Development of quality evaluation sensor for fish freshness control based on KI value

Recently, raw fish, sashimi, is becoming a popular dish in countries other than Japan. Therefore, in order to assure that the raw fish and shellfish are safe for human consumption, a quality evaluation sensor, which shows, at a glance, the quality of sashimi, was developed. The proposed sensor is based on the principle that the freshness of sashimi, which is judged from the KI value, can be determined from the degree of color change of thiazole blue (MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) due to the redox reaction of MTT accompanying the oxidation of hypoxanthine (Hx) by xanthine oxidase (XOD). The proposed sensor consists of 5 ml of 80% ethanol-1 M Tris-HCl buffer (pH 7.8) containing 2.0 mg of Hx, 2.0 mg of MTT and 0.38 unit of XOD. The proposed sensor and fish were kept together at 5, -10 and -20 degrees C, and the freshness of sashimi stored at each temperature was determined from the color change of the sensor. The concept "freshness of sashimi" can be expressed as remaining of validity (RDV), which is described in our previous study. A good relationship was obtained between the KI value and the RDV determined by the proposed sensor. From these results, the proposed sensor system can be used to non-destructively determine the fish freshness and RDV.     

Gluconic acid production from sucrose in an airlift reactor using a multi-enzyme system

Sucrose from sugarcane is produced in abundance in Brazil, which provides an opportunity to manufacture other high-value products. Gluconic acid (GA) can be produced by multi-enzyme conversion of sucrose using the enzymes invertase, glucose oxidase, and catalase. In this process, one of the byproducts is fructose, which has many commercial applications. This work concerns the batch mode production of GA in an airlift reactor fed with sucrose as substrate. Evaluation was made of the influence of temperature and pH, as well as the thermal stability of the enzymes. Operational conditions of 40 °C and pH 6.0 were selected, based on the enzymatic activity profiles and the thermal stabilities. Under these conditions, the experimental data could be accurately described by kinetic models. The maximum yield of GA was achieved within 3.8 h, with total conversion of sucrose and glucose and a volumetric productivity of around 7.0 g L^?1 h^?1.     

Computer vision technique for freshness estimation from segmented eye of fish image

Preserving the quality of fish is a challenging task. Several different cooling methods and materials are used during their storage, transportation purpose. These are responsible factors that decide the freshness of a post harvested fish. In this proposed algorithm, a computer vision-based technique is developed to predict the freshness level of fish from its image. Eyes of the fish are considered as the region of interest, as a good correlation has been observed between the colour of the eye and different duration of storage day. It is segmented from the image of a fish sample and then a strategic framework is used for extraction of the discriminatory features. These extracted features show a degradation pattern which acts as an indicative parameter to determine the level of freshness of sample of fish. The proposed method provides a recognition accuracy of 96.67%. The experimental results indicate that this is an efficient and non-destructive methodology for detecting the fish freshness. The high accuracy of freshness detection and low computation time makes this non-destructive methodology efficient for real-world usage in the fish industry and market.     

Multienzymatic amperometric biosensor based on gold and nanocomposite planar electrodes for glycerol determination in wine

Amperometric biosensors based on gold planar or nanocomposite electrode containing multiwalled carbon nanotubes for determination of glycerol were developed. The biosensors were constructed by immobilization of a novel multienzyme cascade consisting of glycerol kinase/creatine kinase/creatinase/sarcosine oxidase/peroxidase between a chitosan "sandwich." A measuring buffer contained adenosine 5'-triphosphate (ATP), creatine phosphate, and an artificial electrochemical mediator ferrocyanide. The currents proportional to glycerol concentration were measured at working potential of -50 mV against Ag/AgCl reference electrode. The biosensors showed linearity over the ranges of 5-640 μM and 5-566 μM with detection limits of 1.96 and 2.24 μM and sensitivities of 0.80 and 0.81 nA μM(-1), respectively. Both types of biosensors had a response time of 70s. The biosensors demonstrated satisfactory operational stability (no loss of sensitivity after 90 consecutive measurements) and excellent storage stability (90% of the initial sensitivity after 15 months of storage at room temperature). The results obtained from measurements of wines correlated well with those obtained with an enzymatic-spectrophotometric assay. The presented multienzyme cascade can be used also for determination of triglycerides or various kinase substrates when glycerol kinase is replaced by other kinases.     

