Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning.

Four commercial kits, three based on sandwich ELISA techniques and one on latex agglutination were compared for the detection of staphylococcal enterotoxins in foods from outbreaks of food poisoning. Enterotoxin was detected in 14 of 18 foods with the Swiss SET-EIA and in 9 or 10 with the Unipath SET-RPLA and two ELISAs from Transia. The advantages and disadvantages of the four methods are discussed.     

Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder

The growth of S. aureus and the production of staphylococcal enterotoxin A (SEA) in skim milk concentrates stored at inappropriate temperatures in a recovery milk tank (tank for excess concentrated skim milk) used in the manufacture of skimmed milk powder were investigated. Also, it was estimated if a possible outbreak of food poisoning would occur if the contaminated skimmed milk powder was used in the manufacture of processed milk. Skim milk concentrates with milk solid content of 15, 25, and 35% were inoculated with S. aureus at 1-2 log CFU/ml and incubated at 15, 25, or 35 degrees C for 0 to 24 h with or without shaking. Bacterial growth and the level of SEA production were measured. At 35 degrees C with shaking, there was a significant difference (p<0.05) in one way layout analysis of variance, and it was demonstrated that the growth of S. aureus and SEA production could be milk solid content-dependent. Shaking accelerated the growth of S. aureus and SEA production at 35 degrees C. Generally, skim milk powder is produced by mixing a set percentage of skim milk concentrates (recovery milk) from the recovery milk tank into raw milk. If recovery milk contaminated with S. aureus at levels of 1-2 log CFU/ml is kept at 15 to 35 degrees C due to a power failure, it was estimated that processed milk consumption of 670-1200 ml, 420-1500 ml and 18-83 ml would trigger the onset of food poisoning symptoms when skim milk concentrates (recovery milk) are stored at 25 degrees C for 24 h, 35 degrees C for 10 h, and 35 degrees C for 24 h, respectively, during the production of the skim milk powder. Based on these consumption levels, it was concluded that, if recovery milk cannot be refrigerated and is stored at room temperature (25 to 35 degrees C), it must be used within 8 h and preferably within 6 h.     

Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk

This work evaluates a newly developed wavelength modulation-based SPR biosensor for the detection of staphylococcal enterotoxin B (SEB) in milk. Two modes of operation of the SPR biosensor are described: direct detection of SEB and sandwich assay. In the sandwich assay detection mode, secondary antibodies are bound to the already captured toxin to amplify sensor response. Samples including SEB in buffer and SEB in milk were analyzed in this work. The SPR biosensor has been shown to be capable of directly detecting concentrations of SEB in buffer as low as 5 ng/ml. In sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml for both buffer and milk samples. The reported wavelength modulation-based SPR sensor provides a generic platform which can be tailored for detection of various foodborne pathogens and agents for food analysis and testing.     

Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.     

A simple and rapid antibody-capture ELISA for the detection of staphylococcal enterotoxin A in food including a simple extraction step

A sensitive and precise ELISA for the detection of staphylococcal enterotoxin A in food has been developed, using specific IgG anti-toxin antibodies purified from immunized rabbits. an antibody capture format was found to be the most useful following cross-reaction and sensitivity studies. the ELISA was used to investigate concentration of staphylococcal enterotoxin A from food samples by polyethylene glycol dialysis. the procedure was shown to be inefficient and highly variable, as well as time-consuming. Direct analysis of aqueous food extracts without a concentration step was found to be possible. With an assay time of 140 min, the ELISA detection limit was 12.5 pg of toxin (corresponding to a sensitivity of 0.5 ngg^-1 of food sample.     

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.     

