Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

Control of tobacco-specific nitrosamines by the Bacillus siamensis: Strain isolation, genome sequencing, mechanism analysis and genetic engineering.

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16 S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Study on barrier mechanism and application of silico-alkaline sol-modified bentonite cutoff wall materials to lead pollution in lead-zinc tailings pond.

In this paper, the barrier mechanism of silico-alkaline sol-modified bentonite as cutoff wall materials for lead ions in lead­zinc tailings ponds was investigated. Mechanical property tests, adsorption capacity tests, and permeability tests were conducted to assess the performance of the materials. The results indicated that the addition of silico-alkaline sol at a proportion of 20% by weight of bentonite improved the mechanical strength, anti-seepage ability, and adsorption capacity of the materials towards lead ions. The modification process of bentonite using silico-alkaline sol was confirmed through XED analysis. It was observed that silico-alkaline sol particles adsorbed onto the end face of montmorillonite crystal layer, altering its charge properties. This modification enhanced the adsorption capacity of bentonite towards metal cations. The interaction between the cutoff walls and lead ions was primarily governed by ion exchange. Through the utilization of CT and the finite element method (FEM), demonstrated the exiguity of connected flow lines in the vertical direction within the cutoff walls. Furthermore, column tests revealed that lead ions permeating through the cutoff walls gradually transformed into residuals and were immobilized within the wall. Visual Modflow analysis confirmed the effective application of the cutoff wall in remediating contaminated sites and the potential for practical implementation.

Isolation of starch and protein degrading strain Bacillus subtilis FYZ1-3 from tobacco waste and genomic analysis of its tolerance to nicotine and inhibition of fungal growth.

Aerobic fermentation is an effective technique for the large-scale processing of tobacco waste. However, the specificity of the structure and composition of tobacco-derived organic matter and the toxic alkaloids in the material make it currently difficult to directly use microbial agents. In this study, a functional strain FYZ1-3 was isolated and screened from thermophilic phase samples of tobacco waste composting. This strain could withstand temperatures as high as 80°C and grow normally at 0.6% nicotine content. Furthermore, it had a strong decomposition capacity of tobacco-derived starch and protein, with amylase activity of 122.3 U/mL and protease activity and 52.3 U/mL, respectively. To further understand the mechanism of the metabolic transformation of the target, whole genome sequencing was used and the secondary metabolite gene cluster was predicted. The inhibitory effect of the strain on common tobacco fungi was verified using the plate confrontation and agar column methods. The results showed that the strain FYZ1-3 was Bacillus subtilis, with a genome size of 4.17 Mb and GC content of 43.68%; 4,338 coding genes were predicted. The genome was annotated and analyzed using multiple databases to determine its ability to efficiently degrade starch proteins at the molecular level. Moreover, 14 functional genes related to nicotine metabolism were identified, primarily located on the distinct genomic island of FYZ1-3, giving a speculation for its nicotine tolerance capability on the molecular mechanism. By mining the secondary metabolite gene cluster prediction, we found potential synthetic bacteriocin, antimicrobial peptide, and other gene clusters on its chromosome, which may have certain antibacterial properties. Further experiments confirmed that the FYZ1-3 strain was a potent growth inhibitor of Penicillium chrysogenum, Aspergillus sydowii, A. fumigatus, and Talaromyces funiculosus. The creation and industrial use of the functional strains obtained in this study provide a theoretical basis for its industrial use, where it would be of great significance to improve the utilization rate of tobacco waste.

The lipid digestion behavior of oil-in-water emulsions stabilized by different particle-sized insoluble dietary fiber from citrus peel.

In this study, oil-in-water emulsions stabilized by insoluble dietary fibre from citrus peel (CIDF) exhibited an obviously delayed lipid digestion property through gastrointestinal tract (GIT) model. Our results suggested that the rate and extent of lipid digestion greatly relied on particle sizes and concentrations of CIDF, and the inhibition effect of lipolysis was markedly enhanced with decreasing particle sizes and increasing CIDF levels. Furthermore, compared with Tween80-stabilized emulsion, the maximum inhibition extent of lipolysis was 38.77% for CIDF400-stabilized one at 0.4 wt% concentration. Effects of CIDFs on lipid digestion was mainly due to the formation of protective layers around oil droplets, further blocking the entry of lipase to the internal lipids, and/or attributed to the increasing viscosity of emulsions caused by CIDFs, finally limiting the transportation of some substances in the simulated small intestine digestion. Our research would provide useful references for the application of CIDF-stabilized emulsions in low-calorie food.