Flow cytometric determination of genome size for eight commercially important fish species in China

The genome size (C value) of eight commercially important fish species in China was measured using flow cytometry. Chicken (Gallus domesticus) erythrocytes were used as reference cells. When using propidium iodide (PI) as the fluorescent dye, genome sizes were 1.09?±?0.08, 2.75?±?0.12, 1.05?±?0.05, 1.35?±?0.11, 0.99?±?0.05, 0.90?±?0.08, 0.90?±?0.07, and 0.88?±?0.07?pg for Japanese eel (Anguilla japonica), mullet (Myxocyprinus asiaticus), yellowcheek carp (Elopichthys bambusa), blunt snout bream (Megalobrama amblycephala), yellow catfish (Pelteobagrus fulvidraco), ricefield eel (Monopterus albus), mandarin fish (Siniperca chuatsi), and snakehead (Ophicephalus argus), respectively. However, genome sizes were 1.25?±?0.00, 3.08?±?0.02, 1.25?±?0.00, 1.57?±?0.01, 0.96?±?0.01, 1.00?±?0.01, 0.91?±?0.01, and 0.89?±?0.01?pg for these fishes, respectively, when 4??, 6-diamidino-2-phenylindole (DAPI) was used as the fluorescent dye. Regardless of the dye used, the more evolutionarily advanced species had a smaller genome size than those with a lower evolutionary status. For each species, we also measured the size of erythrocytes and their nucleus and evaluated the relationships between erythrocyte size, nucleus size, chromosome number, and genome size. Genome size was positively correlated with erythrocyte nucleus size and chromosome number when using PI as the fluorescent dye, but it was only correlated with erythrocyte nucleus size when DAPI was used.     

Electrochemical sensing of trimethylamine based on polypyrrole-flavin-containing monooxygenase (FMO3) and ferrocene as redox probe for evaluation of fish freshness

Amperometric and impedimetric biosensor for detecting trimethylamine (TMA) which represents good parameters for estimating fish freshness has been developed. The biosensor is based on a conducting polypyrrole substituted with ferrocenyl, where flavin-containing monooxygenase 3 (FMO3) enzyme was immobilised by covalent bonding. FMO3 catalyzes the monooxygenation TMA to trimethylamine N-oxide (TMO). For catalysis FMO require flavin adenine (FAD) as a prosthetic group, NADPH as a cofactor and molecular oxygen as cosubstrate. Ferrocenyl group substituted on the polypyrrole matrix will serve as redox probe for monitoring the response of the biosensor to TMA. The construction of the biosensor was characterized by FT-IR, cyclic voltammetry and impedance measurements. Detection is done through the analysis of the current of oxidation signal of the ferrocenyl groups and compared to the measurement of impedance related to the electrical properties of the layers. Amperometric and impedimetric response were measured as a function of TMA concentration in range of 0.4 μgm L(-1)-80 μgm L(-1) (6.5 μmol L(-1)-1.5 mmol L(-1)). Amperometric measurements show a decrease in current response which is in correlation with the increase of the charge transfer resistance demonstrated by impedance. Calibration curve obtained by impedance spectroscopy shows a high sensitivity with a dynamic range from (0.4 μgm L(-1) to 80 μgm L(-1)). We demonstrated, using ferrocene as redox probe for catalytic reaction of FMO3, that high sensitivity and dynamic range was obtained. The biosensor was stable during 16 days. The biosensor shows high selectivity and its sensitivity to TMA in real samples was evaluated using fish extract after deterioration during storage.     

A periplasmic location for the thiosulphate-oxidizing multi-enzyme system from Thiobacillus versutus

Abstract Evidence is presented to show that the thiosulphate-oxidising multi-enzyme system from Thiobacillus versutus has a periplasmic location, and that the oxygen-binding site of the cytochrome oxidase ( aa ₃) is on the inner surface of the membrane. A scheme for the mechanism of generation of a proton motive force during electron flow from thiosulphate to oxygen via cytochrome c and aa ₃ is proposed.     

Multivessel system for cold-vapor mercury generation determination of mercury in hair and fish

A multivessel system for the determination of mercury (Hg) by cold-vapor atomic absorption spectrometry (CV-AAS) and inductively coupled plasma atomic emission spectrometry (ICP-AES) was developed. The performance of the proposed device was tested by determining total Hg in quality-control samples of hair and fishes following acid digestion. Application of the apparatus to the determination of Hg by CV-AAS following alkaline digestion was studied as well. The detection limit obtained for CV-AAS was 0.11 ng/mL and for ICP-AES 1.39 ng/mL. The results show that the system is appropriate to be used in techniques involving cold-vapor generation of Hg.     