Detection of staphylococcal enterotoxin B in spiked food samples

Contamination of food with infectious agents, intentional or not, is a global concern with far-reaching economic and social impact. Staphylococcal enterotoxins are a major cause of food poisoning, but most methods for the identification of these agents in food require extensive pretreatment or concentration of the sample prior to analysis. The array biosensor was developed as a portable device for the simultaneous analysis of multiple complex samples for multiple targets with minimal sample preparation. In this study, we use an array biosensor to expand and improve on a staphylococcal enterotoxin B (SEB) assay with the ultimate intent of incorporating testing for SEB into a battery of sensitive and convenient assays for food safety validation. In addition to buffer studies, six different types of food samples, including beverages, homogenates of fruit and meat, and carcass washings, were spiked with SEB, incubated for at least 2 h to permit antigen sequestration, and assayed. For all samples, there were differences in fluorescence intensity, but 0.5 ng of SEB per ml could be detected in <20 min with little if any pretreatment and no sample preconcentration.     

Chemiluminescent imaging detection of staphylococcal enterotoxin C1 in milk and water samples

A sensitive, simple and rapid technique for high throughput simultaneous detection of staphylococcal enterotoxin C₁ (SEC₁) has been developed. The proposed method has the advantage of showing the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of luminol-based enhanced chemiluminescence (ECL) assay, and high throughput of chemiluminescence (CL) imaging assay. It was based on a standard sandwich immunoassay format; 96-well ELISA plates were used as solid phase material. A commercial high-sensitivity cooled CCD camera has been applied to image the weak CL. Under the optimum conditions, the increased CL intensity was proportional with the concentration of SEC₁ in the range of 8.0–125.0 ng ml⁻¹ and the detection limit was 0.5 ng ml⁻¹ (3σ). The relative standard deviation (RSD) for eight parallel measurements of 25.0 ng ml⁻¹ SEC₁ was 0.06. The proposed method has been successfully applied to the determination of SEC₁ in milk and water samples. The results obtained compared well with those by ELISA.     

Surface plasmon resonance analysis of staphylococcal enterotoxin B in food

Surface plasmon resonance (SPR) biosensors are electro-optical instruments used for analyzing real-time protein-protein interactions. This work evaluates an SPR biosensor (Biacore 3000) in the detection of staphylococcal enterotoxin B (SEB) in foods. A sandwich SPR immunosensor involving two antibodies was used. The capturing antibody, bound covalently to the surface of the biosensor chip, performs the initial binding of the antigen and a second antibody binds to the captured antigen. The second antibody makes antigen verification possible and amplifies the signal. Pure SEB as well as SEB in spiked foods (milk and meat) were detected with little interference from the food matrix. In the control experiments with uncontaminated food samples no significant signal was detected. The SPR biosensor assay detects SEB at approximately10 ng/ml rapidly, with initial binding within 2 min. The entire measurement cycle (including washing and chip regeneration) may take 5 min using one antibody or 8 min using two antibodies. These results suggest that the SPR biosensor may be a useful tool for real-time analysis of toxin in foods.     

Bacteriological investigation of an outbreak of Clostridium perfringens food poisoning caused by Japanese food without animal protein.

An outbreak of Clostridium perfringens food poisoning occurred in a senior citizen's home in Japan. Japanese food, spinach boiled with fried bean curd, was considered to be the causative food as a result of the detection of the C. perfringens enterotoxin gene by nested PCR. The number of enterotoxin-positive C. perfringens was enumerated as 4.3 x 10(5)/g in the causative food by the MPN method combined with nested PCR. By cultivation, enterotoxin-positive C. perfringens was isolated from all the fecal specimens of patients tested and the causative food. The isolates from patients were serotypable, heat-resistant and the majority produced enterotoxin, however most isolates from the causative food were nonserotypable, enterotoxin-negative and heat-sensitive.     

