Maximum Recovery Diluent Research Articles

Modelling the effect of UV light at different wavelengths and treatment combinations on the inactivation of Campylobacter jejuni

Application of novel decontamination strategies such as Ultraviolet (UV) irradiation are required to mitigate the risks associated with Campylobacter jejuni in food. This study evaluated the use of a light-emitting diode (LED) based technology to inactivate C. jejuni NCTC 11168 in Maximum Recovery Diluent (MRD) at wavelengths of 280, 300 and 365 nm and combinations. To assess the survival curves, two linear (Log linear (LL) and Linear and Shoulder) and two non-linear models (Weibull and Double Weibull) were fitted. UV exposures showed different antimicrobial effects where a combination of 280/300 nm was the most effective treatment with a 4Dt value of 5 s observed in a bacterial suspension of 5 log CFU/mL. Moreover, the LL model was the most robust model to describe the inactivation kinetics of Campylobacter when exposed to UV and therefore, modelling tools could be applied to predict the efficiency of UV light in a model solution.Industrial relevance: Light-based technologies like UV light are identified in the literature as potential alternatives to assure the decontamination of surfaces, liquids and solid food. However, some of these techniques require further investigation. The present study evaluated the use of a LED system and effect of combined wavelengths in the inactivation of Campylobacter through predictive modelling. This technique was observed to predict and explain kinetics of inactivation of Campylobacter and could be key in the scaling-up process of UV light at industrial level.

Characterization of a Novel Atmospheric Air Cold Plasma System for Treatment of Packaged Biomaterials

<abstract><title><italic>Abstract.</italic></title> The technology of atmospheric plasma (ionized gas), widely used in material processing, offers one of the most significant breakthroughs in food and agri-material processing. This technology allows for the generation of bactericidal molecules very efficiently with low power requirements. Non-thermal atmospheric plasma has been used to effectively decontaminate surfaces, but it has received limited investigation for in-package decontamination. This study demonstrates the potential for in-package plasma treatment of model liquids within sealed packages. The advantage of in-package cold plasma treatment is that the bactericidal molecules are generated and contained in the package, allowing extended exposure to bacteria while reverting back to the original package gas within 24 h storage. This study treated liquid food surrogates containing bacteria and oxidation sensitive dye (methylene blue) inside sealed packages. The surrogates were placed in 96-well plates and were packaged in air and exposed directly and indirectly to the plasma field in a 2.2 cm high × 23 cm × 31 cm sealed polypropylene container for up to 5 min. Treatments were carried out using a prototype dielectric barrier discharge (DBD) operating at a voltage of 40 kV and frequency of 50 Hz. The UV-Vis emission spectra of the plasma revealed emission bands for nitrogen and oxygen species, including strong emission lines for excited states of the atomic species O, O⁺, N, and N⁺. The results from the evaluation of methylene blue suggest that direct exposure to the plasma ionization field produced a greater oxidative effect compared to indirect exposure. For 5 min treatment, direct exposure of methylene blue resulted in a 90% reduction in absorbance, and indirect exposure of methylene blue resulted in a 75% reduction in absorbance. These reductions may result from conversion of ozone into hydroxyl radicals, which reduces the methylene blue from dark blue color to clear. This process is irreversible. Additionally, bacterial studies examining treatment of E. coli ATCC 25922 suspended in maximum recovery diluent in 96-well plates found a 7 log₁₀ reduction after 50 s treatment and 24 h storage for both direct and indirect plasma exposure. Ozone concentrations measured immediately after 5 min of ionization were approximately 1600 ppm. The goal of this research is to maximize bacterial reductions and minimize quality (oxidative) loss for biomaterials inside a sealed package.

Atmospheric cold plasma inactivation of Escherichia coli in liquid media inside a sealed package

The main objective of this study was to determine the inactivation efficacy of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) generated inside a sealed package for Escherichia coli ATCC 25922. A plasma discharge was generated between two circular aluminium electrodes at 40 kV. E. coli suspensions (10(7) CFU ml(-1)) in either maximum recovery diluent (MRD) or phosphate buffered saline (PBS) were treated in a 96-well microtitre plate inside a sealed package. The effects of treatment time, post-treatment storage time, either direct or indirect samples exposure to the plasma discharge and suspension media were studied. Regardless of the media tested, 20 s of direct and 45 s of indirect plasma treatment resulted in complete bacterial inactivation (7 log CFU ml(-1)). At the lower plasma treatment times (10-30 s) investigated, the effects of suspension media and mode of exposure on the inactivation efficacy were evident. The inactivation efficacy was also influenced by the post-treatment storage time. It was demonstrated that the novel DBD-ACP can inactivate high concentrations of E. coli suspended in liquids within sealed packages in seconds. A key advantage of this in-package nonthermal novel disinfection approach is the elimination of post-processing contamination.