Miniaturized real-time monitoring system for L-lactate and glucose using microfabricated multi-enzyme sensors.

A miniaturized on-line monitoring system for the detection of L-lactate and glucose is presented. The system is based on a microfabricated multi-enzyme silicon sensor chip with flow channels integrated on the chip. The sensors were fabricated in containment technology. They were characterized in test solutions. The cross-talking behaviour was investigated and was found to be practically negligible. The linear measurement ranges of both glucose and lactate sensors were large enough for most practical applications. As a result of the miniaturization the analyte consumption could be reduced to a few nmol min(-1). The system was equipped with a microdialysis probe whose recovery was 45% for lactate and 37% for glucose in test solutions using a flow rate of 3 microl min(-1). Lower flow rates of 0.5 microl min(-1) resulted in recoveries of over 90%. The long-term stability of the system was acceptable. Initial measurements have also been performed in vitro using human blood serum.     

Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.     

Prioritising the rehabilitation of fish passage in a regulated river system based on fish movement

Environmental rehabilitation budgets are often limited, and management actions need to be prioritised to achieve the best outcomes. Prioritisation can best be done when evidence informs the decision‐making process. We acoustically tagged twenty Golden Perch (Macquaria ambigua) in the Loddon River, Australia, and tracked their movements to gain an understanding on the requirements for fish passage at a major regulating structure, the Box Creek regulator. The movements of these fish were monitored through a network of receivers located throughout the lower Loddon River and Pyramid Creek system. Five fish moved 50–120 km upstream, four of which reached the Box Creek regulator before moving back downstream to the entrance of the Kerang Lakes system. Most long distance upstream movements were associated with an increase in river discharge. The remaining 15 fish moved <20 km, with all fish being detected at least once. This pilot study indicates that Box Creek regulator is acting as a barrier for some fish within the Loddon River system. Movement data also indicate that Golden Perch migration pathways may be influenced by river discharge. The management implications of this work includes the need to reinstate fish passage at Box Creek regulator and the potential use of environmental flows to enhance colonisation of native fish species throughout the Murray Darling Basin.     

Gene amplification system based on double rolling-circle replication as a model for oncogene-type amplification

Gene amplification contributes to a variety of biological phenomena, including malignant progression and drug resistance. However, details of the molecular mechanisms remain to be determined. Here, we have developed a gene amplification system in yeast and mammalian cells that is based on double rolling-circle replication (DRCR). Cre-lox system is used to efficiently induce DRCR utilizing a recombinational process coupled with replication. This system shows distinctive features seen in amplification of oncogenes and drug-resistance genes: (i) intra- and extrachromosomal amplification, (ii) intensive chromosome rearrangement and (iii) scattered-type amplification resembling those seen in cancer cells. This system can serve as a model for amplification of oncogenes and drug-resistance genes, and improve amplification systems used for making pharmaceutical proteins in mammalian cells.     

Designing a reusable system based on nanodiamonds for biochemical determination of urea

A reusable system including urease covalently bound to the surface of modified nanodiamonds (MNDs) has been developed for the multiple determination of urea. The immobilized enzyme exhibits functional activity and catalyzes the hydrolysis of urea to yield ammonia. The presence of ammonia is confirmed by the formation of a colored product after the addition of chemical reagents. It was shown that the MNDs-urease complex can function in a wide range of temperatures and pH as well as in deionized water. The complex provides a linear yield of the product at low analyte concentrations and allows the multiple determination of urea in vitro.     

Novel protein carrier system based on cyanobacterial nano-sized extracellular vesicles for application in fish

Aquaculture has been one of the fastest-growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP-loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.     

纤维素乙醇统合加工过程中的酵母多酶共展示体系研究进展

利用统合生物加工过程(Consolidated bioprocessing,CBP)生产纤维素乙醇是目前国内外的研究热点。CBP需要一种“集成化”微生物,既能生产水解木质纤维素的多种酶类又能利用水解木质纤维素产生的糖类发酵产乙醇。以酿酒酵母表面展示技术为依托,建立CBP菌株多酶共展示体系的研究主要分为以下两个方向：一是直接将纤维素酶展示在细胞表面,即非复合型纤维素酶体系;另一种是通过表面展示纤维小体(Cellulosome)将纤维素酶间接地锚定在细胞表面,即复合型纤维素酶体系,本文主要从以上两个方向阐述了近几年对于纤维素乙醇生物统合加工过程的研究进展。因纤维小体对纤维素的降解能力比非复合型纤维素酶体系更强,所以其在酿酒酵母细胞表面的组装研究受到越来越多的关注,为了更深入透彻地了解纤维小体的酵母展示技术,文中对纤维小体的结构与功能及其在纤维素乙醇发酵中的应用研究进行重点论述,并对该领域的发展方向进行展望。     

