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AIM In this study, the effect of frozen storage on the inhibition of microbial population in coconut neera for shelf life extension was analysed. METHODS AND RESULTS The aliquots of fresh neera were frozen at -6 and -20°C. The microbial reduction and chemical qualities were analysed during storage days, and were compared with the control. The highest reduction in microbes was obtained at -20°C for lactic acid bacteria, yeast and total viable count. Moreover, the fluorescence cell staining and cell density analysis exhibited a decline at -20°C, however it showed higher at -6°C. At the end of 28 days, neera exhibited a pH of 5·37, total soluble solid of 14·0 °Brix and total acidity of 0·82 mg l-1 at -20°C. Besides, color, viscosity, total protein and ethanol showed a less difference with control and better sensory attribute up to 21 days at -20°C. CONCLUSIONS Frozen storage at -20°C resulted in a better inhibition of lactic acid bacteria, which preserves neera from the metabolic conversion. The physicochemical qualities of neera were preserved for a longer period when stored at -20°C than at atmospheric storage. SIGNIFICANCE AND IMPACT OF THE STUDY Frozen storage reduced microbial population and preserves the chemical properties with acceptable sensory attributes and increases the shelf life of coconut neera, making it fit for consumption.

Aims This study aimed to monitor development of spoilage-associated microbiota on high-oxygen modified atmosphere packaged (MAP) minced beef, assess diversity of Pseudomonas sp. therein employing a polyphasic approach and probe their ability to grow anaerobically in the presence of carbon dioxide. Methods and results Headspace atmosphere and total viable count of MAP minced beef were monitored, and spoilage-associated microbiota was identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Pseudomonas spp. represented a major part of the spoilage-associated microbiota throughout the spoilage process and were characterized with a polyphasic approach including MALDI-TOF, randomly amplified polymorphic DNA biotyping, 16S rDNA and rpoD sequence analysis, and carA multiplex polymerase chain reaction. Pseudomonas isolates displayed high diversity and varying assertiveness under conditions employed in MAP minced beef with P. fragi, P. lundensis and P. weihenstephanensis as dominant species. Conclusions The polyphasic approach enabled high-throughput characterization of Pseudomonas sp. Their adapted capability to grow anaerobically and resistance to high levels of CO2 is suggested to be a general feature within the genus, which is hitherto underexplored. Significance and impact of the study This study shows that diverse Pseudomonas generally regarded as strict aerobes and CO2 -sensitive appear well adapted to grow under MAP conditions, leading to high cell counts in minced beef and ultimately contribute to spoilage of the product.

Aims: The results from European standard disinfectant tests are used as one basis to approve the use of disinfectants in Europe. The design of these laboratory-based tests should thus simulate as closely as possible the practical conditions and challenges that the disinfectants would encounter in use. No evidence is available that the organic and microbial loading in these tests simulates actual levels in the food service sector. Methods and Results: Total organic carbon (TOC) and total viable count (TVC) were determined on 17 visibly clean and 45 visibly dirty surfaces in two restaurants and the food preparation surfaces of a large retail store. These values were compared to reference values recovered from surfaces soiled with the organic and microbial loading, following the standard conditions of the European Surface Test for bactericidal efficacy, EN 13697. Conclusions: The TOC reference values for clean and dirty conditions were higher than the data from practice, but cannot be regarded as statistical outliers. This was considered as a conservative assessment; however, as additional nine TOC samples from visibly dirty surfaces were discarded from the analysis, as their loading made them impossible to process. Similarly, the recovery of test organisms from surfaces contaminated according to EN 13697 was higher than the TVC from visibly dirty surfaces in practice; though they could not be regarded as statistical outliers of the whole data field. No correlation was found between TVC and TOC in the sampled data, which re-emphasizes the potential presence of micro-organisms on visibly clean surfaces and thus the need for the same degree of disinfection as visibly dirty surfaces. Significance and Impact of the Study: The organic soil and the microbial burden used in EN disinfectant standards represent a realistic worst-case scenario for disinfectants used in the food service and food-processing areas.

