Monitoring the succession of the biota grown on a selective medium for pseudomonads during storage of minced beef with molecular-based methods

In the present study, the succession of the biota grown on a selective medium for pseudomonads (pseudomonas agar based medium – PAB) during the storage of meat under different conditions was monitored. Thus, minced beef was stored aerobically and under modified atmosphere packaging in the presence (MAP+) and absence (MAP−) of oregano essential oil at 0, 5, 10 and 15 °C. A total of 267 pure cultures were recovered from PAB throughout the storage period and subjected to PCR-Denaturing gradient gel electrophoresis (PCR-DGGE) for their differentiation. In parallel, the direct analysis of the whole cultivable community (WCC) from the same medium was applied. These two approaches were used in order to indicate the lack of selectivity. Fifteen different DGGE fingerprints were obtained after PCR – DGGE analysis of the isolates, which were assigned to Pseudomonas putida (3 fingerprints), Pseudomonas fragi and Pseudomonas fluorescens, Pseudomonas spp., Serratia liquefaciens (2), Citrobacter freundii, Serratia grimesii, Hafnia alvei (3), Rahnella spp. and Morganella morganii. Twelve of them occurred during the direct analysis of the WCC. The biota succession found to be affected from the different storage conditions. However, the outcome of the two strategies was quite different, which is leading to the use of different appropriated molecular approaches in order to widen the knowledge of bacterial succession of meat.     

Effects of water extract of Urtica dioica L. and modified atmosphere packaging on the shelf life of ground beef

Effects of lyophilized Urtica dioica L. water extract (LUWE) and modified atmosphere packaging (MAP) on the quality and shelf life of ground beef were investigated. Ground beef was stored as aerobic control, MAP (80%O₂ + 20% CO₂), MAP + 250 ppm LUWE and MAP and 500 ppm LUWE at 2 ± 0.5 °C for 14 days. MAP and LUWE had significant effects on mesophilic, psychrotrophic and lactic acid bacteria and Pseudomonas counts. Depending on the level of LUWE, Pseudomonas and psychrotrophic counts decreased. Treatment with 500 ppm LUWE + MAP showed the lowest TBARS values compared to other groups during storage. 80% O₂-MAP increased TBARS values. Treatment had no significant effect on L* and b* values of the exterior of the ground beef, but had significant effects on the color of interior sections.     

Potential of a simple HPLC-based approach for the identification of the spoilage status of minced beef stored at various temperatures and packaging systems

The shelf life of minced beef stored (i) aerobically, (ii) under modified atmosphere packaging (MAP), and (iii) under MAP with oregano essential oil (MAP/OEO) at 0, 5, 10, and 15 °C was investigated. The microbial association of meat and the temporal biochemical changes were monitored. Microbiological analyses, including total viable counts (TVC), Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae, and yeasts/moulds, were undertaken, in parallel with sensory assessment, pH measurement and HPLC analysis of the organic acid profiles. Spectral data collected by HPLC were subjected to statistical analysis, including principal component analysis (PCA) and factorial discriminant analysis (FDA). This revealed qualitative discrimination of the samples based on their spoilage status. Partial least squares regression (PLS-R) was used to evaluate quantitative predictions of TVC, Pseudomonas spp., Br. thermosphacta, lactic acid bacteria, Enterobacteriaceae, and yeasts/moulds. Overall, the HPLC analysis of organic acids, was found to be a potential method to evaluate the spoilage and microbial status of a meat sample regardless of the storage conditions. This could be a very useful tool for monitoring the quality of meat batches during transportation and storage in the meat food chain.     

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.     

Effects of carbon monoxide-modified atmosphere packaging and irradiation on E. coli K12 survival and raw beef quality

The objective of this study was to evaluate the combined effects of irradiation and carbon monoxide in modified atmosphere packaging (CO-MAP) on total plate counts, Escherichia coli K12, color, and odor of fresh beef during refrigerated storage. Beef was packaged aerobically or in CO-MAP, and irradiated at 0, 0.5, 1.0, 1.5 or 2.0 kGy then held at 4 °C for 28 days. Raw beef odor decreased and acid/sour, rancid and grassy odors increased starting on day 14. Initially, no difference existed for visual green color scores due to gas atmosphere. After 14 days of storage, aerobically packaged beef was greener and less red than CO-MAP packaged beef. The a∗ value of CO-MAP packaged beef was higher than that of aerobically packaged beef. Red color of CO-MAP packaged samples decreased slightly in some irradiated samples after 14 days of storage. On day 0 and thereafter, no coliforms were detect after irradiation at 1.5 or 2.0 kGy regardless of packaging format. These findings suggest that CO-MAP could be used to preserve beef color irradiated at doses sufficient to reduce microbial loads to safe levels during 28 days of storage.     

