.92, R2adj = 0.89, RMSE = 1.25). Furthermore, the mold and yeast counts and their growth rate in the modified cheese were lower than those for control one; Aforementioned result could be explained by the shortage of the source of energy for the microorganism in the modified cheese. The lactose content of the modified sample was less than 0.2 ± 0.05% at the end of fermentation, while this was 3.7 ± 0.68% in the control sample.

Keywords: non-linear viscoelastic, protein modification, semicircular shape model, ultrafiltered cheese

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Authors: Ž. Vaštag, Lj. Popović, S. Popović

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After cold pressing of pumpkin oil, the defatted oil cake (PUOC) was utilized as raw material for processing of bio-functional hydrolysates. In this study, the in vitro bioactivity of an alcalase (AH) and a pepsin hydrolysate (PH) prepared from the major pumpkin 12S globulin (cucurbitin) are compared. The hydrolysates were produced at optimum reaction conditions (temperature, pH) for the enzymes, during 60min. The bioactivity testing included antioxidant and angiotensin I converting enzyme inhibitory activity assays. The hydrolysates showed high potential as natural antioxidants and possibly antihypertensive agents in functional food or nutraceuticals. Additionally, preliminary studies have shown that both hydrolysates could exhibit modest α-amylase inhibitory activity, which indicates on their hypoglycemic potential.

Keywords: cucurbitin, alcalase, pepsin, protein hydrolysates, in vitro bioactivity

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Authors: Lorena C. Cintra, Izadora M. De Oliveira, Amanda G. Fernandes, Francieli Colussi, Rosália S. A. Jesuíno, Fabrícia P. Faria, Cirano J. Ulhoa

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Xylan is the main component of hemicellulose and for its complete degradation is required cooperative action of a system consisting of several enzymes including endo-xylanases (XYN), β-xylosidases (XYL) and α-L-arabinofuranosidases (ABF). The recombinant hemicellulolytic enzymes an endoxylanase (HXYN2), β-xylosidase (HXYLA), and an α-L-arabinofuranosidase (ABF3) were used in hydrolysis tests. These three enzymes are produced by filamentous fungi and were expressed heterologously and produced in Pichia pastoris previously. The aim of this work was to evaluate the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of sugarcane bagasse (SCB). The interaction between the three recombinant enzymes during SCB pre-treated by steam explosion hydrolysis was performed with different concentrations of HXYN2, HXYLA and ABF3 in different ratios in according to a central composite rotational design (CCRD) 23, including six axial points and six central points, totaling 20 assays. The influence of the factors was assessed by analyzing the main effects and interaction between the factors, calculated using Statistica 8.0 software (StatSoft Inc. Tulsa, OK, USA). The Pareto chart was constructed with this software and showed the values of the Student’s t test for each recombinant enzyme. It was considered as response variable the quantification of reducing sugars by DNS (mg/mL). The Pareto chart showed that the recombinant enzyme ABF3 exerted more significant effect during SCB hydrolysis, with higher concentrations and with the lowest concentration of this enzyme. It was performed analysis of variance according to Fisher method (ANOVA). In ANOVA for the release of reducing sugars (mg/ml) as the variable response, the concentration of ABF3 showed significance during hydrolysis SCB. The result obtained by ANOVA, is in accordance with those presented in the analysis method based on the statistical Student's t (Pareto chart). The degradation of the central chain of xylan by HXYN2 and HXYLA was more strongly influenced by ABF3 action. A model was obtained, and it describes the performance of the interaction of all three enzymes for the release of reducing sugars, and can be used to better explain the results of the statistical analysis. The formulation capable of releasing the higher levels of reducing sugars had the following concentrations: HXYN2 with 600 U/g of substrate, HXYLA with 11.5 U.g-1 and ABF3 with 0.32 U.g-1. In conclusion, the recombinant enzyme that has a more significant effect during SCB hydrolysis was ABF3. It is noteworthy that the xylan present in the SCB is arabinoglucoronoxylan, due to this fact debranching enzymes are important to allow access of enzymes that act on the central chain.

Keywords: experimental design, hydrolysis, recombinant enzymes, sugar cane bagasse

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Authors: T. Sadunishvili, L. Kutateladze, T. Urushadze, R. Khvedelidze, N. Zakariashvili, M. Jobava, G. Kvesitadze