Parentage determination of mandarin fish (Siniperca chuatsi) based on microsatellite DNA markers

In this study, ten microsatellite loci were chosen to estimate the parentage of 260 progeny in five mandarin fish (Siniperca chuatsi) full-sib families. Simulation based on allele frequency date demonstrated the combined exclusion power would be over 97% if the number of loci was up to nine. Based on the information from these nine loci, 98% of progeny were unambiguously allocated to their putative parental pairs in the parentage analysis. The assignment success rate by the real data set was lower than that predicted by the simulations, with 94% of progeny assigned correctly. The discrepancies might be caused by a scoring error or allelic dropout caused by poor quality genomic DNA. Moreover, 69 progeny were selected randomly for the double-blind test, the result indicated that 95% of the progeny can be correctly assigned to their families. This study provided a microsatellite-based approach for parentage assignment in S. chuatsi, and that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.     

Enzyme electrodes based on organic metals

The parameters of enzyme electrodes based on organic metals are presented. Cytochrome b₂ (E.C. 1.1.2.3), glucose oxidase (E.C. 1.1.3.4), xanthine oxidase (E.C. 1.2.3.2) and peroxidase (E.C. 1.11.1.7) were used in electrodes sensitive to l-lactate, glucose, hypoxanthine and hydrogen peroxide. Electrocatalytic oxidation of NADH on organic metals and ethanol and acetaldehyde sensitive electrodes containing alcohol dehydrogenase (E.C. 1.1.1.1) were studied. Biocatalytic charge accumulation, the mechanism of electron exchange between the enzyme active centres and organic metals, and the future application of organic metals are discussed.     

Nylon shavings enzyme reactor for batch determination of urea

The design and performance of an enzyme reactor (enzyme electrode) which features (i) incorporating nylon shavings onto which an enzyme is covalently bonded, (ii) a flat-surface combination pH electrode for proton monitoring, and (iii) a body providing an injection port for sample injection and washing and stirring capabilities is described. The reactor configuration described here offers good diffusional and partition characteristics which result in relatively fast response, good stability, simplicity of operation, low sample and reagent consumption, and adaptability to flow systems. Application to the determination of urea in standards and physiological salt solutions is demonstrated by use of immobilized urease (EC 3.5.1.5).     

基于物质流分析的废纸回收利用体系生态成本研究

废弃物回收利用在一定程度上对缓解资源和环境危机起到积极的作用,已经成为可持续发展的重要举措,但生产过程中消耗的资源、能源,排放的污染物同样也会对自然环境产生负面影响。为解决此问题,以废纸回收利用体系为例,基于物质流分析方法构建了生态成本核算模型,为废弃物回收利用体系优化提供基础。在对生态成本相关研究归纳总结的基础上,定义了生态成本的概念,界定了生态成本的研究内容,并分析基于物质流核算生态成本的可行性。生态成本是对生态负荷的价值化,主要分为资源耗减成本、污染产生和环境保护成本以及生态环境损害成本3部分。污染产生和环境保护成本可以通过将总成本按比例分配给正、负产品的方式求得,资源耗减成本和环境损害成本借助LIME方法核算,总生态成本是回收利用体系内部各项生态成本的总和。生态成本核算是评价生态负荷的重要手段,在废纸回收利用体系物质流动图的基础上,分析各生产流程生态成本的构成情况。提出的生态成本核算模型不仅适用于废纸回收利用体系,其他废弃物也同样适用。通过生态成本的核算,寻找到对生态环境影响较大的工序、流程,为废弃物回收利用体系经济与环境的双赢提供理论与实践指导。     

The staining of fish otoliths for age determination

Attempts were made to find methods for the staining of fish otoliths which would give results comparable to those of the Christensen burning technique. A variety of different histological stains and otoliths of different species were experimented with. Otoliths of sole, turbot, brill and scad gave best results when sectioned and stained in acidified Neutral Red, whereas those of cod, hake, whiting, plaice and grey mullet showed up the annuli better when dyed in aqueous Aniline Blue or Toluidine Blue and then sectioned. Small, translucent otoliths such as those of pelagic species may be enhanced by staining in Eosin Y.