Efficacy of pulsed UV-light for the decontamination of Escherichia coli O157:H7 and Salmonella spp. on raspberries and strawberries

ABSTRACT:  Small fruits are increasingly being implicated in outbreaks of foodborne illness, and fresh produce is now the 2nd leading cause of foodborne illness in the United States. Conventional methods of decontamination are not effective, and there is a need to evaluate novel technologies. Pulsed ultraviolet (UV)-light is one such technology. In this study, pulsed UV-light was applied to strawberries and raspberries at varying UV doses and times. On raspberries, maximum reductions of Escherichia coli O157:H7 and Salmonella were 3.9 and 3.4 log₁₀ CFU/g at 72 and 59.2 J/cm², respectively. On the surfaces of strawberries, maximum reductions were 2.1 and 2.8 log₁₀ CFU/g at 25.7 and 34.2 J/cm², respectively. There was no observable damage to the fruits at these UV doses. The results obtained in this study indicate that pulsed UV-light has the potential to be used as a decontamination method for raspberries and strawberries.     

Analytical Methods for Virus Detection in Water and Food

Potential ways to address the issues that relate to the techniques for analyzing food and environmental samples for the presence of enteric viruses are discussed. It is not the authors?? remit to produce or recommend standard or reference methods but to address specific issues in the analytical procedures. Foods of primary importance are bivalve molluscs, particularly, oysters, clams, and mussels; salad crops such as lettuce, green onions and other greens; and soft fruits such as raspberries and strawberries. All types of water, not only drinking water but also recreational water (fresh, marine, and swimming pool), river water (irrigation water), raw and treated sewage are potential vehicles for virus transmission. Well over 100 different enteric viruses could be food or water contaminants; however, with few exceptions, most well-characterized foodborne or waterborne viral outbreaks are restricted to hepatitis A virus (HAV) and calicivirus, essentially norovirus (NoV). Target viruses for analytical methods include, in addition to NoV and HAV, hepatitis E virus (HEV), enteroviruses (e.g., poliovirus), adenovirus, rotavirus, astrovirus, and any other relevant virus likely to be transmitted by food or water. A survey of the currently available methods for detection of viruses in food and environmental matrices was conducted, gathering information on protocols for extraction of viruses from various matrices and on the various specific detection techniques for each virus type.     

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.     

Efficacy of gaseous chlorine dioxide as a sanitizer for killing Salmonella, yeasts, and molds on blueberries, strawberries, and raspberries

Gaseous chlorine dioxide (ClO2) was tested for its effectiveness in killing Salmonella, yeasts, and molds on blueberries, strawberries, and red raspberries. An inoculum (100 microl, 6.0 to 6.8 log CFU/g of fruit) that contained five serotypes of Salmonella enterica was deposited on the skin, calyx tissue, or stem scar tissue of blueberries, skin or stem scar tissue of strawberries, and skin of red raspberries, dried for 2 h at 22 degrees C, then held for 20 h at 4 degrees C and 2 h at 22 degrees C before treatment. Sachets that contained reactant chemicals were formulated to release gaseous ClO2 at concentrations of 4.1, 6.2, and 8.0 mg/ liter of air within treatment times of 30, 60, and 120 min, respectively, at 23 +/- 1 degrees C. Lethality of ClO2 to Salmonella, yeasts, and molds was measured when fruits were in an atmosphere that contained 75 to 90% relative humidity. Treatment with 8.0 mg/liter of ClO2 significantly (alpha = 0.05) reduced the population of Salmonella on blueberries by 2.4 to 3.7 log CFU/g. Lethality was higher to cells in inoculum placed on the skin compared with the stem scar tissue. Populations of Salmonella on strawberries treated with 8.0 mg/liter of ClO2 were reduced by 3.8 to 4.4 log CFU/g; a significant reduction of 1.5 log CFU/g of raspberries was achieved. Treatment with 4.1 to 8.0 mg/liter of ClO2 caused reductions in populations of yeast and molds on blueberries, strawberries, and raspberries of 1.4 to 2.5, 1.4 to 4.2, and 2.6 to 3.0 log CFU/g, respectively. Treatment with 4.1 mg/liter of ClO2 did not markedly affect the sensory quality of fruits stored for up to 10 days at 8 degrees C. Results indicate that gaseous ClO2 has promise as a sanitizer for small fruits.     