Development of a Purity Certified Reference Material for Vinyl Acetate.

Vinyl acetate is a restricted substance in food products. The quantification of the organic impurities in vinyl acetate is a major problem due to its activity, instability, and volatility. In this paper, while using the mass balance method to determine the purity of vinyl acetate, an improved method was established for the determination of the content of three impurities in vinyl acetate reference material, and the GC-FID peak area normalization for vinyl acetate was calibrated. The three trace organic impurities were identified by gas chromatography tandem high-resolution mass spectrometry to be methyl acetate, ethyl acetate, and vinyl propionate. The content and relative correction factors for the three organic impurities were measured. The purity of vinyl acetate determined by the mass balance method was 99.90% with an expanded uncertainty of 0.30%, and the total content of organic impurities was 0.08% with a relative correction factor of 1.23%. The vinyl acetate reference material has been approved as a national certified reference material in China as GBW (E) 062710.

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens.

Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.

Broiler stunned state detection based on an improved fast region-based convolutional neural network algorithm.

An improved fast region-based convolutional neural network (RCNN) algorithm is proposed to improve the accuracy and efficiency of recognizing broilers in a stunned state. The algorithm recognizes 3 stunned state conditions: insufficiently stunned, moderately stunned, and excessively stunned. Image samples of stunned broilers were collected from a slaughter line using an image acquisition platform. According to the format of PASCAL VOC (pattern analysis, statistical modeling, and computational learning visual object classes) dataset, a dataset for each broiler stunned state condition was obtained using an annotation tool to mark the chicken head and wing area in the original image. A rotation and flip data augmentation method was used to enhance the effectiveness of the datasets. Based on the principle of a residual network, a multi-layer residual module (MRM) was constructed to facilitate more detailed feature extraction. A model was then developed (entitled here Faster-RCNN+MRMnet) and used to detect broiler stunned state conditions. When applied to a reinforcing dataset containing 27,828 images of chickens in a stunned state, the identification accuracy of the model was 98.06%. This was significantly higher than both the established back propagation neural network model (90.11%) and another Faster-RCNN model (96.86%). The proposed algorithm can complete the inspection of the stunned state of more than 40,000 broilers per hour. The approach can be used for online inspection applications to increase efficiency, reduce labor and cost, and yield significant benefits for poultry processing plants.

Inhibition of lignification of Zizania latifolia with radio frequency treatments during postharvest.

Zizania latifolia is easily lignified after harvesting, leading to the degradation of food quality and commercial value. Thus, this study evaluated the effect of radio frequency (RF) treatments on lignification inhibition of Zizania latifolia. The results showed that the lignin content of Zizania latifolia treated with RF decreased significantly compared with the control group. At the 7th day of storage, the phenylalanine ammonia lyase activity of the 90 W RF treatment group decreased by 52.9% compared with the initial value. The activities of peroxidase and polyphenol oxidase in the stems of Zizania latifolia were significantly (p < 0.05) decreased after RF treatments. Besides, a decrease in conversion rate of O2 - and H2O2 to downstream products was observed, indicating that the related invertases were inhibited by RF treatment. All of these showed that RF treatments contribute to inhibit or delay the lignification of Zizania latifolia, providing a better taste and quality for products.

A porous aromatic framework as a versatile fiber coating for solid-phase microextraction of polar and nonpolar aromatic organic compounds.

A porous aromatic framework (PAF) derived from triphenylamine (type PAF-41) was prepared and is shown to be a viable coating for fibrous solid-phase microextraction (SPME). PAF-41 can be easily synthesized and has a high surface area, a rich π-electron structure, and electron-rich nitrogen atoms in its framework. The PAF-41-coated fibrous SPME extractor was combined with a gas chromatographic separation and flame ionization detection. The method was applied to the quantitation of some aromatic organic compounds (AOCs), including polar amphetamine and methamphetamine and nonpolar ethylbenzene, o-, m- and p-xylenes, and styrene. The method was optimized after which a linear response is found for the 10-500 ng·mL-1 amphetamine and methamphetamine concentration ranges. The limits of detection are 1.0 and 0.5 ng·mL-1; and relative standard deviations for six repeated extractions with a single fiber are 5.3 and 6.7%. The method was applied for the determination of amphetamine and methamphetamine in spiked urine samples without any pretreatment except for dilution with water. The PAF-41 modified fiber also was applied to the extraction of styrene, xylenes and ethylbenzene. The enrichment capacities of the extractor for these AOCs were superior to those of commercial SPME extractors. Graphical abstract (a) Schemetic of the PAF-41-coated solid-phase microextraction (SPME) fiber. (b) Scanning electron microscope images of the PAF-41 fiber.

