Development of multiplex PCR assays to identify Escherichia coli pathogenic genes in food

Kyu-Heon Kim, Joon Il Cho, Chi-Yeun Cheung, Jong Mi Lim, Sooyeul Cho, Dae Hyun Cho, Chan Soon Kang, Do Hoon Kim

                                                                       

Abstract

In order to rapidly screen for the virulence factor that produces pathogenic Escherichia coli in food, we have developed multiplex polymerase chain reaction (PCR) assays. The multiplex PCR assays detect 4 pathogenic genes of enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). This allows for the generation of specific fragments 150, 179, 218, 401, 465, 584, and 881 bp for VT1, ST, LT, 16S rRNA, inV, VT2, and eaeA genes, respectively. The detection limit of 3 log CFU/mL for eaeA, LT, VT1, VT2, 4 log CFU/mL for inV, 6 log CFU/mL for ST by single PCR, while 5 log CFU/mL for VT1, VT2, 6 log CFU/mL for eaeA, LT, 7 log CFU/mL for ST, inV by multiplex PCR. This optimized detection method of pathogenic E. coli can be used as supportive data to revise the microbiological analytical manuals for the Korean Food Code.

source: visit here

Keywords :pathogenic gene ,Escherichia coli ,multiplex polymerase chain reaction (PCR) assay.

Rapid method for determination of β-carotene in foods using ultra high performance liquid chromatography

Jaeho Ha, You-Shin Shim, Hye-Young Seo, Hyun-Jin Nam, Masahito Ito, Hiroaki Nakagawa

                                                            

Abstract

The ultra high performance liquid chromatography (u-HPLC) method for determination of β-carotene in foods was validated in terms of precision, accuracy, and linearity. The u-HPLC separation was performed on a reversed column C₁₈ (particle size 2 μm, i.d. 2 mm, length 50 mm), followed by ultra violet (UV) detection at 450 nm. The recovery of β-carotene was more than 84.4% and the limit of detection and limit of quantitation of u-HPLC analysis were 0.28 and 0.85 μg/mL for β-carotene with butylated hydroxytoluene (BHT) and 0.62 and 1.89 μg/mL for β-carotene without BHT, respectively. The calibration graph for β-carotene was linear from 0.1 to 25.0 μg/mL for u-HPLC. The intra- and interday precisions (relative standard deviations) were <7.5 and <7.8%, respectively. Benefits of u-HPLC analysis of β-carotene in foods is reduction of the analysis time to approximately 1/4, saving the volume of solvent to approximately 1/15. It seems that u-HPLC can offer significant improvements in speed, sensitivity, and resolution compared with conventional HPLC, this bodes well for future applications.

source: visit here

Keywords :β-carotene ,rapid determination ,ultra high performance liquid chromatography.

Rapid detection of Vibrio parahaemolyticus strains and virulent factors by loop-mediated isothermal amplification assays

Xihong Zhao, Li Wang, Jin Chu, Yanyan Li, Yanmei Li, Zhenbo Xu, Lin Li, Mark E. Shirtliff, Xiaowei He, Yao Liu, Jihua Wang, Liansheng Yang

                                                              

Abstract

A loop-mediated isothermal amplification (LAMP) method for rapid detection of the foodborne Vibrio parahaemolyticus strains and related virulent factors had been developed and evaluated in this study. Six primers, including outer primers, inner primers, and loop primers, were specially designed for recognizing 8 distinct sequences on 3 target genes, which were tlh, tdh, and trh. The detection limits were found to be 100, 100 fg, and 1 pg DNA/tube for tlh, tdh, and trh, respectively. Application of LAMP assays were performed on 368 foodborne V. parahaemolyticus strains, the sensitivities of LAMP assays for the tlh, tdh, and trh were 100, 95.6, and 96.4%, and the negative predictive values (NPV) were 100, 84.7, and 93.1%, respectively; with a 100% specificity and positive predictive value (PPV) for all 3 target genes.

source: visit here

Keywords :loop-mediated isothermal amplification (LAMP),Vibrio parahaemolyticus ,rapid detection.