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Multiple repeated soil-climatic zones in Georgia determines the diversity of microorganisms. Hundreds of microscopic fungi of different genera have been isolated from different ecological niches, including some extreme environments. Biosynthetic ability of microscopic fungi has been studied. Trichoderma ressei, representative of the Ascomycetes secrete cellulolytic and xylanolytic enzymes that act in synergy to hydrolyze polysaccharide polymers to glucose, xylose and arabinose, which can be fermented to biofuels. The other mesophilic strains producing cellulases are Allesheria terrestris, Chaetomium thermophile, Fusarium oxysporium, Piptoporus betulinus, Penicillium echinulatum, P. purpurogenum, Aspergillus niger, A. wentii, A. versicolor, A. fumigatus etc. In the majority of the cases the cellulases produced by strains of genus Aspergillus usually have high β-glucosidase activity and average endoglucanases levels (with some exceptions), whereas strains representing Trichoderma have high endo enzyme and low β-glucosidase, and hence has limited efficiency in cellulose hydrolysis. Six producers of stable cellulases and xylanases from mesophilic and thermophilic fungi have been selected. By optimization of submerged cultivation conditions, high activities of cellulases and xylanases were obtained. For enzymes purification, their sedimentation by organic solvents such as ethyl alcohol, acetone, isopropanol and by ammonium sulphate in different ratios have been carried out. Best results were obtained with precipitation by ethyl alcohol (1:3.5) and ammonium sulphate. The yields of enzyme according to cellulase activities were 80-85% in both cases. Cellulase activity of enzyme preparation obtained from the strain Trichoderma viride X 33 is 126 U/g, from the strain Penicillium canescence D 85–185U/g and from the strain Sporotrichum pulverulentum T 5-0 110 U/g. Cellulase activity of enzyme preparation obtained from the strain Aspergillus sp. Av10 is 120 U/g, xylanase activity of enzyme preparation obtained from the strain Aspergillus niger A 7-5–1155U/g and from the strain Aspergillus niger Aj 38-1250 U/g. Optimum pH and temperature of operation and thermostability, of the enzyme preparations, were established. The efficiency of hydrolyses of different agricultural residues by the microscopic fungi cellulases has been studied. The glucose yield from the residues as a result of enzymatic hydrolysis is highly determined by the ratio of enzyme to substrate, pH, temperature, and duration of the process. Hydrolysis efficiency was significantly increased as a result of different pretreatment of the residues by different methods. Acknowledgement: The Study was supported by the ISTC project G-2117, funded by Korea.

Keywords: cellulase, xylanase, microscopic fungi, enzymatic hydrolysis

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Authors: Muhammad Rosli Abdullah, Noor Hasmiza Harun

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A microorganism detection system has a potential to be used with the advancement in a biosensor development. The detection system requires an optical sensing system, microfluidic device and biological reagent. Although, the biosensors are available in the market, a label free and a lab-on-chip approach will promote a flexible solution. As a preliminary study of microorganism detection, three mechanisms such as Total Internal Reflection (TIR), Micro Fluidic Channel (MFC) and magnetic-electric field propagation were study and simulated. The objective are to identify the TIR angle, MFC parabolic flow and the wavelength for the microorganism detection. The simulation result indicates that evanescent wave is achieved when TIR angle > 42°, the corner and centre of a parabolic velocity are 0.02 m/s and 0.06 m/s respectively, and a higher energy distribution of a perfect electromagnetic scattering with dipole resonance radiation occurs at 500 nm. This simulation is beneficial to determine the components of the microorganism detection system that does not rely on classical microbiological, immunological and genetic methods which are laborious, time-consuming procedures and confined to specialized laboratories with expensive instrumentation equipment.

Keywords: microorganism, microfluidic, total internal reflection, lab on chip

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Authors: Amira M. Hassanein, Nehal A. Salahuddin, Atsunori Matsuda, Toshiaki Hattori, Mona N. Elfiky

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New insights into the design of highly sensitive, carbon-based electrochemical sensors are presented in this work. This was achieved by exploring the interesting properties of conductive (Mg/Al) layered double hydroxide- Dodecyl Sulphate/Polypyrrole nanocomposites which were synthesized by in-situ polymerization of pyrrole during the assembly of (Mg/Al) layered double hydroxide, and by employing the anionic surfactant Dodecyl sulphate as a modifier. The morphology and surface area of the nanocomposites changed with the percentage of Pyrrole. Under optimal conditions, the modified carbon paste electrode successfully achieved detection limits of 0.057 and 0.134 nmol.L-1 of Terazosin hydrochloride in pharmaceutical formulation and spiked human serum fluid, respectively. Moreover, the sensors are highly stable, reusable, and free from interference by other commonly present excipients in drug formulations.