Aims: Development of a predictive model for the determination of the shelf life of modified atmosphere-packed (MAP) cooked sliced ham in each step of the cold chain. Methods and Results: The growth of lactic acid bacteria (LAB), as well as the development of the total viable count and changes of sensory and pH value parameters in MAP cooked sliced ham, stored under different constant temperature conditions from 2 to 15°C was investigated. As a result of the measurements, the end of the shelf life could be considered as the time when LAB reach more than 7 log10 CFU g−1. Different primary and secondary models were tested and analysed to find the best way to calculate the shelf life. For primary modelling, the modified Gompertz Function and the modified Logistic Function were compared. There was no substantial difference between either model. The effect of temperature on the growth rate was modelled by using the Arrhenius and the Square root model, whereas the Arrhenius equation gave a better result. A combination of the primary and secondary model was used for shelf-life prediction under dynamic conditions. This combination showed the best prediction of microbial counts using the modified Logistic model and the Arrhenius equation. Conclusions: With the developed model, it is possible to predict the shelf life of MAP cooked sliced ham based on the growth of LAB under different temperature conditions. Significance and Impact of the Study: The developed model can be used to calculate the remaining shelf life in different steps of the chain. Thus, it can deliver an important contribution to improve food quality by optimizing the storage management.

AIMS The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5 degrees C or 10 degrees C). METHODS AND RESULTS The influence of low water activity (a(w)) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10 degrees C) in an environmental cabinet. Salmonella counts declined during storage at low a(w) (75% RH) conditions at 5 degrees C or 10 degrees C. Salmonella counts increased during storage at high a(w) (96% RH) at 10 degrees C only. At 5 degrees C, TVCs increased during storage at high a(w), but not at low a(w). TVCs increased on all samples from carcasses stored at high or low a(w) at 10 degrees C, except those samples taken from areas of surface fat. CONCLUSIONS This suggests that substrate composition dictates growth rates under low a(w) conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth. SIGNIFICANCE AND IMPACT OF THE STUDY The data obtained in this study provides useful insights on the influence of a(w) and temperature on pathogen survival on meat surfaces at chill temperature.

Aims: The present study was aimed to evaluate the microbiological and sensory characteristics of biopreserved packed fresh plaice (Pleuronectes platessa), during storage. Methods and Results: Fish samples were treated with a Bifidobacterium bifidum strain and thymol as preservatives, stored at different temperatures (4 and 12°C) and package atmospheres (in air, under vacuum, modified atmosphere). Data about the total viable count (TVC) were used to predict the shelf life of the fish fillets as affected by the preservatives used and the storage conditions. Data obtained revealed that B. bifidum could perform an efficient synergy with thymol, in controlling the hygiene biological markers. Besides, the tested bifidobacterial species, combined with low storage temperature and anoxia/hypoxia, showed a great efficacy against the main fresh packaged fish spoilage species (Pseudomonas spp., Photobacterium phosphoreum). Conclusions: The studied technique could be considered effective in fresh fish spoilage control. Significance and Impact of the Study: Great interest is developing on food biopreservation, because of the ever increasing needs to protect consumers’ and environment's health, and to revalorize some important food characteristics, such as naturalness and authenticity, without leaving the safety aside.

Aims: To evaluate synergetic effect of slight acidic electrolysed water (SAEW)and fumaric acid (FA) on inactivation of total viable count (TVC) andStaphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 andSalmonella Typhimurium in fresh beef and to study shelf life and sensoryquality of beef.Methods and Results: Inoculated samples was dipped for 1, 3 and 5 min andimmersed at 25, 40 and 60°C in SAEW, strong acidic electrolysed water(StAEW) and SAWE + FA. Treated meat was air-packaged and stored at 4 or10°C. During storage, sampling was performed at 2-day intervals formicrobiological and sensory changes. TVC was decontaminated at 40°C for3 min by more than 3 70 log CFU g 1 , and examined pathogens were reducedby more than 2 60 log CFU g 1 with SAEW + FA treatment. This treatmentprolonged shelf life of beef meat up to 9 and 7 days when stored at 4 and10°C, respectively.Conclusion: The combined treatment of SAEW + FA showed greaterbactericidal effect and prolonged shelf life compared with individualtreatments.Significance and impact of the study: Combined treatment of SAEW and FAcan be a suitable hurdle technology reducing bacteria in fresh beef,substantially enhancing their microbial safety and decreasing pathogens growthduring storage.IntroductionDuring slaughtering and processing of all edible carcasstissues, the meat is subjected to internal and externalcontaminations from a variety of sources. Microbialgrowth is generally confined to the outer surfaces wherebacteria irreversibly bind to. Meats contaminated withListeria monocytogenes, Staphylococcus aureus, Escherichiacoli O157:H7 and Salmonella spp. have been reported asthe causal agents of food-borne diseases (Koohmaraieet al. 2005; Jofre et al. 2008; Perez-Rodriguez et al. 2010).The micro-organisms reach the carcass surface where theypenetrate into deeper layers of the meat. By reducing thisprimal surface contamination and avoiding or limitingthe microbial growth, we can considerably reduce theburden of food-borne diseases and extend shelf life offresh meat (Pipek et al. 2005).Although food-borne pathogens are subjected to physi-cal, chemical and nutritional stress during processing andthe improvements are made in hygienic slaughter pro-cessing, they still persist at high level in raw meat (Abbas-si-Ghozzi et al. 2012; Osaili et al. 2013), rendering theirelimination/inhibition a challenge to processors (Pathaniaet al. 2010). Significant advances have been made indeveloping thermal and nonthermal intervention technol-ogies to reduce food-borne pathogens in meat and meatproduct during the slaughtering process. Innovative tech-nologies are needed that use little heat to preserve the