Inhibitory effects of blueberry root methanolic extract on degranulation in KU812F cells

Sun-Yup Shim, Hyeon-Jin Sun, Young-Hwan Song, Hyeung-Rak Kim, Dae-Seok Byun

                                                                       

Abstract

The effect of blueberry (Vaccinium angustifolium) root methanolic extract (BRM) on the A23187 plus phorbol myristate acetate (PMA)-induced degranulation in human basophilic KU812F cells was investigated. The total phenolic content (TPC) was 170±1.9 mg gallic acid equivalents (GAE)/g BRM. BRM reduced levels of histamine and β-hexosaminidase released from stimulated KU812F cells. Intracellular calcium concentration ([Ca²⁺]i) were determined by spectrofluorometric analysis using Fura 2-AM was found to be reduced by BRM with dose-dependent manner. Moreover, Western blot analysis revealed that protein kinase C (PKC) translocation was inhibited by BRM with dose-dependent manner. These results indicated that BRM inhibited histamine and β-hexosaminidase through the suppression of Ca²⁺ influx and PKC translocation. Therefore, we suggest that BRM is potent inhibitor of degranulation in mast cells and basophils, and may be useful in preventing the allergic reactions.

source: visit here

Keywords :Vaccinium angustifolium ,degranulation ,calcium influx ,protein kinase C.

Essential oil composition and antioxidant activities of alkanet (Alkanna tinctoriasubsp. tinctoria)

M. Sabih Ozer, Cengiz Sarikurkcu, Bektas Tepe, Sendil Can

                                                                               

Abstract

This study is designed to examine the chemical composition and in vitro antioxidant activity of the hydrodistillated essential oil and the various extracts of alkanet (Alkanna tinctoria subsp. tinctoria). Gas chromatography (GC) and GC-mass spectrometry (GC-MS) analysis of the essential oil were resulted in the determination of 27 different compounds, representing 93.32% of the total oil. The major compounds detected in the oil, were pulegone (22.27%), 1,8-cineole (13.03%), α-terpinyl acetate (6.87%), and isophytol (6.83%), respectively. Antioxidant activities of the samples were determined by 4 different test systems namely β-carotene/linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power, and chelating effect. In all systems, essential oil showed the weakest activity profile. On the other hand, ethyl acetate and water extracts exhibited excellent antioxidant activities. As well as the antioxidant activities of the extracts, they were evaluated in terms of their total phenolic and flavonoid contents. In parallel to the experiments, ethyl acetate and water extracts were found to be rich-in these phytochemicals.

source: visit here

Keywords :Alkanna tinctoria ,essential oil ,antioxidant activity ,1,1-diphenyl-2-picrylhydrazyl (DPPH).

Cytoprotective activity of lotus (Nelumbo nucifera Gaertner) leaf extracts on the mouse embryonic fibroblast cell

Ju Yeon Ha, Kyueun Lee, Jeongmi Park, Ahyoung Dong, Han-Seung Shin

                                                                          

Abstract

This study was conducted to evaluate the cytoprotective activity of lotus (Nelumbo nuciferaGaertner) leaf extract (LLE) on mouse embryonic fibroblast (MEF) cells. The 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging activities of LLE increased in a concentration dependent manner. The cells, damaged by oxidative stress, decreased their viability following increasing concentration of H₂O₂, but the co-treatment of n-butanol fraction of LLE and H₂O₂ resulted in an increase in cell growth, by about 25%, compared to the cells treated with H₂O₂. The n-butanol fraction of LLE inhibited the cytotoxicity induced by H₂O₂ in a concentration dependent manner. The oxidative damage to the cells, measured by apoptotic and necrotic cell accumulation, was similar with the addition of the n-butanol fraction of LLE to H₂O₂. Taken together, these results suggest that LLE inhibited the cytotoxicity which is induced by H₂O₂, and has a protective effect on MEF cell against oxidative stress.

source: visit here

Keywords :lotus leaf ,proximate composition ,antioxidant activity ,apoptosis ,cell viability.