Keywords: layered double hydroxide, polypyrrole, terazosin hydrochloride, square-wave adsorptive anodic stripping voltammetry

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Authors: Klaudia Chmielewska, Krystyna Dzierzbicka, Iwona Inkielewicz-Stepniak

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Carnosine, an endogenous peptide consisting of β-alanine and L-histidine has a variety of functions to mention: antioxidant, antiglycation, and reducing the toxicity of metal ions. It has therefore been proposed to act as a therapeutic agent for many pathological states, although its therapeutic index is limited by quick enzymatic cleavage. To overcome this limitation, there’s an urge to create new derivatives which might become less potent to hydrolysis, while preserving the therapeutic effect. The poster summarizes the efficiency of two peptide synthesis methods, which were: (1) the mixed anhydride with isobutyl chloroformate and N-methylmorpholine (NMM) and (2) carbodiimide - mediated coupling method via appropriate reagent condensing, here – CDI. The methods were used to obtain dipeptides which were the derivatives of carnosine. Obtained dipeptides were made in the form of methyl esters and their structures will be confirmed 1H NMR, 13C NMR, MS and elemental analysis techniques. Later on, they will be analyzed for their antioxidant properties, in comparison to carnosine.

Keywords: carnosine, method, peptide, synthesis

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Authors: Michelle Belinda S. Weerawardana, Gobika Thiripuranathar, Priyani A. Paranagama

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Most of fresh vegetables and fruits available in the retail markets undergo a physiological disorder in its appearance and coloration, which indeed discourages consumer purchase. A loss of millions of dollars yearly to the food industry had been due to this pronounced color reaction called Enzymatic Browning which is driven due to the catalytic activity by an oxidoreductase enzyme, polyphenol oxidase (PPO). The enzyme oxidizes the phenolic compounds which are abundantly available in fruits and vegetables as substrates into quinones, which could react with proteins in its surrounding to generate black pigments, called melanins, which are highly UV-active compounds. Annona muricata (Katu anoda) and Musa acuminata (Ash plantains) is a fruit and a vegetable consumed by Sri Lankans widely due to their high nutritional values, medicinal properties and economical importance. The objective of the present study was to evaluate and determine the effective natural anti-browning inhibitors that could prevent PPO activity in the selected fruit and vegetable. Enzyme extracts from Annona muricata (Katu anoda) and Musa acuminata (Ash plantains), were prepared by homogenizing with analytical grade acetone, and pH of each enzyme extract was maintained at 7.0 using a phosphate buffer. The extracts of inhibitors were prepared using powdered ginger rhizomes and essential oil from the bark of Cinnamomum zeylanicum. Water extracts of ginger were prepared and the essential oil from Ceylon cinnamon bark was extracted using steam distillation method. Since the essential oil is not soluble in water, 0.1µl of cinnamon bark oil was mixed with 0.1µl of Triton X-100 emulsifier and 5.00 ml of water. The effect of each inhibitor on the PPO activity was investigated using catechol (0.1 mol dm-3) as the substrate and two samples of enzyme extracts prepared. The dosages of the prepared Cinnamon bark oil, and ginger (2 samples) which were used to measure the activity were 0.0035 g/ml, 0.091 g/ml and 0.087 g/ml respectively. The measurements of the inhibitory activity were obtained at a wavelength of 525 nm using the UV-visible spectrophotometer. The results evaluated thus revealed that % inhibition observed with cinnamon bark oil, and ginger for Annona muricata was 51.97%, and 60.90% respectively. The effects of cinnamon bark oil, and ginger extract on PPO activity of Musa acuminata were 49.51%, and 48.10%. The experimental findings thus revealed that Cinnamomum zeylanicum bark oil was a more effective inhibitor for PPO enzyme present in Musa acuminata and ginger was effective for PPO enzyme present in Annona muricata. Overall both the inhibitors were proven to be more effective towards the activities of PPO enzyme present in both samples. These inhibitors can thus be corroborated as effective, natural, non-toxic, anti-browning extracts, which when added to the above fruit and vegetable will increase the shelf life and also the acceptance of the product by the consumers.

Keywords: anti-browning agent, enzymatic browning, inhibitory activity, polyphenol oxidase

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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: Francesca Crivellari, Nicholas Mavrogiannis, Zachary Gagnon