R . J . H O D G S O N , R . J . M . L Y N C H , G . K . W A T S O N , R . L A B A R B E , R . T R E L O A R A N D C . L L I S O N . 2001. Aims: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization. Methods and Results: A seven-organism bacterial consortium was grown in a biofilm mode on enamel and hydroxyapatite (HA) surfaces in a continuous culture system and exposed to repeated sucrose challenges. This produced ‘pH-cycling’ conditions within the system. Populations on HA surfaces were enumerated. Changes in relative proportions of the different populations, and in the total viable count, were observed, between different treatments. Microradiography of the enamel sections showed increasing demineralization with increasing sucrose concentration. The lesions formed were similar to ‘white-spot’ lesions found in vivo. Differences in the quality of biofilms formed were also observed using Confocal Laser Scanning Microscopy. Conclusions: An in vitro model has been validated for the analysis of both physiological and ecological responses to sucrose challenges in bacterial populations, and subsequent changes in enamel mineralization. Significance and Impact of the Study: This model should facilitate the study of changes in bacterial populations in response to application of putative anticaries agents and concomitant changes in enamel mineralization.

The effectiveness of cleaning was investigated through food factory trials and laboratory experiments using a naturally occurring biofilm from a food factory environment and generated biofilms. The efficacy of factory cleaning and disinfection programmes was assessed by swabbing and total viable count (TVC) analysis of surfaces before cleaning, after cleaning and after disinfection. Cleaning produced a 0.91 log reduction in the attached population. Investigation of the effectiveness of a variety of cleaning methods in the removal of a naturally occurring food factory biofilm showed that the high pressure spray and the mechanical floor scrubber, which use a high degree of mechanical action, were most effective. Cleaning trials with biofilms of Pseudomonas aeruginosa or Staphylococcus aureus showed that spraying with water at pressures of 34.5, 51.7 and 68.9 bar did not significantly increase the removal, as assessed by direct epifluorescent microscopy (DEM) and swabbing and TVC analysis, beyond the three log reduction observed at 17.2 bar. The effect of spray time at 17.2 bar showed that increasing spray time from 1 to 10 s did not significantly increase removal of Ps. aeruginosa biofilm. Investigation of the optimum distance of the spray lance from the surface at 17.2 bar was found to be between 125 and 250 mm. The use of an alkaline, acidic or neutral detergent prior to spraying with water at 17.2 bar did not significantly increase the removal of Ps. aeruginosa or Staph. aureus. However, the acidic and alkaline products significantly (P = 0.05) affected the viability of Staph. aureus and Ps. aeruginosa, respectively, thereby minimizing the potential for the spread of contamination.

A device was developed for direct heating of water by solar radiation in a flow-through system of copper pipes. An adjustable thermostat valve prevents water below the chosen temperature from being withdrawn. The results show that it is possible to eliminate coliform and thermotolerant coliform bacteria from naturally contaminated river water by heating to temperatures of 65 degrees C or above. Artificial additions of Salmonella typhimurium, Streptococcus faecalis and Escherichia coli to contaminated river water were also inactivated after heating to 65 degrees C and above. The total viable count could be reduced by a factor of 1000. The heat-resistant bacteria isolated from the Mlalakuva River (Tanzania) were spore-forming bacteria which exhibited greater heat resistance than commonly used test bacteria originating from countries with colder climates. To provide a good safety margin it is recommended that an outlet water temperature of 75 degrees C be used. At that temperature the daily production was about 501 of decontaminated water per m2 of solar panel, an amount that could be doubled by using a heat exchanger to recycle the heat.