Comparison of Different Sampling Strategies and Laboratory Methods for the Detection of C. jejuni and C. coli from Broiler Flocks at Primary Production

The objective of the study was to evaluate the performance of different combinations of sample type, transport medium and culture methods for the recovery of Campylobacter jejuni and C. coli from broiler flocks at primary production. Boot swabs moistened with one of four different transport media [maximum recovery diluent (n=120), Exeter broth (EX) (n=120), buffered peptone water (n=120) and modified semi-solid Cary-Blair (n=120)], caecal samples (n=40) and faecal samples (n=120) from 40 broiler flocks were compared and sensitivity estimates obtained using a Bayesian model. Samples were cultured onto mCCDA before and after enrichment in EX and incubated microaerobically at 41.5°C. Campylobacter suspect colonies were identified to the species level by multiplex PCR. Results from the Bayesian model indicated that boot swabs after enrichment had higher sensitivity (90-94%) than caecal contents before or after enrichment (84% and 89%, respectively) and faecal samples after enrichment (82%) for the detection of Campylobacter spp., although these differences were not statistically significant. Enrichment significantly increased the sensitivity of boot swab and caecal samples for detection of Campylobacter spp. and C. jejuni, respectively. However, the enrichment of caecal samples resulted in a significant decrease in the sensitivity of these samples for detection of C. coli. There was much greater variation in the sensitivity estimates of the methods for detecting C. coli than for C. jejuni, and the ranking of methods was different between the two species. Boot swabs gave the best sensitivity values for detection of C. jejuni, and enrichment culture of faecal samples was the most sensitive method for detection of C. coli.

Efficacy of UV light treatment for the microbiological decontamination of chicken, associated packaging, and contact surfaces.

UV light was investigated for the decontamination of raw chicken, associated packaging, and contact surfaces. The UV susceptibilities of a number of Campylobacter isolates (seven Campylobacter jejuni isolates and three Campylobacter coli isolates), Escherichia coli ATCC 25922, and Salmonella enterica serovar Enteritidis ATCC 10376 in liquid media were also investigated. From an initial level of 7 log CFU/ml, no viable Campylobacter cells were detected following exposure to the most intense UV dose (0.192 J/cm(2)) in liquid media (skim milk subjected to ultrahigh-temperature treatment and diluted 1:4 with maximum recovery diluent). Maximum reductions of 4.8 and 6.2 log CFU/ml were achieved for E. coli and serovar Enteritidis, respectively, in liquid media. Considerable differences in susceptibilities were found between the Campylobacter isolates examined, with variations of up to 4 log CFU/ml being observed. UV treatment of raw chicken fillet (0.192 J/cm(2)) reduced C. jejuni, E. coli, serovar Enteritidis, total viable counts, and Enterobacteriaceae by 0.76, 0.98, 1.34, 1.76, and 1.29 log CFU/g, respectively. Following UV treatment of packaging and surface materials, reductions of up to 3.97, 4.50, and 4.20 log CFU/cm(2) were obtained for C. jejuni, E. coli, and serovar Enteritidis, respectively (P < 0.05). Overall, the color of UV-treated chicken was not significantly affected (P ≥ 0.05). The findings of this study indicate that Campylobacter is susceptible to UV technology and that differences in sensitivities exist between investigated isolates. Overall, UV could be used for improving the microbiological quality of raw chicken and for decontaminating associated packaging and surface materials.

