Characterization of α-carrageenan solution behavior by field-flow fractionation and multiangle light scattering

The salt mediated molecular conformation change of alpha (α)-carrageenan was studied in 0.1 M solutions of NaCl, NaI, and KCl. Asymmetric Field-Flow Fractionation with multiangle laser light scattering (AFFF/MALLS) detection was used to determine the average molecular weight, radius of gyration, and hydrodynamic radius which were in turn used to calculate the molecular density. In the presence of 0.1 M NaCl, an inert salt that does not promote gelation, α-carrageenan has a denser structure compared to κ-carrageenan of a similar molecular weight. A distinct and dramatic increase in the molecular weight (factor of 2) was observed for α-carrageenan in 0.1 M NaI compared to 0.1 M NaCl. This combined with only a slight change in the radius of gyration, suggests intermolecular interaction to a more compact structure (e.g., coaxial helices). A similar increase in molecular weight is observed in 0.1 M KCl, accompanied with an approximate 50% increase in the radius of gyration as well as an increase in polydispersity. This may also be attributed to intermolecular interaction with helix formation (coaxial or lateral) or may be due to K⁺ cations interacting with naturally occurring residual ι-carrageenan in the sample. As previously reported for other carrageenans the random coil to helix transition of α-carrageenan appears to be stabilized by K⁺ cation or I⁻ anion in an aqueous environment.     

Enterotoxigenic Profiling of Emetic Toxin‐ and Enterotoxin‐Producing Bacillus cereus,Isolated from Food,Environmental, and Clinical Samples by Multiplex PCR

Bacillus cereus comprises the largest group of endospore‐forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin‐ (8 patterns) and enterotoxin‐producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.     

Purification of Polyphenoloxidase from the Purple‐fleshed Potato (Solanum tuberosum Jasim) and its Secondary Structure

ABSTRACT: Polyphenoloxidase (PPO) was purified from purple‐fleshed potatoes (Solanum tuberosum Jasim) using membrane concentration, ammonium sulfate fractionation, Resource Q ion exchange chromatography, and Sephacryl S‐200 HR gel permeation chromatography. PPO was purified 78‐fold from a crude extract. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results showed that the purified enzyme has a major subunit molecular weight of 40 kDa. To elucidate the secondary structure of the purified PPO, circular dichroism (CD) was performed. The CD spectrum of the purified enzyme showed that PPO contains 35% α‐helix, 30% β‐turn, and 35% random coil structure.     

Diverse Profiles of N‐acyl Homoserine l‐Lactones,Biofilm, Virulence Genes and Integrons in Food‐Borne Aeromonas Isolates

Aeromonas are regarded as opportunistic as well as primary pathogens of humans and fish, and are associated with gastroenteritis and septicemia in humans. Production of N‐acyl‐homoserine lactone (AHL) signal molecules and biofilm was determined in 22 Aeromonas isolates, from different food products in India, using thin‐layer chromatography (TLC) analysis and microtiter‐plate assay, respectively. Overall, highly heterogeneous patterns of AHL production were observed, with the production of N‐butanoyl homoserine lactone (C4‐HSL) and N‐hexanoyl homoserine lactone (C6‐HSL) by the majority (81.8%) of Aeromonas food isolates. Moreover, putative N‐pentanoyl homoserine lactone (C5‐HSL), N‐heptanoyl homoserine lactone (C7‐HSL), and N‐octanoyl homoserine lactone (C8‐HSL) were produced by 72.7%, 27.3%, and 9.1% of isolates, respectively. This is the 1st report of production of C7‐HSL by Aeromonas species. Aeromonas food isolates were highly variable in their biofilm forming abilities with majority of them as weak biofilm producers in 2 different media, TSB and M9 minimal medium supplemented with 0.4% glucose. The genes encoding for putative virulence factors, glycerophospholipid cholesterol acyltransferase (gcat), heat‐labile cytotonic enterotoxin (alt), heat‐stable cytotonic enterotoxin (ast), serine protease (ser), polar flagella (fla), and lateral flagella (lafA) were present in 95.5%, 59.1%, 22.7%, 81.8%, 77.3%, and 22.7% of the strains, respectively. Class 1 integrons (100 to 3000 bp) were found in 68.2% of food isolates; whereas, 50% isolates contained class 2 integrons (150 to 1600 bp). This study provides a baseline data on the diversity of AHLs, biofilm forming ability and presence of virulence genes and integrons in Aeromonas food isolates from India.     