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Portable diagnostic methods have become increasingly important for a number of different purposes: point-of-care screening in developing nations, environmental contamination studies, bio/chemical warfare agent detection, and end-user use for commercial health monitoring. The cheapest and most portable methods currently available are paper-based – lateral flow and dipstick methods are widely available in drug stores for use in pregnancy detection and blood glucose monitoring. These tests are successful because they are cheap to produce, easy to use, and require minimally invasive sampling. While adequate for their intended uses, in the realm of blood-borne pathogens and numerous cancers, these paper-based methods become unreliable, as they lack the nM/pM sensitivity currently achieved by clinical diagnostic methods. Clinical diagnostics, however, utilize techniques involving surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISAs), which are expensive and unfeasible in terms of portability. To develop a better, competitive biosensor, we must reduce the cost of one, or increase the sensitivity of the other. Electric fields are commonly utilized in microfluidic devices to manipulate particles, biomolecules, and cells. Applications in this area, however, are primarily limited to interfaces formed between immiscible interfaces. Miscible, liquid-liquid interfaces are common in microfluidic devices, and are easily reproduced with simple geometries. Here, we demonstrate the use of electrical fields at liquid-liquid electrical interfaces, known as fluidic dielectrophoresis, (fDEP) for biodetection in a microfluidic device. In this work, we apply an AC electric field across concurrent laminar streams with differing conductivities and permittivities to polarize the interface and induce a discernible, near-immediate, frequency-dependent interfacial tilt. We design this aqueous electrical interface, which becomes the biosensing “substrate,” to be intelligent – it “moves” only when a target of interest is present. This motion requires neither labels nor expensive electrical equipment, so the biosensor is inexpensive and portable, yet still capable of sensitive detection. Nanoparticles, due to their high surface-area-to-volume ratio, are often incorporated to enhance detection capabilities of schemes like SPR and fluorimetric assays. Most studies currently investigate binding at an immobilized solid-liquid or solid-gas interface, where particles are adsorbed onto a planar surface, functionalized with a receptor to create a reactive substrate, and subsequently flushed with a fluid or gas with the relevant analyte. These typically involve many preparation and rinsing steps, and are susceptible to surface fouling. Our microfluidic device is continuously flowing and renewing the “substrate,” and is thus not subject to fouling. In this work, we demonstrate the ability to electrokinetically detect biomolecules binding to functionalized nanoparticles at liquid-liquid interfaces using fDEP. In biotin-streptavidin experiments, we report binding detection limits on the order of 1-10 pM, without amplifying signals or concentrating samples. We also demonstrate the ability to detect this interfacial motion, and thus the presence of binding, using impedance spectroscopy, allowing this scheme to become non-optical, in addition to being label-free.

Keywords: biodetection, dielectrophoresis, microfluidics, nanoparticles

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Authors: Natalia Caldeira De Carvalho, Tassia Batista Pessato, Luis Gustavo R. Fernandes, Ricardo L. Zollner, Flavia Maria Netto

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Protein hydrolysates are ingredients of enteral diets and hypoallergenic formulas. Enzymatic hydrolysis is the most commonly used method for reducing the antigenicity of milk protein. The antigenicity and physicochemical characteristics of the protein hydrolysates depend on the reaction parameters. Among them, pH has been pointed out as of the major importance. Hydrolysis reaction in laboratory scale is commonly carried out under controlled pH (pH-stat). However, from the industrial point of view, controlling pH during hydrolysis reaction may be infeasible. This study evaluated the impact of pH control on the physicochemical properties and antigenicity of the hydrolysates of whey proteins with Alcalase. Whey protein isolate (WPI) solutions containing 3 and 7 % protein (w/v) were hydrolyzed with Alcalase 50 and 100 U g-1 protein at 60°C for 180 min. The reactions were carried out under controlled and uncontrolled pH conditions. Hydrolyses performed under controlled pH (pH-stat) were initially adjusted and maintained at pH 8.5. Hydrolyses carried out without pH control were initially adjusted to pH 8.5. Degree of hydrolysis (DH) was determined by OPA method, peptides profile was evaluated by HPLC-RP, and molecular mass distribution by SDS-PAGE/Tricine. The residual α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) concentrations were determined using commercial ELISA kits. The specific IgE and IgG binding capacity of hydrolysates was evaluated by ELISA technique, using polyclonal antibodies obtained by immunization of female BALB/c mice with α-La, β-Lg and BSA. In hydrolysis under uncontrolled pH, the pH dropped from 8.5 to 7.0 during the first 15 min, remaining constant throughout the process. No significant difference was observed between the DH of the hydrolysates obtained under controlled and uncontrolled pH conditions. Although all hydrolysates showed hydrophilic character and low molecular mass peptides, hydrolysates obtained with and without pH control exhibited different chromatographic profiles. Hydrolysis under uncontrolled pH released, predominantly, peptides between 3.5 and 6.5 kDa, while hydrolysis under controlled pH released peptides smaller than 3.5 kDa. Hydrolysis with Alcalase under all conditions studied decreased by 99.9% the α-La and β-Lg concentrations in the hydrolysates detected by commercial kits. In general, β-Lg concentrations detected in the hydrolysates obtained under uncontrolled pH were significantly higher (p<0.05) than those detected in hydrolysates produced with pH control. The anti-α-La and anti-β-Lg IgE and IgG responses to all hydrolysates decreased significantly compared to WPI. Levels of specific IgE and IgG to the hydrolysates were below 25 and 12 ng ml-1, respectively. Despite the differences in peptide composition and α-La and β-Lg concentrations, no significant difference was found between IgE and IgG binding capacity of hydrolysates obtained with or without pH control. These results highlight the impact of pH on the hydrolysates characteristics and their concentrations of antigenic protein. Divergence between the antigen detection by commercial ELISA kits and specific IgE and IgG binding response was found in this study. This result shows that lower protein detection does not imply in lower protein antigenicity. Thus, the use of commercial kits for allergen contamination analysis should be cautious.

