Genotypic characterization of Brochothrix thermosphacta isolated during storage of minced pork under aerobic or modified atmosphere packaging conditions

A total of 306 colonies were isolated from the selective medium for Brochothrix spp., during the spoilage of minced pork stored at 0, 5, 10 and 15°C and packed aerobically and under modified atmosphere packaging conditions (MAP). Brochothrix biodiversity was assessed by Pulsed Field Gel Electrophoresis (PFGE), and representative strains were further analysed by Rep-PCR using primer (GTG)(5) and Sau-PCR with primers SAG(1)and SAG(2). Although, different results were obtained from the different methods, a significant diversity among isolates recovered from aerobic conditions was observed. On the contrary, isolates from MAP showed a lower degree of heterogeneity. The storage conditions affected the Brochothrix diversity, the strains isolated in the initial stage being different from the ones present at the final stage of storage at chill temperatures. A representative number of isolates, based on the results of the clustering by molecular methods, were subjected to 16S rRNA gene sequencing, revealing that all belonged to Brochothrix thermosphacta.     

Lactic acid bacteria population dynamics during minced beef storage under aerobic or modified atmosphere packaging conditions

A total of 266 lactic acid bacteria (LAB) have been isolated from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO₂–30% O₂–30% N₂ in the presence MAP (+) and absence MAP (−) of oregano essential oil. Sequencing of their 16S rRNA gene along with presence of the katA gene demonstrated dominance of the LAB microbiota by Leuconostoc spp. during aerobic storage at 5, 10 and 15 °C, as well as during MAP (−) and MAP (+) storage at 10 and 15 °C; Lactobacillus sakei prevailed during aerobic storage at 0 °C, as well as at MAP (−) and MAP (+) storage at 0 and 5 °C. The sporadic presence of other species such as Leuconostoc mesenteroides, Weisella viridescens, Lactobacillus casei and Lactobacillus curvatus has also been determined. Pulsed-Field Gel Electrophoresis of high molecular weight genomic DNA revealed the dynamics of the isolated LAB strains. Prevalence of Leuconostoc spp. was attributed to one strain only. On the other hand, packaging conditions affected Lb. sakei strain spoilage dynamics.     

Characterization by culture-dependent and culture-independent methods of the bacterial population of suckling-lamb packaged in different atmospheres

This study offers insight into the dynamics of bacterial populations in fresh cuts of suckling lamb under four different atmospheric conditions: air (A), and three Modified Atmosphere Packaging (MAP) environments, 15%O₂/30%CO₂/55%N₂ (C, commercial), 70%O₂/30%CO₂ (O), and 15%O₂/85%CO₂ (H) for 18 days. Microbial analyses by both conventional methods and PCR-DGGE were performed. Controversial and surprising results emerged from comparing both methods in relation to the genus Pseudomonas. Thus, conventional methods detected the presence of high numbers of Pseudomonas colonies, although PCR-DGGE only detected this genus in air-packaged samples. PCR-DGGE detected higher microbial diversity in the control samples (A) than in the modified atmospheres (C, O, H), having atmosphere H the fewest number of species. Brochothrix thermosphacta, LAB (Carnobacterium divergens and Lactobacillus sakei), and Escherichia spp. were detected in all the atmospheres throughout storage. Moreover, previously undescribed bacteria from lamb meat such as Enterobacter hormaechei, Staphylococcus equorum and Jeotgalicoccus spp. were also isolated in this study by DGGE. Additionally, qPCR analysis was used to detect and characterize strains of Escherichia coli. Virulence genes (stx₁, stx₂ and eae) were detected throughout storage in 97% of the samples. A high CO₂ atmosphere was the most effective packaging combination doubling storage time in comparison with commercial atmosphere.     

The characterisation of lactic acid bacteria during the fermentation of an artisan Serbian sausage (Petrovská Klobása)

Petrovská Klobása is an artisan Serbian sausage made only from meat and spices without any additives or starter cultures. In order to characterise lactic acid bacteria (LAB) microflora, a total number of 404 LAB strains were isolated from 15 samples collected during 90 days of the fermentation and 120 days of storage of one batch of Petrovská Klobása. The isolates were preliminarily identified by phenotypic tests and subjected to (GTG)₅-PCR fingerprinting. Representatives of each group were identified by 16S rDNA sequencing. The results showed that among the isolates, Lactobacillus sakei and Leuconostoc mesenteroides predominate with 36.4% and 37.1% of total LAB strains, respectively. Pediococcus pentosaceus was also isolated in high proportion (18.4%) whereas Enterococcus durans and Enterococcus caseliflavus made only 1% and 6% of the total isolates, correspondingly. The analysis of vacuum packed and modified atmosphere packed (MAP) samples showed higher presence of L. mesenteroides and L. sakei in the total microflora.     