再生丝素蛋白固定过氧化氢酶传感器的研究

从莴苣种子中提取过氧化氢粗酶,利用再生丝素蛋白在甲醇作用下,其分子结构由可溶性randomcoil向不溶性β-sheet发生转变,从而将过氧化氢粗酶固定在的β-sheet所特有的分子氢键中,制得过氧化氢传感器。该传感器在pH=6.5的KH2PO4-NaOH工作介质中,过氧化氢浓度在5.0×10-5～3.0×10-3mol/L范围内呈良好线性关系。     

Evolution of oxidative and structural characteristics of proteins,especially lipid transfer protein 1 (LTP1) in beer during forced-ageing

The evolution of oxidative and structural characteristics of proteins, especially lipid transfer protein 1 (LTP1), in beer during forced-ageing was examined. The oxidative characteristics of beer and proteins were evaluated by DPPH radical scavenging activity and ABTS radical cation scavenging activity. Results showed that the levels of proteins, thiols, LTP1 and antioxidant activity decreased gradually. This was accompanied by the degradation of macromolecular proteins in beer during forced-ageing. Results from circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), surface hydrophobicity (S₀) and ζ-potential further indicated that the secondary and tertiary structure of LTP1 changed drastically during forced-ageing, with the reduction of the S₀, α-helix and β-sheet contents and the increase in negative ζ-potential and random coil. Thus, the proteins, especially LTP1, might play important roles in maintaining oxidative stability of beer.     

Factors Affecting Enterotoxin Production by Thermonuclease Positive Streptococcus faecium IF-100 Isolated from an Infant Food

Among the different media tested for optimal production of enterotoxin by Streptococcus faecium IF-100, trypticase soy broth was the best for maximal enterotixin production by Streptococcus faecium IF-100 at 37°C after 8 hr incubation in agitated cultures. The pH optimum was 8.0. Although addition of 1% casamino acid enhanced enterotoxin production, yeast extract did not affect enterotoxin production appreciably. Dextrose, at 0.5%, was inhibitory towards enterotoxin production. Production of enterotoxin was also inhibited by addition of acridine orange, suggesting possible involvement of some extra chromsomal factor in the production of this metabolite in S. faecium IF-100. A supplemented medium for maximum enterotoxin production is described.     

STUDIES ON OCCURRENCE AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS FROM SELECT MEATS

A study was undertaken to assess the prevalence of Clostridium perfringens in meat and to characterize the isolates obtained in the study for virulence factors. A total of 211 meat samples of different animals (70 each of buffalo and goat and 71 of poultry) were screened and the highest occurrence of C. perfringens was observed in goat (91.4%) followed by poultry (70.4%) and buffalo (65.7%). Among the 116 isolates (buffalo‐32, goat‐37 and poultry‐45) of C. perfringens screened for the presence of enterotoxin gene by PCR, 9.3, 32.4 and 15.5% isolates of buffalo, goat and poultry, respectively, were found to possess enterotoxin gene. Screening of 15 enterotoxin gene possessing isolates for verocytotoxicity revealed that 12 isolates exhibited cytopathic effect while 3 isolates did not show any cytopathic effect in spite of the presence of enterotoxin gene. A total of 115 C. perfringens isolates were screened for other virulence markers, i.e., lecithinase and hemolysin. The results revealed that the majority of the isolates expressed these activities. Antibiogram studies of C. perfringens isolates using 16 antibiotics displayed multidrug resistance. The isolates showed resistance to streptomycin, ceftazidime, colistin sulfate, cephalothin, ampicillin and gentamicin. Whereas 100% sensitivity to ciprofloxacin, ofloxacin and nitrofurantoin was seen, moderate sensitivity was observed with tetracycline and sulfatriad.     