Effectiveness of High Intensity Light Pulses (HILP) treatments for the control of Escherichia coli and Listeria innocua in apple juice, orange juice and milk

High Intensity Light Pulses (HILP) represent an emerging processing technology which uses short (100–400 μs) light pulses (200–1100 nm) for product decontamination. In this study, model and real foods of differing transparencies (maximum recovery diluent (MRD), apple and orange juices and milk) were exposed to HILP in a batch system for 0, 2, 4 or 8 s at a frequency of 3 Hz. After treatment, inactivation of Escherichia coli or Listeria innocua was evaluated in pre-inoculated samples. Sensory and other quality attributes (colour, pH, Brix, titratable acidity, non-enzymatic browning, total phenols and antioxidant capacity (TEAC)) were assessed in apple juice. Microbial kill decreased with decreasing transparency of the medium. In apple juice (the most transparent beverage) E. coli decreased by 2.65 and 4.5 after exposure times of 2 or 4 s, respectively. No cell recovery was observed after 48 h storage at 4 °C. No significant differences were observed in quality parameters, excepting TEAC and flavour score, where 8 s exposure caused a significant decrease ( p < 0.05). Based on these results, HILP with short exposure times could represent a potential alternative to thermal processing to eliminate undesirable microorganisms, while maintaining product quality, in transparent fruit juices.

Reconstitution conditions for dried probiotic powders represent a critical step in determining cell viability

Resuscitation of dried cultures represents a critical control point in obtaining active and effective probiotic strains. This study examined the effects of various rehydration conditions on the viability of Bifidobacterium longum NCC3001 and Lactobacillus johnsonii La1. Reconstitution conditions for these strains were optimized using a multivariate experimental design approach. Furthermore, using flow cytometry, the cell integrity was followed during reconstitution. By adjusting the pH, availability of a metabolizable sugar, reconstitution duration, powder matrix and ratio of powder to reconstitution solution, the recovery of Bif. longum NCC3001 and Lact. johnsonii La1 following reconstitution was increased eight- and two-fold, respectively, over standard reconstitution in maximum recovery diluent. It was shown that pH had a significant effect on the recovery of Bif. longum NCC3001 and Lact. johnsonii La1. The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. The maximum recovery of 11.7 (10)log CFU g(-1) Bif. longum NCC3001 was achieved at 30-min reconstitution at pH 8, in the presence of 2% L-arabinose and a ratio of 1:100 of powder to diluent. Lact. johnsonii La1 showed highest recovery (9.3 (10)log CFU g(-1)) after reconstitution, when mixed with maltodextrin at pH 4. To achieve accurate viable probiotic numbers from dried probiotic cultures, the reconstitution conditions should be optimized for the strain used.

Survival of Escherichia coli O157:H7 and non-pathogenic E. coli on irradiated and non-irradiated beef surfaces

This study examined changes in numbers of pathogenic (PEC) and non-pathogenic (NPEC) Escherichia coli during storage at 10 °C on the surfaces of irradiated (IR) and non-irradiated (NIR) meat pieces excised from the neck, brisket and rump of beef carcasses and in Brain Heart Infusion Broth (BHI) and Maximum Recovery Diluent (MRD). On irradiated meat pieces, there were significant differences between mean PEC and NPEC counts at all sites. Differences in counts were also observed between IR and NIR surfaces and among the three meat sites for both E. coli types. These differences occurred only on IR samples, suggesting that the irradiation associated reductions in normal beef surface flora influenced survival of both E. coli types. PEC and NPEC counts increased during storage in BHI, but only NPEC counts increased in MRD. The results of this study highlight the impact of meat surface type and the presence/absence of the normal beef carcass surface flora on E. coli survival and/or growth during meat storage. Such previously unreported effects, and their precise mechanisms, have direct implications in the development and application of accurate models for the prediction of the safety and shelf life of stored meat.

Radio frequency heating of comminuted meats - considerations in relation to microbial challenge studies

Bacillus cereus and Clostridium perfringens vegetative cell and spore cocktails in maximum recovery diluent (MRD) were inoculated into pork luncheon meat to challenge a previously developed radio frequency (RF) cooking protocol. After RF cooking and cooling microbial enumeration results showed a reduction in B. cereus vegetative cell and spores of 5.4 and 1.8 log 10 cfu g −1, respectively while the corresponding reduction for C. perfringens vegetative cells and spores were 6.8 and 4.1 log 10 cfu g −1, respectively. However, post cooking temperatures within the product were lower than anticipated. Subsequent analysis of product thermal and dielectric properties indicated that MRD addition and compositional variations within meat ingredients altered thermal and dielectric properties which in turn contributed to reduced and less uniform temperatures. The study shows that for RF microbial challenge studies, adjustment of product formulation prior to MRD addition is critical to ensure a similar composition to the normal product and a true picture of microbial inactivation.