Anaerobic Microbiology of Fresh Beef Packaged Under Modified Atmosphere or Vacuum

Beef was packaged under CO₂ containing < 500 ppm O₂ (MAP), vacuum, or air and stored at 0, 2, or 4°C. Samples were analyzed weekly for bacterial numbers using anaerobic plate count (pre-reduced medium and anaerobic chamber), -aerobic plate count, and anaerobic jar plate count (MAP samples only) methods. For both MAP and vacuum packaged samples, the anaerobic plate count was consistently greater than the aerobic plate count and for MAP samples the anaerobic plate count was consistently higher than the anaerobic jar plate count. Differences between plating methods were most frequent during the latter third of 0 and 2°C storage. Anaerobic isolates from MAP samples were most often lactic acid cocci and staphylococci. No clostridia were isolated from any of the treatments.     

Preliminary selection for potential probiotic Bifidobacterium isolated from subjects of different Chinese ethnic groups and evaluation of their fermentation and storage characteristics in bovine milk

A total of 29 strains of Bifidobacterium were isolated from 18 samples of human feces in different ethnic minority regions of China. All isolates were identified as Bifidobacterium longum (9 strains) and Bifidobacterium pseudocatenulatum (20 strains) based on 16S rRNA gene sequencing and phylogenetic analysis. These strains were preliminarily tested for their suitability to become probiotics by assessing their ability to survive adequately at low pH conditions and their tolerance of different concentrations of bile salts and simulated gastrointestinal juices. In vitro tests were sequentially used to predict the survival of these strains in the simulated conditions in the human gastrointestinal tract. These strains were first exposed to pH 2.5 for 3 h, and 7 out of the 29 strains were discriminated from the others by their high survival rates. Out of these 7 strains, 4 were found to grow and survive well at an even lower pH of 2.0 and in high bile salt concentration. Apart from the gastrointestinal survival capacity, both fermentation efficiency and storage characteristics are important criteria for selecting for suitable potential probiotic strains. Therefore, the fermentation efficiency in bovine milk and the bacterial viability during the storage in the resultant fermented milk were also evaluated for these 4 selected strains. In this study, we isolated and identified 29 novel Bifidobacterium strains. Based on our initial evaluation, at least 4 of them may serve as valuable resources for further dairy probiotic strain selection.     

Characterization of the Enterobacteriaceae community that developed during storage of minced beef under aerobic or modified atmosphere packaging conditions

The whole cell protein and macrorestriction analysis of DNA of Enterobacteriaceae isolates recovered from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO₂-30% O₂-30% N₂ in the presence (MAP+) and absence (MAP−) of oregano essential oil were studied. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles obtained from whole cell protein analysis of the Enterobacteriaceae isolates revealed seven groups. Moreover, application of a modified PFGE protocol with XbaI restriction, resulted into 19 different fingerprints. The Enterobacteriaceae community of fresh meat consisted of Serratia liquefaciens and Serratia proteamaculans. S. liquefaciens strain VK23 was the dominant isolate of Enterobacteriaceae for the most conditions adopted, except 10 °C and 15 °C under MAP + and 10 °C under MAP−. In the latter cases, Hafnia alvei represented the dominant fingerprint. Citrobacter freundii was recovered from minced beef stored aerobically, while H. alvei and Proteus vulgaris were recovered under MAP. Storage conditions affected the Enterobacteriaceae community; modified atmosphere packaging increased both species and strain diversity.     

Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium

The major objectives of this study were to determine the incidence, diversity and characteristics of Bacillus cereus group spp. isolated from food products marketed in Belgium. The food products investigated in this study included cooked pasta, lasagna, béchamel sauce, bolognaise sauce, fresh minced beef, fresh-cut vegetables and raw basmati rice. B. cereus group spp. were detected in 56.3% (324 of 575) of the samples giving rise to 380 strains. The highest incidence (100%) occurred in the raw basmati rice. Although only 10 (2.6%) of the 380 isolates were determined to be psychrotolerant (able to grow at ≤ 7 °C), 25 (6.2%), 189 (49.7%) and 334 (87.9%) isolates were able to grow at mild temperature abuse conditions of 8 °C, 9 °C and 10 °C, respectively. The large diversity of the isolates obtained (overall and between isolates obtained from the same product type) was highlighted by the results of the (GTG)₅ PCR fingerprinting of 80 selected isolates. Sixty-one of these 80 isolates belonged to 15 distinct clusters (≥ 85% Pearson correlation) whereas the remaining 19 were each clustered separately. Further diversity was also found in the distribution of toxin genes as 16 different profiles were observed in the 80 selected isolates. Whilst none of 80 selected strains harboured the ces gene required for the production of the emetic toxin cereulide, 42 strains (52.5%) carried all seven genes required for the production of the diarrhoeal enterotoxins: haemolytic BL, non-haemolytic enterotoxin and cytotoxin K. The results of this study highlight not only the omnipresence but also the highly diverse ecology of B. cereus spp. within and across several food product types available on the retail market in Belgium. They should also provide the impetus for more studies to enable detailed risk assessment studies to be performed.     