Keywords: allergy, enzymatic hydrolysis, milk protein, pH conditions, physicochemical characteristics

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Authors: Khalida Boutemak, Nasssima Benali, Nadji Moulai-Mostefa

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Effect of enzyme on the yield and chemical composition of Artemisia campestris essential oil is reported in the present study. It was demonstrated that enzyme facilitated the extraction of essential oil with increase in oil yield and did not affect any noticeable change in flavour profile of the volatile oil. Essential oil was tested for antibacterial activity using Escherichia coli; which was extremely sensitive against control with the largest inhibition (29mm), whereas Staphylococcus aureus was the most sensitive against essential oil obtained from enzymatic pre-treatment with the largest inhibition zone (25mm). The antioxidant activity of the essential oil with hemicellulase pre-treatment (EO2) and control sample (EO1) was determined through reducing power. It was significantly lower than the standard drug (vitamin C) in this order: vitamin C˃EO2˃EO1.

Keywords: Artemisia campestris, enzyme pre-treatment, hemicellulase, antibacterial activity, antioxidant activity

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Authors: Muhammad Ali Syed, Arshad Saleem Bhatti, Chen-zhong Li, Habib Ali Bokhari

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Surface plasmon resonance (SPR) biosensors have emerged as a promising technique for bioanalysis as well as microbial detection and identification. Real time, sensitive, cost effective, and label free detection of biomolecules from complex samples is required for early and accurate diagnosis of infectious diseases. Like many other types of optical techniques, SPR biosensors may also be successfully utilized for microbial detection for accurate, point of care, and rapid results. In the present study, we have utilized a commercially available automated SPR biosensor of BI company to study the microbial detection form water samples spiked with different concentration of Staphylococcus aureus bacterial cells. The gold thin film sensor surface was functionalized to react with proteins such as protein G, which was used for directed immobilization of monoclonal antibodies against Staphylococcus aureus. The results of our work reveal that this immunosensor can be used to detect very small number of bacterial cells with higher sensitivity and specificity. In our case 10^3 cells/ml of water have been successfully detected. Therefore, it may be concluded that this technique has a strong potential to be used in microbial detection and identification.

Keywords: surface plasmon resonance (SPR), Staphylococcus aureus, biosensors, microbial detection

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Authors: Kanchan Vishwakarma, Shivesh Sharma, Nitin Kumar

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Soil is incoherent matter on Earth’s surface having organic and mineral content. The spatial variation of 4 soil enzyme activities and microbial biomass were assessed for two seasons’ viz. monsoon and winter along the latitudinal gradient in North-central India as the area of this study is fettered with respect to national status. The study was facilitated to encompass the effect of climate change, enzyme activity and biomass on nutrient cycling. Top soils were sampled from 4 sites in North-India. There were significant correlations found between organic C, N & P wrt to latitude gradient in two seasons. This distribution of enzyme activities and microbial biomass was consequence of alterations in temperature and moisture of soil because of which soil properties change along the latitude transect.

Keywords: latitude gradient, microbial biomass, moisture, soil, organic carbon, temperature

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Authors: Indu Verma, Santanu Kumar Pal

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Interactions between DNA and adsorbed Poly (L-lysine) (PLL) on liquid crystal (LC) droplets were investigated using polarizing optical microcopy (POM) and epi-fluorescence microscopy. Earlier, we demonstrated that adsorption of PLL to the LC/aqueous interface resulted in homeotropic orientation of the LC and thus exhibited a radial configuration of the LC confined within the droplets. Subsequent adsorption of DNA (single stranded DNA/double stranded DNA) at PLL coated LC droplets was found to trigger a LC reorientation within the droplets leading to pre-radial/bipolar configuration of those droplets. To our surprise, subsequent exposure of complementary ssDNA (c-ssDNA) to ssDNA/ adsorbed PLL modified LC droplets did not cause the LC reorientation. This is likely due to the formation of polyplexes (DNA-PLL complex) as confirmed by fluorescence microscopy and atomic force microscopy. In addition, dsDNA adsorbed PLL droplets have been found to be effectively used to displace (controlled release) propidium iodide (a model drug) encapsulated within dsDNA over time. These observations suggest the potential for a label free droplet based LC detection system that can respond to DNA and may provide a simple method to develop DNA-based drug nano-carriers.