The effects of packaging method (vacuum pouch vs. plastic tray) on spoilage in a cook-chill pork-based dish kept under refrigeration

The effects of two packaging methods on the spoilage of a cook-chill pork-based dish kept under refrigeration were studied. Raw pork cuts and pre-cooked tomato sauce were packed under vacuum “sous vide” in polyamide–polypropylene pouches (SV) or into translucent polypropylene trays under modified atmosphere (80% N₂ + 20% CO₂) and sealed with a top film (PT). Samples were cooked inside the pack at an oven temperature/time of 70 °C/7 h, chilled at 3 °C and stored at 2 °C for up to 90 days. Microbial (psychrotrophs, lactic-acid bacteria, Enterobacteriaceae, moulds and yeasts), physical–chemical (pH, water activity and total acidity) and sensory (colour, odour, flavour, texture and acceptance) parameters were determined. Heat penetration was faster in SV (2 °C/min) than in PT (1 °C/min) (core temperature). Both packaging methods were equally effective in protecting against microbial spoilage for 90 day at 2 °C. Minor counts were only detected for lactic-acid bacteria and anaerobic psychrotrophs in SV. No Enterobacteriaceae growth was found. Slight differences between SV and PT in pH and total acidity were observed. SV and PT had similar effects on the sensory preservation of the dishes. A gradual loss of acceptance of the cooked pork and tomato sauce was observed. Rancid flavour in PT and warmed-over-flavour in SV were noted in the final stages of storage. According to acceptance scores, the shelf-life of both SV and PT was 56 days at 2 °C. Both packaging methods can be used to manufacture sous vide meat-based dishes subsequently stored under refrigeration for catering use.     

Rapid monitoring of the spoilage of minced beef stored under conventionally and active packaging conditions using Fourier transform infrared spectroscopy in tandem with chemometrics

Fourier transform infrared (FTIR) spectroscopy was exploited to measure biochemical changes within fresh minced beef in an attempt to rapidly monitor beef spoilage. Minced beef packaged either aerobically, under modified atmosphere and using an active packaging were held from freshness to spoilage at 0, 5, 10, and 15 °C. Frequent FTIR measurements were collected directly from the sample surface using attenuated total reflectance, in parallel the total viable counts of bacteria, the sensory quality and the pH were also determined.     

Packaging-specific influence of chitosan on color stability and lipid oxidation in refrigerated ground beef

We examined the influence of chitosan on lipid oxidation and color stability of ground beef stored in different modified atmosphere packaging (MAP) systems. Ground beef patties with chitosan (1%) or without chitosan (control) were packaged either in high-oxygen MAP (HIOX; 80% O₂ + 20% CO₂), carbon monoxide MAP (CO; 0.4% CO + 19.6% CO₂ + 80% N₂), vacuum (VP), or aerobic packaging (PVC) and stored at 1 °C. Chitosan increased (P < 0.05) redness of patties stored in PVC and CO, whereas it had no effect (P > 0.05) in HIOX. Chitosan patties demonstrated lower (P < 0.05) lipid oxidation than controls in all packaging. Control patties in PVC and HIOX exhibited greater (P < 0.05) lipid oxidation than those in VP and CO, whereas chitosan patties in different packaging systems were not different (P > 0.05) from each other. Our findings suggested that antioxidant effects of chitosan on ground beef are packaging-specific.     

Color-stabilizing effect of lactate on ground beef is packaging-dependent

Previous research on lactate-induced color stability in ground beef did not address the potential influence of packaging. The objective of the present study was to examine the effects of lactate on the color stability of ground beef patties stored in different modified atmosphere packaging (MAP) systems. Ground beef patties with either 2.5% potassium lactate or no lactate were packaged in vacuum (VP), high-oxygen MAP (HIOX; 80% O₂ + 20% CO₂), carbon monoxide MAP (CO; 0.4% CO + 19.6% CO₂ + 80% N₂), or aerobic packaging (PVC) and stored for 0, 2, or 4 days at 2 °C. Lactate-treated patties were darker (P < 0.05; lower L∗ values) than control patties. Surface redness (a∗ values) was greater (P < 0.05) for lactate patties than the controls when stored in PVC, HIOX, and VP. However, lactate’s effects on a∗ values were not evident when packaged in CO (P > 0.05). The color-stabilizing effect of CO could have masked lactate’s effect on surface redness. While lactate patties in PVC and VP demonstrated lower (P < 0.05) discoloration than controls, no differences (P > 0.05) existed between controls and lactate samples in CO and HIOX. Our results indicated that the effects of lactate on ground beef color are dependent on packaging.     