Effect of modified atmosphere packaging on the growth of spoilage microorganisms and Listeria monocytogenes on fresh cheese

Queso Fresco has a limited shelf life and has been shown to support the rapid growth of Listeria monocytogenes during refrigerated storage. In addition to improving quality and extending shelf life, modified atmosphere packaging (MAP) has been used to control the growth of pathogenic microorganisms in foods. The objectives of this study were to determine the effects of MAP conditions on the survival and growth of spoilage microorganisms and L. monocytogenes during storage of Queso Fresco manufactured without starter cultures. For L. monocytogenes experiments, cheeses were surface inoculated at ～4 log₁₀ cfu/g before packaging. Inoculated and uninoculated (shelf life experiments) cheeses were placed in 75-µm high-barrier pouches, packaged under 1 of 7 conditions including air, vacuum, or combinations of N₂ and CO₂ [100% N₂ (MAP1), 30% CO₂:70% N₂ (MAP2), 50% CO₂:50% N₂ (MAP3), or 70% CO₂:30% N₂ (MAP4), 100% CO₂ (MAP5)], and stored at 7°C. Samples were removed weekly through 35 d of storage. Listeria monocytogenes counts were determined for inoculated samples. Uninoculated samples were assayed for mesophilic and psychrotolerant counts, lactic acid bacteria, coliforms, and yeast and mold. In general, cheeses packaged under conditions consisting of higher contents of CO₂ had lower pH levels during storage compared with those stored in conditions with lower levels or no CO₂ at all. Similarly, the antimicrobial efficacy of MAP in controlling spoilage microorganisms increased with increasing CO₂ content, whereas conditions consisting of 100% N₂, vacuum, or air were less effective. Mean L. monocytogenes counts remained near inoculation levels for all treatments at d 1 but increased ～2 log₁₀ cfu/g on cheeses packaged in air, vacuum, and 100% N₂ (MAP1) conditions at d 7 and an additional ～1.5 log₁₀ cfu/g at d 14 where they remained through 35 d. In contrast, treatments consisting of 70% CO₂ (MAP4) and 100% CO₂ (MAP5) limited increases in mean L. monocytogenes counts to <1 log₁₀ cfu/g through 14 d and ～1.5 log₁₀ cfu/g by d 21. Mean L. monocytogenes counts increased to levels significantly higher than inoculation (d 0) on cheeses stored in MAP2 and MAP3 on d 21, on d 28 for MAP4, and on d 35 for cheeses stored under MAP5 conditions. Overall, significant treatment × time interactions were observed between air, vacuum, and MAP1 when each was compared with MAP2, MAP3, MAP4, and MAP5. These data demonstrate that packaging fresh cheese under modified atmospheres containing CO₂ may be a promising approach to extend shelf life while limiting L. monocytogenes growth during cold storage.     

Prevalence and characterization of Listeria monocytogenes isolated from soft cheese

A survey was made in 1995–1996 for Listeria spp. in 63 soft cheeses, made from raw ewe's milk using traditional methods, in the Province of Beira Baixa (Portugal). Listeria spp. were isolated from 47 (75%) of the cheeses, L.monocytogenes was isolated from 29 (46%), and L.innocua but not L.monocytogenes from 18 (29%). Of 24 isolates of L.monocytogenes that were serotyped, 20 were serotype 4b, three were serotype 1/2b and one was serotype 1/2a. Phage typing of isolates of L.monocytogenes and L.innocua showed that in some cases a particular phage type was associated with cheese from a particular source. Twenty four strains of L.monocytogenes tested were able to grow at 30°C in culture medium adjusted with HCl to a pH in the range from 4.4 to 6.0 within 3 days; in the pH range 4.4–6.8 a representative strain grew most rapidly at pH 6.8. The pH range in the cheeses during maturation was between about 5.2–6.4. Whether L.monocytogenes could multiply in the cheeses would depend on factors such as concentration of organic acids and of salt, and storage temperature.     