Study on controlling of cadmium pollution with fly ash-bentonite blocking wall.

A novel blocking wall was synthesized using fly ash and bentonite with strong adsorption capability. The optimized material ratio of the blocking wall was determined by penetration tests, shear tests and adsorption tests. The morphologies and stabilities of blocking wall samples with optimized material ratio were investigated by scanning electron microscopy (SEM), energy spectrum analysis (EDS), and erosion tests, the adsorption capability of blocking wall with optimized material ratio was investigated by adsorption kinetic and isotherm tests, the migration and penetration time of Cd pollutants in blocking wall with optimized material ratio were investigated by penetration tests and numerical simulations. Results indicated that the optimized fly ash/bentonite ratio (in mass) of blocking wall was 5:1. The permeability coefficient was 1.11 × 10-8 m s-1 and the maximum adsorption rate was 98.38%. Meanwhile, Cd2+ adsorbed on the blocking wall homogeneously; the alkali corrosion resistance of the blocking wall was higher than its acid corrosion resistance, and its resistance to organic pollutants and solutions with high Cd2+ concentrations were high. The adsorption process can be described by the Quasi-first-order kinetics and the Freundlich equation. Specifically, the overall adsorption efficiency was simultaneously affected by liquid membrane diffusion and particle diffusion and the adsorption process is an endothermic one dominated by physical adsorption. Additionally, Cd2+ penetration through the wall was dominated by molecular diffusion. The variation of permeability coefficient was inversely proportional to the initial concentration of Cd2+. The penetration of Cd2+ at initial concentration of 500 mg L-1 through the blocking wall needs 40 years.

Adsorption of fluoride from aqueous solution by fly ash cenospheres modified with paper mill lime mud: Experimental and modeling.

Fluoride removal from aqueous solution by adsorption using fly ash cenospheres (FAC) modified with paper mill lime mud (LM) as composite adsorbent had been investigated. The characterization of FAC and composite adsorbent were analyzed by Scanning electron spectroscope (SEM), Energy dispersive spectrometer (EDS), Brunauer emmett teller (BET) and Fourier transform infrared (FTIR), which demonstrated that the porous structure of composite adsorbent was obtained after surface modification. Adsorption of fluoride on modified fly ash cenospheres was fitted with pseudo-second-order model and Langmuir model. Response surface methodology (RSM) was employed to investigate the effects of F- concentration, pH, adsorbent dosage and temperature on the removal efficiency. Analysis of variance (ANOVA) was used to test the adequacy of the mathematical models. The Nonelectrostatic model of modified fly ash cenospheres adsorbing fluoride was built through the Generalized composite method, indicating that two inner-spherical complexes, ≡SF and ≡SOHF-, were formed in the adsorption process by means of the ligand exchange and surface complexation. Optimization of the adsorption conditions enabled the realization of the practical needs for fluoride contaminated water.

Characteristics and management modes of domestic waste in rural areas of developing countries: a case study of China.

A huge accumulation of domestic waste has caused serious environmental contamination in rural areas of developing countries (RADIC). The characteristics and management of domestic waste are carefully discussed, based on field surveys and a literature review. The results indicate that the generation in most of RADIC is less than the median of 0.521 kg day-1 per capita in China, and much smaller than in rural areas of developed countries (RADEC). Organic waste and inert waste with an accumulative mass percentage of 72.31% are dominant components of domestic waste in the rural areas of China. There are trends of increasing amounts of kitchen waste, paper/cardboard, and plastic/rubber and a decreasing trend of ash waste. The RADIC composition of domestic waste had a high content of organic waste and a low content of recyclable waste compared to the RADEC. Domestic waste has good compressibility and a light bulk density ranging from 40 to 650 kg m-3. The moisture, ash, combustible, and calorific values of domestic waste were 53.31%, 18.03%, 28.67%, and 5368 kJ kg-1, respectively. The domestic waste has an abundance of nutrients including organic matter (39.05%), nitrogen (1.02%), phosphorus (0.50%), and potassium (1.42%). In RADIC, domestic waste can be used as an agricultural manure only after it has been collected and sorted for the potential risk of heavy metal accumulation. Based on these characteristics of domestic waste and the different situations of rural areas, four waste management modes including centralized treatment, decentralized treatment, group treatment, and mobile treatment are designed and discussed.