Amino acid,structure and antioxidant properties of Haematococcus pluvialis protein hydrolysates produced by different proteases

The impact of different protease hydrolysis on the amino acid, structure and antioxidant properties of H. pluvialis protein (HP) was investigated. Results showed that the hydrolysate obtained by Alcalase exhibited the highest degree of hydrolysis (20.59%) and peptide yield (92.64%). The essential amino acid, hydrophobic, sulphur and aromatic amino acid contents of enzyme hydrolysates were significantly higher than HP (P < 0.05). FTIR spectra showed that the β-sheet proportion of HP hydrolysates were higher compared with HP, the proportion of random coil structure was lower. The α-helix content of the hydrolysate obtained by Alcalase was the highest, while the turn proportion was the lowest. The Trypsin derived hydrolysate presented the best DPPH and ABTS scavenging ability, and ferric reducing antioxidant power than other HPHs. These results suggested that HP hydrolysates have a great potential as natural functional ingredients in food manufacture.     

Occurrence and characterization of enterotoxigenic Staphylococcus aureus isolated from minimally processed vegetables and sprouts in Korea

Staphylococcus aureus has long been recognized as an important pathogen in food-borne disease in the world. Minimally processed vegetables and sprouts are often contaminated with enterotoxigenic strains of this bacterium. This paper reports the results of a 3-year survey (2006–2008) on the occurrence of S. aureus in minimally processed vegetables and sprouts. Of 345 examined samples, 40 samples (11.6%) were contaminated with S. aureus. A total of 25 enterotoxigenic S. aureus strains were biotyped and their resistance to antibiotics was examined. Most isolated strains produced Staphylococcal enterotoxin A (SEA) (n=23) followed by Staphylococcal enterotoxin I (SEI) and Staphylococcal enterotoxin G (SEG) and mainly belonged to the human biotype (88%). At least 96.1% of the analyzed strains showed antibiotic resistance properties, while 56% of the analyzed strains exhibited multiple antibiotic resistance to the antibiotics tested. Two of the analyzed strains were resistant to methicillin. Moreover, a strain which had multi-resistance to 6 antibiotics was found. The results indicate that enterotoxigenic, antibioticresistant strains of S. aureus are widely proliferated in minimally processed vegetables and sprouts.     

Application of Fourier transform infrared spectroscopy to legume seed flour analysis

The secondary structure of legume (Phaseolus vulgaris L. and Lens culinaris L.) proteins was investigated by studying the amide I infrared absorption band in whole seed flours, before and after dry heating and autoclaving thermal treatments. The analysis procedure, set up on 7S and different model proteins, shows that the content of β-sheet structures in lentil is higher than in common bean (47% vs. 32%). The dry heating does not appreciably affect secondary structures in lentil, while it causes a reduction of β-sheets (to 13%), an increase of aggregates, and the appearance of random coil structures in common bean. The autoclaving treatment produces high amounts of aggregates in both legumes. However, in lentil, random coil structures are lower than in common bean and some β-sheet structures are still detectable. These results indicate that multimeric heat-induced complexes of legume proteins have a high stability because of the high content in β-sheet structures, in particular in lentil, which may adversely affect protein utilization.     

Staphylococcus aureus Growth and Enterotoxin Production in Mushrooms

An enterotoxin A (SEA) producing strain of Staphylococcus aureus was inoculated onto mushrooms and incubated under simulated pre-and post-canning conditions. S. aureus grew and produced SEA in mushrooms incubated at 25°C in 10% to 20% NaCl. Growth and SEA production occurred when mushrooms were stored in plastic bags at 37°C, but not at 25°C. Mushrooms heat-processed at 121.1°C supported S. aureus growth and SEA production. No SEA was recovered from mushrooms inoculated with 1 or 10 ng SEA/g and heated at 121.1°C for 4.5 min. At higher inoculated SEA levels (100 and 1,000 ng/g), SEA was detectable after 10 min of heating. Staphylococcal enterotoxin could be present in canned mushrooms as a result of pre-, but more likely post-processing contamination.     