Keywords: DNA biosensor, drug delivery, interfaces, liquid crystal droplets

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Authors: Ivana Brzonova, Asina Fnu, Alena Kubatova, Evguenii Kozliak, Yun Ji

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Bio-decomposition of lignin by Basidiomycetes in the presence of dimethyl sulfoxide (DMSO) was investigated. The addition of 3-5 vol% DMSO to lignin aqueous media significantly increased the lignin solubility based on UV absorbance. After being dissolved in DMSO, the thermal evolution profile also changed significantly, yielding more high-MW organic carbon at the expense of recalcitrant elemental carbon. Medical fungi C. versicolor, G. lucidum and P. pulmonarius, were observed to grow on the lignin in media containing up to 15 vol. % DMSO. Further detailed product characterization by chromatographic methods corroborated these observations, as more low-MW phenolic products were observed with DMSO as a co-solvent. These results may be explained by the high solubility of lignin in DMSO; thus, the addition of DMSO to the medium increases the lignin availability for microorganisms. Some of these low-MW phenolic products host a big potential to be used in medicine. No significant inhibition of enzymatic activity (laccase, MnP, LiP) was observed by the addition of up to 3 vol% DMSO.

Keywords: basidiomycetes, bio-degradation, dimethyl sulfoxide, lignin

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Authors: Venkat Garigapati

Abstract:

To date, more than a dozen oligonucleotide products are approved as injectable products for clinical use. However, there is no single oligo nucleotide product approved for clinical use. Oral delivery of oligo nucleotides is patient friendly administration however, many challenges involved in the development of oral formulation. Over the course of last twenty plus years, the research in this space aimed to address these challenges. This paper describes the issues involved in solubility, stability, enzymatic (nuclease) induced degradation, and permeation of nucleotides in the Gastrointestinal (GI) and how to overcome these challenges. Also, the translation of in vitro data to in vivo models hinders the formulation development. This paper describes the challenges involved in the development of Oligo Nucleotide products for oral administration. It also discusses the chemistry and formulation strategies for oral administration of oligonucleotides.

Keywords: oral adminstration, oligo nucleotides, stability, permeation, gastrointestinal tract

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Authors: Nusrat Masood, Vijaya Dubey, Suaib Luqman

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Caspase 3, a member of cysteine-aspartic acid protease family, is an imperative indicator for cell death particularly when substantiating apoptosis. Thus, caspase 3 is an interesting target for the discovery and development of anticancer agent. We adopted a four level assessment of both terpenoids and flavonoids and thus experimentally performed the enzymatic assay in cell free system as well as in cancer cell line which was validated through real time expression and molecular interaction studies. A significant difference was observed with both the class of natural products indicating terpenoids as better activators of caspase 3 compared to flavonoids both in the cell free system as well as in cell lines. The expression analysis, activation constant and binding energy also correlate well with the enzyme activity. Overall, terpenoids had an unswerving effect on caspase 3 in all the tested system while flavonoids indirectly affect enzyme activity.

Keywords: Caspase 3, terpenoids, flavonoids, activation constant, binding energy

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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: Preshanthan Moodley

Abstract:

This study compares two different pretreatments for sugarcane leaf waste (SLW): steam salt-alkali (SSA) and microwave salt-alkali (MSA). The two pretreatment types were modelled, optimized, and validated with R² > 0.97. Reducing sugar yields of 1.21g/g were obtained with optimized SSA pretreatment using 1.73M ZnCl₂, 1.36M NaOH and 9.69% solid loading, and 1.17g/g with optimized MSA pretreatment using 1.67M ZnCl₂, 1.52M NaOH at 400W for 10min. A lower pretreatment time (10min) was required for the MSA model (83% lower). The structure of pretreated SLW was assessed using scanning electron microscopy (SEM) and Fourier Transform Infrared analysis (FTIR). The optimized SSA and MSA models showed lignin removal of 80.5 and 73% respectively. The MSA pretreatment was further examined on sorghum leaves and Napier grass and showed yield improvements of 1.9- and 2.8-fold compared to recent reports. The developed pretreatment methods demonstrated high efficiency at enhancing enzymatic hydrolysis on various lignocellulosic substrates.

Keywords: lignocellulosic biomass, pretreatment, salt, sugarcane leaves

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Authors: Abirami Baleswaran, Christel Couderc, Loubnah Belahcen, Jean Dayde, Hélène Tormo, Gwénaëlle Jard