Survival of Listeria innocua on hot and cold beef carcass surfaces

This study aimed to determine the survival and growth of Listeria innocua on hot and cold beef carcass surfaces. Four sites, the neck, outside round, brisket and foreshank/brisket, were inoculated with L. innocua (i) immediately after dressing while hot and (ii) when cold after chilling. After inoculation, all carcasses were stored at 4 degrees C for 72 h. Survival of L. innocua on cold surfaces declined during storage and was less than on hot carcasses at all times. Data on the survival of L. innocua in broth (maximum recovery diluent) indicated that counts could not be compared with those on carcasses, in particular on cold carcasses. The results indicate that L. innocua survives on hot carcass surfaces during chilling, but declines over time on cold surfaces. The decrease in L. innocua counts on cold surfaces may be related to a synergy between the combined stresses of low available water (a(w)) and low temperature. This study is the first to determine the effect of chilling on the survival and growth of Listeria on beef carcass surfaces. The information can potentially be used to determine the survival and growth of the pathogen, L. monocytogenes on beef surfaces.

Survival of surface ripening cultures during storage and monitoring their development on cheese

To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.

The comparative effect of novel Pelargonium essential oils and their corresponding hydrosols as antimicrobial agents in a model food system.

Essential oils and their corresponding hydrosols, obtained after distillation of various scented Pelargonium (Geraniaceae) leaves were assessed for their antimicrobial activity in a model food system. Both the essential oils and hydrosols were used at 1000 ppm in broccoli soup, previously inoculated with Enterobacter aerogenes (at 10(5) cfu g(-1)) and Staphylococcus aureus (at 10(4) cfu g(-1)). The results showed a complete inhibition of S. aureus in the broccoli soup by the essential oils of 'Sweet Mimosa', 'Mabel Grey', P. graveolens, 'Atomic Snowflake', 'Royal Oak', 'Attar of Roses' and a lesser effect by 'Chocolate Peppermint' and 'Clorinda'; the hydrosols, however, had a potentiating effect on the bacterial population in the food. Both extracts showed a complete inhibition of S. aureus in the Maximum Recovery Diluent (MRD). Antibacterial activity against E. aerogenes in the broccoli soup was generally very much reduced: only the essential oil of 'Mabel Grey' showed complete inhibition and virtually no reductions in colonies were seen with the other essential oils; the hydrosols again caused an increase in bacterial colonies. All the essential oils, bar Chocolate Peppermint showed complete inhibition of E. aerogenes in MRD, but the hydrosols showed no effect. The results strongly suggest that the residual hydrosols from distillation of these plant essential oils have no potential as antibacterial agents in foods, in contrast to most of the essential oils, which show potential against some micro-organisms, but only in some food systems. The problem of food component interference and its possible management is discussed.

Use of Steam Condensing at Subatmospheric Pressures To Reduce Escherichia coli O157:H7 Numbers on Bovine Hide

This study used a laboratory-scale apparatus to apply subatmospheric steam to bovine hide pieces inoculated with Escherichia coli O157:H7 in maximum recovery diluent (MRD) and in high–liquid content and low–liquid content fecal suspensions (HLC fecal and LLC fecal, respectively). The survival of the organism in fecal clods, which were stored for 24 days in a desiccated state, was assessed. Inoculated fecal clods were also treated with subatmospheric steam. Steam treatment at 80 ± 2°C for 20 s reduced E. coli O157:H7 concentrations on hide inoculated to initial concentrations of approximately 7 log10 CFU/g by 5.46 (MRD inoculum), 4.17 (HLC fecal inoculum), and 5.99 (LLC fecal inoculum) log10 CFU/g. The reductions achieved in samples inoculated with LLC feces were larger than in samples inoculated with HLC feces (P < 0.05). Treatment at 80 ± 2°C for 10 s resulted in significantly smaller reductions (P < 0.05) on hide pieces of 2.54 (MRD), 1.94 (HLC fecal), and 2.15 (LLC fecal) log10 CFU/g. There were no significant differences among the reductions observed in all inoculum types in samples treated for 10 s. E. coli O157:H7 inoculated in fecal clods to 7.78 log10 CFU/g and stored at 4 or 15°C survived for at least 24 days. Steam treatment (20 s) of 3-day-old clods reduced surviving E. coli O157:H7 numbers from 4.20 log10 CFU/g to below the limit of detection of the assay used (1.20 log10 CFU/g). This study shows that steam condensing at or below 80 ± 2°C can reduce E. coli O157:H7 when present on bovine hide, reducing the risk of cross contamination to the carcass during slaughter and dressing.