A water resistant solid-phase microextraction fiber with high selectivity prepared by a metal organic framework with perfluorinated pores.

A novel solid-phase microextraction (SPME) fiber was fabricated by the construction of fluorous metal organic frameworks (FMOF) and a polyimide (PI) composite strategy. As an auxiliary material, PI was expected to help FMOF particles form well-knit film on the surface of stainless steel wire and reinforce the coating, and FMOF was expected to afford a special structure to absorb, extract and enrich. Furthermore, it was explored for the headspace SPME (HS-SPME) of six volatile aromatic compounds (VACs) from water samples followed by gas chromatographic (GC) separation with flame ionization detection. Under the optimized conditions, the fiber afforded wide linear ranges (1-1000 µgL(-1)), low detection limits (0.15-0.9 µgL(-1)) and acceptable repeatability (<4.6%) and reproducibility (<7.3%). The FMOF@PI coated fiber not only offered large enhancement factors for benzene (1227) but also exhibited high extraction selectivity for benzene to other benzene homologues, hydrocarbons and phenols; for example, the extraction ratio of benzene to toluene, n-hexane and phenol could be as high as 10.2, 64.1 and 32.3, respectively. Moreover, the FMOF@PI-coated fiber afforded good thermal, water and organic solvent stabilities, and a long lifetime (over 200 times). The developed method was successfully applied to the determination of VACs in wastewater samples.

[Determination of three isothiazolinone biocides in water-borne adhesives by high performance liquid chromatography].

A rapid high performance liquid chromatography (HPLC) method was developed for the quantitative analysis of three isothiazolinone biocides (2-methyl-4-isothiazolin-3-one (MI), 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 1, 2-benzylisothiazolin-3-one (BIT)) in water-borne adhesives. The sample was extracted with methanol-water (1:1, v/v), and purified by centrifugation and filtration. The isothiazolones were separated on a C18 column with methanol-water as mobile phases under gradient elution and detected with a diode array detector (DAD). The pretreatment factors such as extraction solvent, extraction method, dilution ratio, extraction time were optimized. Under the optimized conditions, the targets had good linearities (r2H > or = 0.9992) in the range of 0.25-10.0 mg/L. The recoveries were between 92% and 103% with the relative standard deviations not more than 4%. The limits of detection (LODs) were between 0.43 mg/kg and 1.14 mg/kg. The limits of quantification (LOQs) were between 1.44 mg/kg and 3.81 mg/kg. The results showed that the method can achieve the purpose of quantitative detection. The analyses of real samples verified the reliability of this method.

[A dual-band algorithm to detect contaminants with low visibility on chicken carcass surface].

A novel dual-band algorithm for detecting contaminants with low visibility on chicken carcass surface based on hyperspectral image was proposed. Firstly, The 675 nm band image, in which the identity of the intensity within ROI (Region of Interest) is the best and the spectrum difference between ROI and the edge of the ROI is the biggest, was chosen from the hyperspectral data for binarization and the mask was extracted by using region growing on the biggest connected area. Then the "and" operation between the mask and the 400 nm band image with the largest discriminability of contaminants was carried out. The max ROI which can self adapt according to the position and shape of the chicken carcass was obtained. Finally, the labeling method was used to recognize if there are contaminations within the segmented ROI. The results showed that through the proposed method, the max ROIs which could self adapt to the position and shape of the chicken carcass were extracted and the average size of the ROI was bigger than 176% compared to that by existing methods. The average correct identification rate of contaminations such as blood, bile and feces was 81.6%.

Extraction, preliminary characterization and antioxidant activity of Se-enriched Maitake polysaccharide.

A Se-enriched Grifola frondosa polysaccharide (Se-GP) was obtained from G. frondosa enriched with Se by spraying an Na2SeO3 solution during fruit body growth using a Box-Behnken design and compared to G. frondosa polysaccharide (GP) for preliminary characterization and determination of the antioxidant activity. Under optimal conditions, polysaccharide yields and both the Se-GP and GP contents do not differ; however, the Se content of Se-GP (17.52 µg/g) was 48.7 times that of GP. Three homogenous Se-GPs (Se-GP11, Se-GP22 and Se-GP33) or GPs (GP11, GP22 and GP33) were obtained via DEAE-52 and Sephacryl S-400 purification. Their molecular weight and polysaccharide content of these compounds were not obviously different, whereas the Se content of Se-GP11, Se-GP22 and Se-GP33 was 9.41, 6.59 and 16.25 times that of GP11, GP22 and GP33, respectively. The antioxidant activity of Se-GP for the DPPH, ABTS and hydroxyl radicals was higher than that of GP and was highest for the hydroxyl radical.