Lipase Mediated Synthesis of Low Molecular Weight Flavor Esters

Screening 27 commercial lipases showed that enzymes from Candida cylindracea, Pseudomonas fluorescens and Mucor miehei (immobilized) promoted synthesis of selected low molecular weight esters in nonaqueous systems. Maximum production after 24 hr incubation was obtained with substrate concentrations of 0.05 mol/L for isopentyl acetate, 0.2 mol/L for ethyl butyrate and 0.3 mol/L for isopentyl butyrate. Yield of butyl butryate was almost 100% at acid substrate greater than 0.2 mol/L. Substrate inhibition was observed with P. fluorescens lipase but not with C. cylindracea or M. miehei lipases, up to 1 mol/L. Hexane, octane and decane could be used as reaction media except for ethyl butyrate synthesis where hexane was the medium of choice. Poor synthesis was achieved when methylene chloride was used.     

Formation and stabilization of emulsion with A1, A2 and B β-casein genetic variants

The milk protein β-casein (β-CN) is a flexible, approximately random coil protein with a molecular mass of ∼24 kDa. It contains distinct charged and hydrophobic, non-charged regions in the primary structure. The strongly amphiphilic nature of β-casein causes it to concentrate at an interface, which is a prerequisite to form and stabilize emulsions. The emulsifying (particle-size distribution, turbidity measurement) and structural (surface coverage, CD) properties of β-casein genetic variants were studied at pH 6.7 and I = 75 mM (representative pH value for milk). β-Casein B variant produced bigger emulsion droplets than the A¹ and A² variant, having better emulsion-stabilizing properties than A² and A¹ variants. Significant differences were observed in the surface coverage of teflon droplets made with β-casein genetic variants. A relationship between the surface load and emulsion-stabilizing properties was found. An increased content of secondary structure, especially for β-CN B and A¹ variants, was found after adsorption onto the hydrophobic surface.     

Characterization of Thermal Denaturation Structure and Morphology of Soy Glycinin by FTIR and SEM

The conformation and microstructure of soy glycinin were investigated by Fourier transform infrared spectroscopy and scanning electron microscopy after dry heating and autoclaving thermal treatments. The changes in frequency and signal intensity of infrared bands revealed the thermal denaturation on the solid-state structure of glycinin. The Fourier transform infrared spectral changes were subsequently assessed using the second derivative spectroscopy in the amide I region (1700–1600 cm^?1). The bands at 1618 cm^?1 and 1682 cm^?1 were considered to reflect the formation of intermolecular and intramolecular aggregates (A1 and A2), and the contents of β -sheet indicated the degree of denaturation. In autoclaved samples, the contents of the glycinin α -helix, turn, random coil, and A2 significantly increased (P < 0.05), while the contents of the glycinin β -sheet and A1 significantly decreased (P < 0.05). The dry heating slightly affected the secondary structures of glycinin. The scanning electron microscopy results showed that the autoclaving treatment glycinin had denser and more uniform gel with homogeneous pores and heterogeneous granular structure, and the dry heating glycinin had crumbled multi-layered sheet like structure. The data suggested that the conformations of the autoclaved glycinins had more changes than dry thermally treated glycinins. While both dry thermally treated and autoclaved treated samples showed a high content in β -sheet structures which may adversely affect protein utilization.     

Isolation and Characterization of Spore‐Forming Bacilli (SFB) from Shepherd's Purse (Capsella bursa‐pastoris)

Shepherd's purse (Capsella bursa‐pastoris), native to Europe, is commonly consumed fresh and sometimes inadequately washed before consumption in Korea. The objective of this study was to characterize isolates of spore‐forming bacilli (SFB) in samples of fresh Shepherd's purse. Three genera were identified: Bacillus (9 species), Paenibacillus (3 species), and Brevibacillus (1 species). None of the genes of the hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) complexes, or of the emetic toxin, was detected in the 25 SFB isolates, except for 2 Bacillus pseudomycoides isolates, where all 3 genes of the HBL enterotoxin complex were detected. There were significant sequence variations between the 2 species (Bacillus cereus and B. pseudomycoides) in the 3 genes of the HBL enterotoxin complex. These findings may provide insights into the diverse characteristics of the B. pseudomycoides HBL enterotoxin complex. Antibiotic resistance was assessed using 8 antibiotics. Among the 25 SFB isolates, 11 showed resistance to antibiotics, of which 5 were multiresistant. Assessment of the spoilage potential showed that all 25 SFB isolates could produce enzymes that can cause spoilage of foods. In conclusion, our findings may serve as integrative information for food research and industrial sectors.     