Inhibitory activities of Rubi Fructus on digestive enzymes

Jung Sung Kim, Woo Jin Jeon, Hyun Ju You, Myung Soo Park, Geun Eog Ji

                                                              

Abstract

Control of postprandial glucose level is important in the management of diabetes. Thus, carbohydrate-hydrolyzing enzymes have been the targets to delay absorption of glucose. In this study, water extracts of Rubi Fructus (WERF) showed inhibition on both α-glucosidase from baker’s yeast and α-amylase from porcine pancreas. The IC₅₀ value of WERF on α-amylase was 59.4 μg/mL. The inhibitory activities of WERF were considerably decreased during incubation at 25, 37, and 45°C for 24 hr. For the assessment of the active components tannin fraction was separated from WERF using Sephadex LH-20. Whereas LH-unbound fraction showed minimal inhibition on α-amylase LH-bound fraction containing enriched tannins showed higher inhibition (IC₅₀ 8.9 μg/mL) than WERF (IC₅₀ 59.4 μg/mL). These results suggested that tannin enriched fraction of WERF contributed to the inhibitory activity of WERF on α-amylase.

source:visit here

Keywords :Rubi Fructus ,α-glucosidase inhibitor ,α-amylase inhibitor ,tannin.

Influence of fructo oligosaccharides and garlic on formation of heterocyclic amines in fried ground beef patties

Kyunghee Jung, Kyueun Lee, Jeongmi Park, Ahyoung Dong, Han-Seung Shin

                                                                                  

Abstract

The effects of fructooligosaccharide (FOS) and garlic on the formation of 15 heterocyclic amines (HCAs) were evaluated in fried beef patties. The HCAs were extracted from the fried meat samples and purified using a solid-phase extraction method and then analyzed on a liquid chromatography-mass spectrometry. Among the 15 HCAs, 3-amino-1, 4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido [1,2-a:3′,2′-d]imidazole (Glu-P-1), 2-aminodipyrido [1,2-a:3′,2′-d]imidazole (Glu-P-2), 9H-pyrido [3,4-b]indole (norharman), 1-methyl-9H-pyrido [3,4-b]indole (harman), 2-amino-9H-pyrido [2,3-b]indole (AαC), 2-amino-3,8 dimethylimidazo [4,5-f]-quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) were detected in all of the cooked beef patties. Analysis of variance revealed that the addition of 1 or 3 g of FOS significantly reduced the formation of total amino-carboline type HCAs in the cooked beef patties, and adding the 1 or 3 g of FOS to ground beef patties reduced levels of PhIP and MeIQx (amino-imidazo-azaarenes; AIAs) in the patties. When it is compared with the HCA formation in control, additions of minced garlic (5.0, 10.0, and 15.0 g) to the ground beef patties (100 g) reduced HCA formation in the range of 14 to 100%.

source: visit here

Keywords :heterocyclic amine ,liquid chromatographymass spectrometry ,fructooligosaccharides ,garlic ,beef patty.

Characteristics of Monascus sp. isolated from Monascus fermentation products

Ji Yeun Kim, Hae-Jin Kim, Jee-Hwan Oh, Inhyung Lee

                                                                                       

Abstract

To screen for the strains suitable for industrial applications, 15 Monacus sp. were isolated from various Monascus fermentation products. The isolated strains were belonging to Monascus pilosus, Monascus purpureus, Monascus albidulus, and Monascus femeus based on the ITS sequence and morphological characteristics. They produced globular and hyaline conidia, and red, orange, or hyaline ascomata except for M. fumeus, in which ascomata was not observed in malt extract media. Each strain produced monacolin K and citrinin at different levels on different media: 15.79–20.05 mg/L of monacolin K and 0–255 μg/L of citrinin in submerged culture of monacolin K producing media, 1.27–2.69 mg/L and about 507.4 μg/L in YES medium, 0–0.78 g/kg and 0–9.7 mg/kg inMonascus red rice products, suggesting that it is necessary to adopt the similar conditions to a specific application for screening as well. These various isolated strains would be great resources for industrial application to develop Monascus fermented functional products.

source: visit here

Keywords :Monascus sp. ,monacolin K ,citrinin ,Monascus fermentation product.