Campylobacter spp. in the kitchen: spread and persistence.

Campylobacter spp., principally C. jejuni, continue to be major bacterial enteropathogens, causing over 50 000 confirmed cases of infection in England and Wales each year. Most infections are sporadic and occur at home. In the farm to fork' approach to food safety, the consumer has an important role in protecting themselves and their colleagues. It is important, however, that this role is not overemphasized. For example, contaminated poultry meat is an important vehicle for infection with Campylobacter spp. and studies have shown that carcases can carry over 10 8 Campylobacter cells. The bacteria can also be isolated from the outer wrapping of chickens purchased from retail outlets. It can be difficult for consumers to control the spread of these bacteria. The preparation of meals using raw chicken will result in the widespread dissemination of Campylobacter spp. and many hand- and chicken-contact sites will become contaminated. It was thought that these bacteria were unable to persist in the kitchen but this may have been a manifestation of the recovery techniques used. If more sensitive methods are applied, Campylobacter spp. can be recovered from surfaces 24 h after contamination and from dry materials such as hand and tea towels. These and other issues will be discussed.

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy.

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.

High voltage electrical stimulation: its effect on microbial contamination of lamb carcases in a commercial abattoir

This study was designed to establish the effect of an abattoir's commercial high voltage electrical stimulator on lamb carcase microbial contamination, including TVC, coliform and Campylobacter colony counts. Over eight visits, batches of six lamb carcases were sampled using the wet and dry swab technique both before and after passage through a high voltage electrical stimulator. On each visit three of the carcases were sent through the electrical stimulator while it was operational (the treatment group) and three carcases were sent through when the electrical stimulator had its power turned off (the control group). The swabs were placed into maximum recovery diluent, transported ‘on-ice’ and held at 0°C overnight before dilutions and plating out the following day. Too few coliform colonies were recovered for a meaningful analysis and no campylobacters were recovered. The total viable counts of the electrically stimulated and the control carcases were found not to be significantly different.

Osmotic stress on dilution of acid injuredEscherichia coliO157:H7

To determine surviving numbers of Escherichia coli from cultures or food systems, dilution with 0.1% peptone is regularly used. Higher numbers of survivors could be obtained from an acid-treated culture if 0.5 mol l-1 sucrose was added to the 0.1% peptone. Sorbitol, glucose or sodium chloride, but not glycerol, could be used in place of sucrose. Using electron microscopy distinct differences could be seen between acid-treated and untreated cells. The osmolarity of the diluents ranged from 5 to 500 mosmol kg-1 H2O for the 0.5 mol l-1 sugar or glycerol solutions, to about 1000 mosmol kg-1 H2O for the salt solution. Maximum recovery diluent has an osmolarity of about 300 mosmol kg-1 H2O and resulted in recovery of similar numbers of injured cells as a 0.5 mol l-1 solution of sugar in 0.1% peptone. Taking into account the observed damage to acid-treated cells and the differences in osmolarity of the diluents, it is likely that dilution in 0.1% peptone imposed additional stress on the acid-injured cells which caused further cell damage. Dilution in a more osmotically stable solution alleviated this osmotic stress.

Recovery of sub-lethally injured bacteria from frozen foods

Using a ‘solid-repair’ method, recovery of the natural microflora in frozen chicken, minced beef, peas, beans and prawns was investigated with particular reference to the detection of sub-lethally injured bacteria. Plate counts following resuscitation in: Hall's Resuscitation Agar (HRA), Trypticase Soy Agar (TSA), Minerals Modified Glutamate Agar (MMGA) and Plate Count Agar (PCA), were compared to conventional aerobic plate counts (APC). HRA and MMGA recovered significantly more cells than the normal APC ( P ⩽ 0·05), though HRA was effective for all foods and consistently recovered greater numbers of cells than MMGA. No improvements in recovery occurred following resuscitation in either TSA or PCA. None of the resuscitation times was optimal for recovery, and repair was comparable at 20°C and 30°C. Maximum Recovery Diluent without salt or supplemented with 0·5% and 1·0% ( w v ) salt did not affect recovery. Overlayers of PCA containing salt at the same concentration as present naturally in the food, when applied on top of HRA produced maximal recovery levels from prawns and, to a lesser extent, from chicken.