草莓中甲型肝炎病毒检测

目的：建立草莓中甲型肝炎病毒（hapatitis A virus,HAV）的有效富集方法及病毒RNA提取方法,用于草莓中HAV检测。方法：利用甲肝减毒疫苗对已知阴性的草莓样品进行人工污染,通过有效富集条件的优化及RNA提取后,采用实时荧光逆转录-聚合酶链式反应进行检测。结果：病毒富集选择Tris-甘氨酸-1 g/100 mL牛肉浸提物缓冲液洗脱、果胶酶30 U、氯仿-正丁醇为抑制剂去除剂、聚乙二醇沉淀、5 ℃孵育1 h等优化条件,检测灵敏度较高;最优RNA提取方法为美国ABI公司生产的RNA提取试剂盒。采用优化后的方法对草莓样品中HAV病毒的检测显示,该病毒粒子的检测灵敏度可以达31.36 CCID50/20 g样品。同时对50 份送检样品进行检测,结果均为阴性。结论：所建立的病毒富集方法和核酸提取方法更适合草莓样品中HAV的检测,灵敏度较高。     

Utilization of gaseous ozone for the decontamination of Escherichia coli O157:H7 and Salmonella on raspberries and strawberries

Each year in the United States, there are approximately 76 million foodborne illnesses, and fresh produce is the second most common vehicle for such illnesses. Before going to market, small fruits are not washed or treated in any manner to extend their shelf life. Washing alone is not a viable option, and the use of novel technologies should be investigated. One such technology is ozone treatment, which has been used with drinking water since the late 19th century. The efficacy of gaseous ozone for killing pathogens on strawberries and raspberries, which were used as a model for small fruits, was investigated in this study. Strawberries and raspberries were artificially contaminated with five strains of Escherichia coli O157:H7 and Salmonella enterica. Fruits were treated with four ozone treatments: (i) continuous ozone flow (5%, wt/wt) for 2, 4, 8, 16, 32, and 64 min; (ii) pressurized ozone (83 kPa) for 2, 4, 8, 16, 32, and 64 min; (iii) continuous ozone (64 min) followed by pressurized ozone (64 min); and (iv) vacuum followed by 64 min of pressurized ozone. Maximum reductions for both strawberries and raspberries were achieved with the third treatment scenario. On strawberries, 2.60- and 2.96-log reductions were achieved for Salmonella and E. coli O157:H7, respectively. For raspberries, 3.55- and 3.75-log reductions were achieved for Salmonella and E. coli O157:H7, respectively. These results indicate that gaseous ozone should be a useful treatment for decontamination of small fruits.     

A predictive microbiology approach for thermal inactivation of Hepatitis A virus in acidified berries

Hepatitis A virus (HAV) is a food-borne enteric virus responsible for outbreaks of hepatitis associated with consumption of raw vegetables. Soft fruits, such as red berries, exposed to faecal contamination are increasingly responsible for collective food-borne illnesses associated with HAV, when eaten raw or used in unprocessed foods. Heat is the most effective measure for the inactivation of HAV. Thermal treatments are used on fruits as a decontamination method, but they have to be adapted to product characteristics; indeed, factors such as sugar or pH may have an impact on the viral sensitivity to thermal treatments. A model was developed for the inactivation of HAV in red berries without supplemented sugar and with different pH values. Nonlinear inactivation curves in acidified raspberries were modelled using an integrated model, with a single equation nesting secondary models of temperature and pH in the primary model. Model predictions were then confronted to experimental results obtained in another laboratory on other berries with different pH values. Excellent predictions were obtained in most cases, while failed predictions provided safe results, with the model predicting higher residual virus titres than what was observed.     