Multiple headspace solid-phase microextraction after matrix modification for avoiding matrix effect in the determination of ethyl carbamate in bread.

This study presents the potential of multiple headspace solid-phase microextraction (multiple HS-SPME) for the quantification of analytes in solid samples. Multiple HS-SPME shares the same advantages as SPME. It also enables a complete recovery of the target compound and therefore the matrix effect, which commonly appears in SPME-based analysis, is avoided. A method based on multiple HS-SPME for the determination of the toxic contaminant ethyl carbamate (EC) in bread samples has been developed and validated, using gas chromatography with flame ionization detector. A novel polyethylene glycol/hydroxy-terminated silicone oil fiber was prepared for the first time and subsequently used instead of commercial ones because of its high extraction ability and good operational stability. An important problem still remained in multiple HS-SPME of EC in fresh bread samples. The adsorption of EC by water in the samples caused low transport of analyte to the headspace, which made multiple HS-SPME invalidated. Mixing with anhydrous sodium sulphate, the sensitivity of the method was improved and the problem was solved. The proposed method showed satisfactory linearity (0.15-1500 µg g(-1)), precision (1.6%, n=5) and limit of detection (0.041 µg g(-1)). Good recoveries, from 92.5 to 103.4%, were observed at three spiking levels. The method was applied to 14 bread samples. The multiple HS-SPME technique offers several advantages including reducing the manipulation time and cost, and avoiding analyte losses, especially in the analysis of a large number of samples in different matrices.

[Multiple headspace solid-phase microextraction after matrix modification for the determination of ethyl carbamate in alcoholic beverages using gas chromatography].

A method based on multiple headspace solid-phase microextraction (MHS-SPME) combined with gas chromatography for determining ethyl carbamate (EC) in various alcoholic beverages was established. A novel polyethylene glycol/hydroxy-terminated silicone oil fiber was used instead of commercial ones because of its high extraction ability. Anhydrous sodium sulphate was added to modify the matrix and the extraction efficiency of EC was greatly improved. The optimum conditions for MHS-SPME were as follows: extraction time, 10 min; extraction temperature, 35 degrees C; Na2SO4 addition, 4.0 mg Na2SO4 per microliter of sample; volume of sample, 20 microL. The proposed method was linear in the range of 0.04 to 100 mg/L with a correlation coefficient of 0.999 7. The limit of detection was 34 microg/L and the repeatability of six replicates was 2.19%. The method was used to determine EC in various alcoholic beverages. The concentrations obtained were compared with those obtained by standard addition method and no statistically significant differences were observed. The application of MHS-SPME avoids the matrix effect, which commonly appears in SPME-based analysis. The results indicate that MHS-SPME has a great potential for EC quantification of complex samples due to its simplicity, sensitivity, reliability, ease of operation and environmental protection, especially for the analysis of a large number of samples in different matrices.

Multiple headspace solid-phase microextraction of ethyl carbamate from different alcoholic beverages employing drying agent based matrix modification.

Multiple headspace solid-phase microextraction (MHS-SPME) combined with gas chromatography-nitrogen phosphorus detector is proposed to determine the toxic contaminant ethyl carbamate (EC) in various alcoholic beverages after matrix modification. The remarkable feature of this method is that matrix effect, which commonly appears in SPME-based analysis, is avoided by determining the total amount of the analyte in the sample. To increase the sensitivity of the method, a novel polyethylene glycol/hydroxy-terminated silicone oil fiber was developed by sol-gel technique and applied for the analysis. Owing to the high polarity and hydrophilia of EC, an important problem still remains because the adsorption by sample matrix causes low transport of EC to the headspace and thus invalidates MHS-SPME for quantification. Mixing with anhydrous sodium sulphate, the sensitivity of the method can be improved. A Taguchi's L(16) (4(5)) orthogonal array design was employed to evaluate potentially significant factors and screen the optimum conditions for MHS-SPME of EC. Under the optimized conditions, limit of detection of 0.034 mg L(-1) was obtained. Relative standard deviation of replicate samples (n=6) was 2.19%. The proposed method was linear in the range of 0.04-100 mg L(-1), and the coefficient of determination was 0.9997. The method was used to determine EC in various alcoholic beverages. The concentrations obtained were compared with those obtained by standard addition method and no statistically significant differences were observed.