Quality characteristics of light pork burgers fortified with soy protein isolate

Adisak Akesowan

                                                                                

Abstract

Effect of soy protein isolate (SPI) (0.5 to 3%, w/w) on physical, chemical, sensory, and microstructure properties of light pork burgers containing added water incorporated with 0.7% mixed gum (konjac/gellan gum= 3:1) was carried out. Increasing of SPI levels resulted in significantly (p<0.05) higher cooking yield, lower reduction in diameter, and darker color of light pork burgers in relation to the control. Textural characteristics including cohesiveness, springiness, and chewiness significantly increased (p<0.05) with increasing of SPI up to 2% level; however, decreased these parameters were evident at 3% SPI used. Sensory results indicated that the 2% SPI light formulation showed significantly higher (p<0.05) scores for texture and juiciness than those of the control. The product was considered nutritious and provided the reduction of fat and total caloric content about 62.3 and 43.1% of the full-fat product, respectively.

source: visit here

Keywords :pork burger .low-fat meat product .soy protein isolate .konjac flour ,quality characteristics.

Optimum conditions for the enzymatic hydrolysis of citron waste juice using response surface methodology (RSM)

Ji Young Lim, Hyang-Sik Yoon, Kwang-Yup Kim, Ki-Sik Kim, Jae Goan Noh, In Gyu Song

                                                                         

Abstract

This study investigated the optimum conditions, including pH, temperature, incubation time, and enzyme concentration, for the enzymatic manufacture of citron waste juice by pectinase and cellulase using response surface methodology (RSM). A central composite design was used as an experimental design for the allocation of treatment combination. The highest yield was 71.1% at pH 5.00, 51.15°C, 53.74 min, and 0.44%, whereas the lowest turbidity was 0.20 at pH 4.87, 50.21°C, 49.99 min, and 0.44%. The highest hunter’s L (lightness), a (redness), and b- (yellowness) values were 47.39 (pH 4.86, 50.72°C, 51.60 min, and 0.44%), −1.34 (pH 5.48, 49.36°C, 46.29 min, and 0.21%), and 13.18 (pH 5.49, 50.05°C, 43.31 min, and 0.23%), respectively. The highest electron donating activity was 68.36% at pH 4.51, 50.62°C, 47.00 min, and 0.23%. The highest limonin and nomilin contents were 3.49 mg/100 g (pH 4.51, 50.30°C, 48.34 min, and 0.21%) and 1.56 mg/100 g (pH 4.59, 50.08°C, 66.07 min, and 0.30%).

source: visit here

Keywords :itron waste ,enzymatic treatment ,yield ,response surface methodology.

Alternative woods for aging distillates-an insight into their phenolic profiles and antioxidant activities

Roberto Rodríguez Madrera, Belén Suárez Valles, Yolanda Diñeiro García, Paula del Valle Argüelles, Anna Picinelli Lobo

                                                                                

Abstract

Work has commenced on the study and characterization of wood native species (oak, chestnut, cherry, alder, ash, and beech), employed to a minor extent in enology, for their application in the elaboration of distillates. To this end, furanic and low molecular weight phenol compounds, total phenolic content, and antioxidant activity in alcoholic extracts by maceration of wood chips at 2 toasting levels were studied. Significant differences between samples, technologically relevant, were due both to species and toasting time. Furthermore, extracts showed interesting antioxidant activities equivalent in some cases to than those reported for alcoholic beverages aged in wood.

source: visit here

Keywords :wood ,enology ,furanics ,phenolics ,total phenolic content ,antioxidant activity.