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Cheeses are often reported to be spoiled by Pseudomonas spp., responsible for defects in appearance, texture, taste, and smell, leading to their non-marketing and even their destruction. Despite preventive actions, problems linked to Pseudomonas spp. are difficult to control by the lack of knowledge and control of these contaminants during the cheese manufacturing. Lactic goat cheese producers are not spared by this problem and are looking for solutions to decrease the number of spoiled cheeses. To explore different hypotheses, experiments are needed. However, cheese-making experiments at the pilot scale are expensive and time consuming. Thus, there is a real need to develop a miniature cheeses model system under controlled conditions. In a previous study, several miniature cheese models corresponding to different type of commercial cheeses have been developed for different purposes. The models were, for example, used to study the influence of milk, starters cultures, pathogen inhibiting additives, enzymatic reactions, microflora, freezing process on cheese. Nevertheless, no miniature model was described on the lactic goat cheese. The aim of this work was to develop a miniature cheese model system under controlled laboratory conditions which resembles commercial lactic goat cheese to study Pseudomonas spp. spoilage during the manufacturing and ripening process. First, a protocol for the preparation of miniature cheeses (3.5 times smaller than a commercial one) was designed based on the cheese factorymanufacturing process. The process was adapted from “Rocamadour” technology and involves maturation of pasteurized milk, coagulation, removal of whey by centrifugation, moulding, and ripening in a little scale cellar. Microbiological (total bacterial count, yeast, molds) and physicochemical (pH, saltinmoisture, moisture in fat-free)analyses were performed on four key stages of the process (before salting, after salting, 1st day of ripening, and end of ripening). Factory and miniature cheeses volatilomewere also obtained after full scan Sift-MS cheese analysis. Then, Pseudomonas spp. strains isolated from contaminated cheeses were selected on their origin, their ability to produce pigments, and their enzymatic activities (proteolytic, lecithinasic, and lipolytic). Factory and miniature curds were inoculated by spotting selected strains on the cheese surface. The expression of cheese spoilage was evaluated by counting the level of Pseudomonas spp. during the ripening and by visual observation and under UVlamp. The physicochemical and microbiological compositions of miniature cheeses permitted to assess that miniature process resembles factory process. As expected, differences involatilomes were observed, probably due to the fact that miniature cheeses are made usingpasteurized milk to better control the microbiological conditions and also because the little format of cheese induced probably a difference during the ripening even if the humidity and temperature in the cellar were quite similar. The spoilage expression of Pseudomonas spp. was observed in miniature and factory cheeses. It confirms that the proposed model is suitable for the preparation of miniature cheese specimens in the spoilage study of Pseudomonas spp. in lactic cheeses. This kind of model could be deployed for other applications and other type of cheese.

Keywords: cheese, miniature, model, pseudomonas spp, spoilage

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Authors: Rezvan Najafi, Korosh Heydari, Massoud Saidijam

Abstract:

5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.

Keywords: colorectal cancer, miR-200c, 5-FU resistance, E-cadherin, PTEN

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Authors: Paweł Stączek, Tomasz Plech, Aleksandra Strzelczyk, Katarzyna Dzitko, Monika Wujec, Edyta Kuśmierz, Piotr Paneth, Agata Paneth

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In this contribution, we will describe the inhibitory potency of nine thiosemicarbazide derivatives against bacterial type IIA topoisomerases, their antibacterial profile, and molecular modeling evaluation. We have found that one of the tested compounds, 4-benzoyl-1-(2-methyl-furan-3-ylcarbonyl) thiosemicarbazide, remarkably inhibits the activity of S. aureus DNA gyrase with the IC50 below 5 μM. Besides, this compound displays antibacterial activity on Staphylococcus spp. and E. faecalis at non-cytotoxic concentrations in mammalian cells, with minimal inhibitory concentrations (MICs) values at 25 μg/mL. Based on the enzymatic and molecular modeling studies we propose two factors, i.e. geometry of molecule and hydrophobic/hydrophilic balance as important molecular properties for developing thiosemicarbazide derivatives as potent Staphylococcus aureus DNA gyrase inhibitors.

Keywords: bioactivity, drug design, topoisomerase, molecular modeling

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Authors: Shweta Kumari, Parmjit S. Panesar, Manab B. Bera

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The hydrolysis of lactose using β-galactosidase is one of the most promising biotechnological applications, which has wide range of potential applications in food processing industries. However, due to intracellular location of the yeast enzyme, and expensive extraction methods, the industrial applications of enzymatic hydrolysis processes are being hampered. The use of permeabilization technique can help to overcome the problems associated with enzyme extraction and purification of yeast cells and to develop the economically viable process for the utilization of whole cell biocatalysts in food industries. In the present investigation, standardization of permeabilization process of novel yeast isolate was carried out using a statistical model approach known as Response Surface Methodology (RSM) to achieve maximal b-galactosidase activity. The optimum operating conditions for permeabilization process for optimal β-galactosidase activity obtained by RSM were 1:1 ratio of toluene (25%, v/v) and ethanol (50%, v/v), 25.0 oC temperature and treatment time of 12 min, which displayed enzyme activity of 1.71 IU /mg DW.