The influence of carbon load on autochthonous water microflora population distribution and diversity, and on the persistence of Campylobacter jejuni, was examined with a two-stage aquatic biofilm model. Serine was chosen since it is a carbon source utilised by C. jejuni and concentrations were chosen to reflect upper limits of amino acid load reported in surface water. The total viable count of the autochthonous biofilm microflora increased with increasing serine concentration (10-fold and 20-fold with 5 nM and 5 μM serine, respectively), as did the counts of the microflora in the planktonic phase. Differences in biofilm species distribution as determined by culture were small with changes in temperature or the addition of serine; but was markedly affected by serine as determined by light microscopy, becoming more luxuriant and dominated by long filamentous cells. The addition of serine to the water significantly and progressively reduced the persistence of C. jejuni, which decreased by 25% and 50% with serine concentrations of 5 nM and 5 μM respectively. We have demonstrated that carbon load affects the species diversity and density of both the planktonic and biofilm phase of aquatic autochthonous microflora. Although the survival of C. jejuni in water in a culturable form was sufficient for this to be an important vehicle for its transmission, carbon load significantly influenced survival; an increase in serine concentration significantly reduced survival.

A.E. GOULAS, I. CHOULIARA, E. NESSI, M.G. KONTOMINAS AND I.N. SAVVAIDIS. 2004. Aims: To determine the microbiological, biochemical and sensory changes of mussels during storage under aerobic, vacuum packaging (VP) and modified atmosphere packaging (MAP) conditions at 4� C, and to determine shelf-life of mussels under the same packaging conditions using the above assessment parameters. Methods and Results: Aqua-cultured mussels (Mytilus galloprovincialis) were obtained from a local culture farm, packaged aerobically under VP and MAP (50%/50% CO2/N2: M1, 80%/20% CO2/N2: M2, 40%/30%/30% CO2/N2/O2: M3), and stored at 4� C. Quality evaluation was carried out using microbiological, chemical and sensory analyses. Microbiological results revealed that the M2 and VP delayed microbial growth compared with that of air-packaged samples. The effect was more pronounced for total viable count (TVC), Pseudomonas spp., lactic acid bacteria (LAB) and H2S-producing bacteria. TVC was reduced by 0AE9‐1AE0, Pseudomonas spp. by 0AE7‐0AE8, LAB by 1AE0‐2AE2, H2S-producing bacteria by 0AE7‐1AE2. Enterobacteriaceae were not significantly affected by MAP conditions. Of the chemical indices determined, the total volatile basic nitrogen and trimethylamine nitrogen values remained lower than the proposed acceptability limits of 35 mg N 100 g )1 and 12 mg N 100 g )1 , respectively, after 15 days of storage. Both the VP and air-packaged mussel samples exceeded these limits. The thiobarbituric acid value of all MAP and VP mussels remained lower than the proposed acceptability limit of 1 mg malondialdehyde kg )1 . The air-packaged samples exceeded this limit. All samples retained desirable sensory characteristics during the first 8 days of storage. Conclusions: Based on odour and taste evaluation, the M1 and M3 samples remained acceptable until ca day 11‐12, the M2 samples remained acceptable until ca day 14‐15 days while the VP and air-packaged mussel samples remained acceptable until ca days 10‐11 and 8‐9 of storage respectively. Based primarily on sensory, but also on biochemical and microbiological parameters determined, M2 gas mixture was the most effective for mussel preservation achieving a shelf-life of ca 14‐15 days. Significance and Impact of the Study: MAP (M2) can be used to increase the shelf-life of refrigerated mussels. A shelf-life extension of refrigerated mussels by ca 5‐6 days under MAP may be obtained.

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7.00, 6.40, 4.76 and 3.13), acids (citric and HCl) and temperature (25 degrees, 30 degrees and 37 degrees C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect (P < 0.05) with higher numbers of Listeria adhering at low pH values (4.76). Culture type was also important with significantly higher numbers of Listeria (P < 0.05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30 degrees C but not at 25 degrees or 37 degrees C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25 degrees C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.