Microbial risk assessment of staphylococcal food poisoning in Korean kimbab

The objective of this study was to conduct a preliminary microbial risk assessment for Staphylococcus aureus in kimbab, and then propose appropriate guidelines for its preparation and storage. Kimbab is a ready-to-eat product, popular in Korea, made of various foods surrounded by rice and seaweed. Because it is usually prepared by hand and stored at room temperature, it can be contaminated with S. aureus. Data on the prevalence, concentration and growth of S. aureus in kimbab as well as typical ambient temperatures in Seoul were collected; probability distributions were then selected to describe these data. A Monte Carlo simulation model was created using @risk. When current Korean retail data on S. aureus prevalence and concentration were used as inputs, the simulation predicted that the S. aureus concentration would be between 4.0 and 4.9 log CFU/g after 1 h of storage at ambient temperature during the warmest part of the year (March through November). Conversely, by assuming a starting concentration of 1 cell/g, the simulation predicted that the S. aureus concentration would reach 4.4 log CFU/g after 5 h of ambient storage in May and 3.4 log CFU/g in October. Our results suggest that, given the current prevalence and concentration of S. aureus found in kimbab at retail outlets, the product should be consumed within 1 h of purchase. Our results also indicate that if Korean consumers wish to safely store kimbab for 5 h at ambient temperature, S. aureus concentration should not exceed 1 CFU/g at the time of preparation.     

乳中金黄色葡萄球菌及其肠毒素的PCR检测

介绍了PCR技术在检测乳中金黄色葡萄球菌及其肠毒素方面的研究进展，并对其今后的发展方向和前景进行了展望。     

Managing the risk of staphylococcal food poisoning from cream-filled baked goods to meet a food safety objective

The International Commission on Microbiological Specifications for Foods (ICMSF) has recently proposed a scheme for the management of microbial hazards for foods that involves the concept of food safety objectives (FSOs). FSOs are intended to specify the maximum levels of hazardous agents required to meet a given public health goal. This scheme offers flexibility for the food industry in terms of allowing the use of alternative but equivalent means for achieving a given FSO. This paper illustrates the application of the ICMSF model via the analysis of the microbiological hazard of Staphylococcus aureus in cream-filled baked goods. Cream-filled baked goods have a notorious history as vehicles for foodborne illness, particularly staphylococcal food poisoning. Although the numbers of cases reported in the United States and Europe have declined in recent years, staphylococcal food poisoning may be much more common than is recognized, particularly in other countries. The ICMSF principles for setting FSOs and the use of performance criteria, process criteria, and validation in relation to hazard analysis critical control point and good hygiene practice plans for managing S. aureus in cream-filled baked goods are described.     

Feasibility of a reference material for staphylococcal enterotoxin A.

A reference material for staphylococcal enterotoxin A (SEA), was produced by spray-drying the toxin in milk. With this procedure the SEA was distributed homogeneously in the material. For ease of handling the reference material was encased in gelatin capsules, each containing 405 ng of SEA. Simply dissolving the milk powder in distilled water resulted in a 100% recovery of the SEA present. The reference material would appear suitable for testing laboratory performance, comparison of detection methods and to validation of extraction procedures.     

中老年低乳糖低脂营养牛奶的研制

针对中老年人的生理特点及营养需求,合理补充钙铁锌硒及维生素等,适当调整脂肪含量,并通过添加乳糖酶将乳糖部分水解,缓解＂乳糖不耐受症＂,采用合理的杀菌方式,全面保持牛奶的营养成分.     

Significance of the presence of bovine milk beta-glucuronidase in mastitis detection

The presence of beta-glucuronidase enzyme in bovine milk was related both to the existence of major and minor pathogens and to somatic cell counts. The detection of this enzyme in whole milk was made possible by the use of p-nitrophenyl-beta-glucuronide as a substrate. This detection allowed us to determine abnormal udder secretions with a high degree of specificity and sensitivity. The particular method of enzyme determination was considered important for mastitis detection because beta-D-glucuronidase, the most significant enzyme in inflammatory processes, is released selectively. The relationship between enzyme, presence of pathogens, and somatic cell counts was established in 220 milk samples obtained at random from individual quarters of apparently healthy udders of cows from four local dairy farms (Santiago del Estero and Tucuman, Argentina). Four of these samples were from cows of recent parturition and two from cows with severe clinical mastitis. Only 17% of the milk samples were normal with somatic cell counts 500,000 cells/ml or less. This ratio is the usual one throughout the area, and the remaining 83% showed higher somatic cell counts. Taking the latter as 100%, the presence of beta-glucuronidase and the positive bacteriological analyses represented 76 and 74%, respectively.