Keywords: β-galactosidase, optimization, permeabilization, response surface methodology, yeast

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Authors: Esma Kocaoglu, Oktay Talaz, Huseyin Cavdar, Murat Senturk, Deniz Eki̇nci̇

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Glutathione reductase (GR) is a crucial antioxidant enzyme which is responsible for the maintenance of the antioxidant GSH (glutathione) molecule. Antimalarial effects of some chemical molecules are attributed to their inhibition of GR; thus inhibitors of this enzyme are expected to be promising candidates for the treatment of malaria. In this work, GR inhibitory properties of N-Methylpyrrole derivatives are reported. Firstly, GR was purified by means of affinity chromatography using 2’,5’-ADP-Sepharose 4B as ligand. Enzymatic activity was measured by Beutler’s method. Synthesis of the compounds was approved by thin layer chromatography and column chromatography. Different inhibitor concentrations were used and all compounds were tested in triplicate at each concentration used. It was found that all compounds have better inhibitory activity than the strong GR inhibitor N,N-bis(2-chloroethyl)-N-nitrosourea, especially three molecules, 8m, 8n, and 8q, are the best among them with low micromolar I₅₀ values. Findings of our study indicate that these Schiff base derivatives are strong GR inhibitors which can be used as leads for designation of novel antimalaria candidates.

Keywords: glutathione reductase, antimalaria, inhibitor, enzyme

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Authors: Preshanthan Moodley, E. B. Gueguim-Kana

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The objective of this study was to develop a method to pretreat sugarcane leaf waste using microwave-assisted (MA) inorganic salt. The effects of process parameters of salt concentration, microwave power intensity and pretreatment time on reducing sugar yield from enzymatically hydrolysed sugarcane leaf waste were investigated. Pretreatment models based on MA-NaCl, MA-ZnCl2 and MA-FeCl3 were developed. Maximum reducing sugar yield of 0.406 g/g was obtained with 2 M FeCl3 at 700W for 3.5 min. Scanning electron microscopy (SEM) and Fourier Transform Infrared analysis (FTIR) showed major changes in lignocellulosic structure after MA-FeCl3 pretreatment with 71.5 % hemicellulose solubilization. This pretreatment was further assessed on sorghum leaves and Napier grass under optimal MA-FeCl3 conditions. A 2 fold and 3.1-fold increase in sugar yield respectively were observed compared to previous reports. This pretreatment was highly effective for enhancing enzymatic saccharification of lignocellulosic biomass.

Keywords: acid, pretreatment, salt, sugarcane leaves

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Authors: T. Bettaieb, S. Soufi, S. Arbaoui

Abstract:

Stevia rebaudiana Bert. is a natural sweet plant. The leaves contain diterpene glycosides stevioside, rebaudiosides A-F, steviolbioside and dulcoside, which are responsible for its sweet taste and have commercial value all over the world as sugar substitute in foods and medicines. Stevia rebaudiana Bert. is sensitive temperature lower than 9°C. The possibility of its outdoor culture in Tunisian conditions demand genotypes tolerant to low temperatures. In order to evaluate the low temperature tolerance of eight genotypes of Stevia rebaudiana, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalases (CAT) were measured. Before carrying out the analyses, three genotypes of Stevia were exposed for 1 month at a temperature regime of 18°C during the day and 7°C at night similar to winter conditions in Tunisia. In response to the stress generated by low temperature, antioxidant enzymes activity revealed on native gel and quantified by spectrophotometry showed variable levels according to their degree of tolerance to low temperatures.

Keywords: chilling tolerance, enzymatic activity, stevia rebaudiana bert, low thermal stress

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Authors: Deokyeong Choe, Keonwook Nam, Young Hoon Roh

Abstract:

Wood waste is a potentially significant resource for economic and environment-friendly recycling. Wood waste represents a key sustainable source of biomass for transformation into bioethanol. Unfortunately, wood waste is highly recalcitrant for biotransformation, which limits its use and prevents economically viable conversion into bioethanol. As a result, an effective pretreatment is necessary to degrade cellulose of the wood waste, which improves the accessibility of cellulase. In this work, a H₃PO₄-acetone pretreatment was selected among the various pretreatment methods and used to dissolve cellulose and lignin. When the H₃PO₄ and acetone were used, 5–6% of the wood waste was found to be very appropriate for saccharification. Also, when the enzymatic saccharification was conducted in the mixture of the wood waste and 0.05 M citrate buffer solution, glucose and xylose were measured to be 80.2 g/L and 9.2 g/L respectively. Furthermore, ethanol obtained after 70 h of fermentation by S. cerevisiae was 30.4 g/L. As a result, the conversion yield from wood waste to bioethanol was calculated to be 57.4%. These results show that the pretreated wood waste can be used as good feedstocks for bioethanol production and that the H₃PO₄-acetone pretreatment can effectively increase the yield of ethanol production.

Keywords: wood waste, H₃PO₄-acetone, bioethanol, fermentation

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