Structural characterization of globulin from common buckwheat (Fagopyrum esculentum Moench) using circular dichroism and Raman spectroscopy

Raman and far-UV circular dichroism (CD) spectroscopy was used to study the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under the influence of various buffer environments and heat treatments. Secondary structural analysis of BWG by CD spectroscopy yielded 15.0% α-helical, 25.8% β-sheet, 28.9% β-turn and 30.3% random coil contents. Raman spectrum also showed β-sheets as the major secondary structure in native BWG. Chaotropic salts caused band shifts and intensity changes in Raman amide III vibration, indicating transitions from β-sheet to disordered structure following the lyotropic series of anions. Extreme pHs and several protein structure perturbants led to changes in CD and Raman spectral characteristics, demonstrating protein unfolding and denaturation. Increasing heating time at 100 °C induced the appearance of anti-parallel β-sheet (1235–1237 cm⁻¹) and caused a progressive increase in random coil content, suggesting protein denaturation and aggregation. Both non-covalent and covalent interactions play important roles in stabilizing the conformation of BWG.     

Growth and enterotoxin production of Staphylococcus aureus in Iranian ultra‐filtered white cheese

This study aimed to investigate the growth and enterotoxin production of Staphylococcus aureus during the ripening and storage of Iranian ultra‐filtered white cheese (IUFWC). According to the results, the addition of starter culture had significant inhibitory effects on the growth and enterotoxin production of S. aureus. Moreover, prolong survival and enterotoxin production were observed in the samples with the highest inoculum (5 log CFU/g) of S. aureus. It was concluded that proper activity of the starter culture together with the refrigeration storage of IUFWC was the key element in inhibiting the growth and enterotoxin production of S. aureus.     

Distribution of newly described enterotoxin-like genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from ready-to-eat foods in Korea

To investigate the distribution of staphylococcal enterotoxin-like (SEl) genes in Staphylococcus aureus from food, a total of 154 S. aureus isolates from ready-to-eat (RTE) foods in Korea were analyzed by mutiplex PCR for the detection of the following 9 staphylococcal enterotoxin-like genes; sek, sel, sem, sen, seo, sep, seq, ser, and seu. Seventy-nine isolates (51.3%) were found to have at least one of SEl genes. The major SEl genes were sek, sem, sen, and seq. Other SEl genes found in the isolates were seo (21 isolates, 13.6%), seu (12 isolates, 7.8%), sep (8 isolates, 5.2%), sel (7 isolates, 4.5%), and ser (2 isolates, 1.3%). Most (95%) of the isolates with staphylococcal enterotoxin (SE) genes were also carried SEl genes. The genes seg, sei, sem, and sen were all detected in the same isolates. Ninety-seven % of isolates with seg+sei+sem+sen also contained seo or seu. Ninety-four % of isolates with sea+seh were found to coexist with sek+seq. The toxic shock syndrome toxin gene, tst-1, was found in all isolates with egc-2, including seg, sei, sem, sen, and seu. The coexistence of SE and SEl genes in S. aureus isolates from RTE foods can be explained by the mobile genetic elements. Because of the mobile genetic element, SE and SEl genes of S. aureus in foods may be transferable to nontoxigenic S. aureus and other food pathogens. Additional studies must be conducted to prevent spread of pathogenic genes such as enterotoxin gene.     

Heat-stable proteases from psychrotrophic pseudomonads: secondary structure and heat stability

Circular dichroism (CD) spectral analysis of a purified protease of Pseudomonas fluorescens T16 was carried out at different temperatures to determine the relationship between its secondary structure and its heat-stability. The protease protein was found to be 33% β-structure and 67% random coil. The protein lacked α-helical structure. Unfolding of the enzyme molecule was increased from 25°C, with maximum unfolding at 45°C. The enzyme exhibited maximum inactivation (low temperature inactivation [LTI] phenomenon) at temperatures between 45 and 55°C. At higher temperatures (60–95°C) the protease appears to undergo an additional conformational change to a more ordered stable structure. Metal ions such as Ca²⁺ appeared to be involved in structural stabilization and are required for protection against heat inactivation. The comparisons of amino acid composition, sequence homology, hydrophobicity index and polarities of heat-stable proteases were made to predict their heat-stabilities.