Mould and yeast flora in fresh berries, grapes and citrus fruits

Fresh fruits are prone to fungal contamination in the field, during harvest, transport, marketing, and with the consumer. It is important to identify fungal contaminants in fresh fruits because some moulds can grow and produce mycotoxins on these commodities while certain yeasts and moulds can cause infections or allergies. In this study, 251 fresh fruit samples including several varieties of grapes, strawberries, blueberries, raspberries, blackberries, and various citrus fruits were surface-disinfected, incubated at room temperature for up to 14 days without supplemental media, and subsequently examined for mould and yeast growth. The level of contamination (percent of contaminated items/sample) varied depending on the type of fruit. All raspberry and blackberry samples were contaminated at levels ranging from 33% to 100%, whereas 95% of the blueberry samples supported mould growth at levels between 10% and 100% of the tested berries, and 97% of strawberry samples showed fungal growth on 33-100% of tested berries. The most common moulds isolated from these commodities were Botrytis cinerea, Rhizopus (in strawberries), Alternaria, Penicillium, Cladosporium and Fusarium followed by yeasts, Trichoderma and Aureobasidium. Thirty-five percent of the grape samples tested were contaminated and supported fungal growth; the levels of contamination ranged from 9% to 80%. The most common fungi spoiling grapes were Alternaria, B. cinerea and Cladosporium. Eighty-three percent of the citrus fruit samples showed fungal growth at levels ranging from 25% to 100% of tested fruits. The most common fungi in citrus fruits were Alternaria, Cladosporium, Penicillium, Fusarium and yeasts. Less common were Trichoderma, Geotrichum and Rhizopus.     

Analysis of volatile fruit components by headspace solid-phase microextraction

A method based on headspace solid-phase microextraction (HS-SPME) has been developed for the analysis of volatile compounds in fruits. Conditions of sampling have been tuned. Repeatability and recoveries obtained with this method have been determined by using a mixture of typical components of fruit aroma. Several fruits, such as raspberries, strawberries, blackberries, banana and mango, have been analysed by the proposed method. The patterns obtained include compounds typically found in fresh fruits and compounds formed during processing or storage. The results showed the use of the technique for fruit characterization and its potential as a routine method for analyzing changes in key flavour compounds during different fruit processing regimes.     

A new concept for the measurement of total volatile compounds of food

 The aim of our work was to develop a rapid and reliable method for the evaluation of the total volatile fraction of fruits (strawberries, raspberries, tomatoes and apples). Our method consists of trapping the volatile compounds of fruits on a solid-phase microextraction (SPME) fibre and determining the total amount of the adsorbed substances after desorption in a GC system, without performing any separation. The patterns obtained by using several types of SPME fibre permitted us to differentiate between the total volatile compounds present in the sample depending upon their chemical nature. Using strawberries as a model, we could show that our method: (1) leads to easily reproducible results; (2) allows differentiation between six varieties in a way which is consistent with an hedonic evaluation of these varieties; (3) shows the variation in total volatile compounds between individual fruits. The technique is rapid, practical, cheap, and promising for an objective evaluation of the volatile fraction of fruits.     

Efficacy of cold atmospheric plasma for inactivation of viruses on raspberries

In this study, the effectiveness of cold atmospheric plasma (CAP) in inactivating murine norovirus (MNV/human norovirus surrogate) and hepatitis A virus (HAV) on aerosol-inoculated dark red Willamette raspberries was explored. Pulsed positive corona discharge system fed by synthetic air was used for the production of CAP. Raspberries were treated for 1, 3, 5, 7, and 10 min at 25 W. Application of CAP enabled a 4 log₁₀ infectivity reduction in <5 min for MNV and approximately 10 min for HAV (from starting level of 6.91 and 7.84 log₁₀ PFU/mL, respectively). Viral genome copies reduction of 3.18 log₁₀ for MNV and 4.32 for HAV were found from starting level of 5.76 and 6.47 log₁₀ gc/μL, respectively. CAP treatment did not result in significant degradation of fruit color, an important quality attribute. The study demonstrated CAP as an efficient post-harvest decontamination method to reduce viral load in raspberries without significantly affecting its quality parameters.Industrial relevanceDue to the fast-processing paces required in the raspberry industry, it is difficult to assure the complete microbiological safety of this fruit. Cold atmospheric plasma is a practical, environmentally-friendly, non-thermal tool for the effective reduction of microbial pathogens. The model developed in this study demonstrated that CAP treatment of fresh raspberries not only inactivated hazardous enteric viruses in a short time (10 min) but also unaffected fruit color stability. The simplicity of described CAP design and low-cost inputs (air and electricity) enable the commercial application of inexpensive plasma chambers for continuous surface decontamination of large volumes of raspberries without bringing processing to a standstill.     