Biodiversity of indigenous staphylococci of naturally fermented dry sausages and manufacturing environments of small-scale processing units

The staphylococcal community of the environments of nine French small-scale processing units and their naturally fermented meat products was identified by analyzing 676 isolates. Fifteen species were accurately identified using validated molecular methods. The three prevalent species were Staphylococcus equorum (58.4%), Staphylococcus saprophyticus (15.7%) and Staphylococcus xylosus (9.3%). S. equorum was isolated in all the processing units in similar proportion in meat and environmental samples. S. saprophyticus was also isolated in all the processing units with a higher percentage in environmental samples. S. xylosus was present sporadically in the processing units and its prevalence was higher in meat samples. The genetic diversity of the strains within the three species isolated from one processing unit was studied by PFGE and revealed a high diversity for S. equorum and S. saprophyticus both in the environment and the meat isolates. The genetic diversity remained high through the manufacturing steps. A small percentage of the strains of the two species share the two ecological niches. These results highlight that some strains, probably introduced by the meat, will persist in the manufacturing environment, while other strains are more adapted to the meat products.     

Characterization of lactobacilli involved in the ripening of soppressata molisana,a typical southern Italy fermented sausage

Lactobacillusspecies (183 strains) isolated during the ripening of soppressata molisana, a typical fermented sausage from Molise, were characterized. Of these isolates the large majority (125 strains) was classified asLactobacillus sake. Few strains were obligately heterofermentative and were identified asLb. brevis. All strains were able to grow in the presence of 8% NaCl and a large majority was able to grow in the presence of 10% NaCl. No strains were able to hydrolyse pork fat at 18°C. Several isolates were H₂O₂and acetoin producers. Few strains were responsible for slime production. All the isolates also showed high resistance to nitrites.     

The microflora of fresh and spoiled sardines (Sardina pilchardus) caught in Adriatic (Mediterranean) Sea and stored in ice

A total of 1500 strains were isolated from the skin and gills of fresh, ice-stored (4 days) and spoiled (8 days) Adriatic sardines, and were identified at different taxonomic levels. Fresh sardine microflora found included mostly non-fermenting Gram-negative bacteria, (Pseudomonas, Flavobacterium, Psychrobacter, Acineobacter, Shewanella), and a minor proportion of Enterobacteriaceae and Gram-positive bacteria. The highest increase in microflora frequency, after 8 days in ice, was observed for thePseudomonas fragigroup (8–30·8%) andShewanella putrefaciens(1·8–11%). A significant presence was also noted for fluorescentPseudomonas(15·6–18·4%) andPsychrobacter immobilis(16·4–23·4%). The isolation frequency of the other groups decreased during storage. The most important proteolytic species werePseudomonas fluorescensandShewanella putrefaciensand the most lipolytic bacteria werePsychrobacter immobilisand again,P. fluorescensandS. putrefaciens. A total of 288 isolates, representative of the main groups, were tested for potential spoiling activity (H₂S and off-odour production, TMAO reduction).Shewanella putrefacienswas the strongest spoiler, followed byPseudomonas fluorescens. A weaker activity was observed for strains ofPseudomonas fragi, enterobacteriaceae and non-saccharolytic pseudomonads. The contribution of weak spoiler bacteria can be undermined by the activity of the strongest spoilers, but in some cases the considerable incidence of the former group suggests their effective role.Morganella morganiiwas the only histamine-producing species among 57 screened strains representative of different taxa.     

Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR-TTGE

In order to characterise the spoilage related to microbiota of raw salmon, a combination of culture-dependent and -independent methods, including PCR–TTGE, was used to analyse 3 raw salmon batches stored for 3 days at chilled temperature in modified atmosphere packaging (MAP) (50% CO₂/50% N₂) or under vacuum. Sensory evaluation, microbiological enumeration and chemical analysis were performed after 3, 7 and 10 days of storage. At the onset of spoilage, 65 bacterial isolates were picked from the plates. Thus, 13 different genera or species were identified by phenotypic and molecular tests: Serratia spp., Photobacterium phosphoreum, Yersinia intermedia, Hafnia alvei, Buttiauxella gaviniae, Pseudomonas sp., Carnobacterium maltaromaticum, Carnobacterium divergens, Lactococcus piscium, Lactobacillus fuchuensis, Vagococcus carniphilus, Leuconostoc gasicomitatum and Brochothrix thermosphacta. The PCR–TTGE profiles and band identification enabled a shift of the dominant populations during the storage to be visualised for all the batches, probably due to the temperature change and the packaging. At the beginning of storage, Pseudomonas sp. dominated the raw salmon microbiota while in the following days (7 and 10), P. phosphoreum and L. piscium were identified as the main bacterial groups. This study enhances the knowledge of MAP and vacuum-packed raw salmon spoilage microbiota.     

Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk

The aim of this study was to use molecular techniques to assess the microbiota of eight raw cow's milk samples at biotype and species level. Sixty-six isolates from raw milk samples were screened by Randomly amplified polymorphic DNA–PCR (RAPD–PCR) biotyping and representative strains of RAPD–PCR profiles were identified by 16S rRNA gene sequencing. Pseudomonas spp. were the most commonly occurring contaminants along with Enterobacteriaceae such as Hafnia alvei, Serratia marcescens and Citrobacter freundii. Moreover, Gram-positive isolates belonging to the genera Staphylococcus and Lactococcus were also found. Experiments of growth at different temperatures showed that more than 50% of the Gram-negative isolates could grow at chill temperatures and that 65% of the Pseudomonas spp. strains grew at 7 °C within 5 days. Only 13 Gram-negative isolates displayed proteolytic activity on milk agar, suggesting that not all the biotypes of milk contaminating species are able to perform this spoilage-associated activity. Among the Gram negative, the proteolytic strains were mainly Peudomonas spp. that displayed the activity at both 7 °C and 20 °C. A reliable molecular identification of raw milk microbiota is important for the study of the microbiological quality of raw milks and for the assessment of the ecology at species level in order to develop improved systems, preventing contamination and having the best conditions for the storage of milk.     

Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese

Eleven strains of lactococci isolated from a farmhouse starter-free cheese manufactured from raw cow's milk were analysed in detail for some technologically-related properties. Large phenotypic differences were encountered between the isolates, some of which could be of practical relevance. Several strains produced lactic acid at a rate and at a final concentration suitable for large-scale cheesemaking. The enzymatic capabilities assayed with the API ZYM system showed that all strains possess similar profiles with weak proteinase and moderate leucine-arylamidase and esterase-lipase activities. Interestingly, two related strains presented a strong β -galactosidase activity. Plasmid and chromosomal analyses indicated a high degree of diversity among wild strains and showed low homology with some well-known Lactococcus lactis strains. Under highly stringent conditions, only one plasmid from a single strain gave a clear positive hybridization signal with lactococcal-derived probes for the β -phospho- galactosidase and the proteinase genes. In general, wild strains produced more odorous compounds and in higher amounts than the reference strains.     

The effects of partial replacement of fish meal by vegetable protein sources in the diet of rainbow trout (Onchorynchus mykiss) on post mortem spoilage of fillets

Five test diets were formulated with decreasing levels of fish meal (up to 50%) replaced by alternative protein sources. Rainbow trout were fed the experimental diets for 12 weeks.The effects of feed ingredients on spoilage of Oncorhynchus mykiss in ice and under MAP/ice (40% CO₂, 30% N₂ and 30% O₂) were investigated in terms of sensory, chemical and microbiological analyses. The results showed that the trout in MAP/ice was rejected at 14 days, after sensory analysis, due to excessive drip, whereas trout in ice were found to be acceptable even after 14 days of storage. However, cooked trout fillets, under both storage conditions, were rejected at 17 days. Fish in ice produced higher K values and higher concentrations of biogenic amines during the storage period of 17 days than the fish in MAP/ice. Bacteria grew more quickly in rainbow trout kept in ice than in MAP/ice. MAP/ice storage extended the shelf life of rainbow trout by approximately 2 days compared to ice storage alone in terms of microbiological analyses.     

Characterization of Fusarium verticillioides strains isolated from maize in Italy: fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.     

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate).Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance.A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy.All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearson's correlation coefficients (r) showed close agreement between all three methods.Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.     

Storage Potential of Tomatoes Harvested at the Breaker Stage using Modified Atmosphere Packaging

Breaker tomatoes sealed in polymeric film (MAP) were stored at 15°C 23 days. A steady state of about 3.5-4.0 % O₂ and CO₂ was established. Mean concentrations of the gases within 24 hr of packaging were minimum 2.5 % O₂ and maximum 8.0% CO₂- Thereafter gas concentration moved gradually to a steady state; no evidence of anoxic conditions occurred. After 23 days of MAP storage fruit ripened normally under ambient conditions. Quality evaluations demonstrated that 15°C MAP storage permitted harvesting of breaker stage of ripeness tomatoes without reducing storage life to an unacceptable duration. MAP delayed changes in acidity, soluble solids, texture, color and polygalacturonase activity and resulted in substantial reduction in fruit weight loss and spoilage as compared to breaker fruit without film packaging.