Fluctuations in the Levels of Antioxidant Compounds and Antioxidant Capacity of Ten Small Fruits During One Year of Frozen Storage

The fluctuations in antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl, 2,2´-azinobis (3-ethylbenzthiazoline-6-sulphonic acid), and ferric reducing/antioxidant power assays), total phenol, total flavonoid, and total anthocyanin contents of 10 small fruits (sweet cherries, sour cherries, strawberries, red currants, raspberries, blackberries, hawthorn, cornelian cherries, and red and white grapes) were monitored during storage at ?20°C. After one year in storage, all varieties except hawthorn and white grapes retained equal or slightly greater total phenol content in comparison to the initial values. The total flavonoid and total anthocyanin levels also remained stable or even increased after 12 months in all fruits exept hawthorn and strawberries. Red currants and raspberries exhibited optimal preservation of antioxidant capacity, while hawthorn showed a decrease in the antioxidant capacity during different time points in storage as well as at the end of storage. Analyzed fruits were classified into four main groups based on hierarchical cluster analysis.     

Chlorine disinfection of produce to inactivate hepatitis A virus and coliphage MS2

Disinfection of produce is principally used to inactivate spoilage microbes and may also reduce the risk of consumer exposure to enteric pathogens. However, the rate and extent of enteric virus inactivation by free chlorine on produce has not been adequately characterized. Experiments were performed to determine the kinetics of free chlorine inactivation of hepatitis A virus (HAV) and the indicator virus coliphage MS2 on strawberries (SBs), cherry tomatoes (CTs), and head lettuce (HL). The oxidant demand of these produce items also was determined. When produce items were exposed to approximately 20 parts per million (ppm) solution of free chlorine for 5-10 min, HAV and MS2 were inactivated by 90-99% and in some cases virus inactivation was >/=99%. Exposure of strawberries to approximately 200 ppm free chlorine resulted in more rapid and extensive inactivation of both viruses. The produce items tested in this study exhibited a demand for chlorine which varied by produce type, and chlorine residuals declined over time. These results demonstrate the potential for chlorine to reduce the levels of infectious viruses on different produce types, but adequate contact time and chlorine residual are required to achieve maximum virus inactivation. The difference in chlorine demand between SBs, CTs, and HL suggests that varying disinfection practices are needed for the wide variety of processed fruits and vegetables. The inactivation kinetics of MS2 and HAV were similar, suggesting that MS2 and perhaps other similar bacterial viruses may be used as process indicators and surrogates for determining the disinfection efficacy of produce in the laboratory or in actual practice.     

Efficacy of aqueous ozone for the decontamination of Escherichia coli O157:H7 and Salmonella on raspberries and strawberries

The efficacy of ozone as a water additive for washing raspberries and strawberries was investigated. Pathogen-inoculated fruits were treated with aqueous ozone concentrations of 1.7 to 8.9 mg/liter at 20 degrees C for 2 to 64 min, with an aqueous ozone concentration of 21 mg/liter at 4 degrees C for 64 min, or with water as a control. Maximum pathogen reductions on raspberries were 5.6 and 4.5 log CFU/g for Escherichia coli O157:H7 and Salmonella, respectively, at 4 degrees C, whereas reductions on strawberries were 2.9 and 3.3 log CFU/g for E. coli O157:H7 and Salmonella, respectively, at 20 degrees C after 64 min. Washing with water (sparging with air as control) resulted in reductions of approximately 1 log CFU/g. The results presented here indicate that aqueous ozone may be useful as a decontaminant for small fruits.     

Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qβ). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process.