Effect of lutein,sesamol, ellagic acid and olive leaf extract on the quality and shelf-life stability of packaged raw minced beef patties

The effects of lutein (100 and 200 μg/g muscle), sesamol (250 and 500 μg/g muscle), ellagic acid (300 and 600 μg/g muscle) and olive leaf extract (100 and 200 μg/g muscle) on total viable counts (TVCs), lipid oxidation (thiobarbituric acid-reactive substances, TBARS), colour, oxymyoglobin oxidation, pH, water-holding capacity (WHC), sensorial properties of raw beef patties (M. longissimus thoracis et lumborum) stored in modified atmosphere packs (80% O₂:20% CO₂) (MAP) aerobically at 4 °C for up to 8 and 12 days, respectively, were examined. All the nutraceuticals reduced (P < 0.001) TVCs. The addition of sesamol, ellagic acid and olive leaf extract reduced (P < 0.001) TBARS in raw beef patties in both packaging systems. Sesamol addition to beef resulted in lower (P < 0.01) a* redness values and increased oxymyoglobin oxidation. Conversely, lutein and olive leaf extract reduced (P < 0.001) oxymyoglobin oxidation relative to the control. The graded addition of ellagic acid and olive leaf extract improved (P < 0.001) WHC.     

Shelf-stability enhancement of precooked red claw crayfish (Cherax quadricarinatus) tails by modified CO2/O2/N2 gas packaging

Three types of packaging systems were compared for their influence on the storage stability (2 °C) of precooked and peeled red claw crayfish tails. Modified atmosphere packaging (MAP; with 80%CO₂/10%O₂/10%N₂) almost totally inhibited the growth of aerobic bacteria and coliforms when compared with aerobic polyvinylchloride packaging (PVCP) and vacuum packaging (VP). MAP also prevented a pH rise, purge loss, and texture toughening or softening of stored red claw when compared with PVCP or VP (P<0.05). Lipid oxidation was minimal in all three packaging systems. Thus, MAP offers an attractive means to preserve the shelf life of precooked red claw stored under refrigerated retail display conditions.     

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 SAG₁and SAG₂. 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.     

Effect of modified atmosphere packaging on visual quality and glucosinolates of broccoli florets

Broccoli (Brassica oleracea var. italica) florets were packaged in polyethylene bags with no holes (M₀), two microholes (M₁), and four macroholes (M₂), and then stored at 4 or 20 °C. The effects of modified atmosphere packaging (MAP) treatments on visual quality and glucosinolate contents were determined by comparing with non-wrapped florets. The results showed that MAP treatments, especially with M₀ and M₁, extended the shelf life and reduced the postharvest deterioration of broccoli florets stored at 4 and 20 °C. All three MAP treatments reduced the decreasing concentration rates of individual, total aliphatic and indole glucosinolates in broccoli florets when compared to those in the control, with M₀ being the most significant, followed by M₁ and M₂ during 23 days of storage at 4 °C or 5 days of storage at 20 °C. Broccoli florets with M₀ treatment maintained the visual quality and glucosinolate contents for 13 days at 4 °C and 3 days at 20 °C.     

The effects of high oxygen modified atmosphere packaging on protein oxidation of bovine M. longissimus dorsi muscle during chilled storage

Modified atmosphere packaging (MAP) extends the shelf life of beef, especially in the context of visual colour. High O₂ MAP (70-80%) may cause quality deterioration through lipid and protein oxidation, the latter linked to a reduction in meat tenderness. The objective of this study was to investigate the effects of MAP in comparison to vacuum packaging (VP) on protein oxidation and subsequent tenderness of LD (M. longissimus dorsi) beef steaks during chilled storage (4 °C) of periods up to 14 days. Steaks were analysed for carbonyl content, free thiol groups, drip loss, Warner-Bratzler shear force (WBSF) and SDS-PAGE of extracted myofibrillar proteins. Free thiol groups were lower in high O₂ MAP (80% O₂/20% CO₂) steaks, after 14 days of storage, indicating protein oxidation compared to VP steaks. SDS-PAGE (non reducing conditions) showed the presence of high molecular weight cross linked myosin heavy chain aggregates in the high O₂ atmosphere packaged steaks, which were absent in VP steaks.     

Plant extracts combined with vitamin E in PUFA-rich diets of cull cows protect processed beef against lipid oxidation

The effect of supplementing PUFA-rich cull cow diets with vitamin E (2.8 g/animal/day) or vitamin E plus plant extracts rich in polyphenols (PERP) (126 g/animal/day), for 101 ± 3 days preceding slaughter, on the oxidative stability of longissimus thoracis (LT) and semitendinosus (ST) steaks was evaluated after ageing (for 12 d at 4 °C either in carcass or under-vacuum) and packaging (14 d under-vacuum (V), 4 d aerobic (A) and 7 d under modified atmosphere (70:30, O₂/CO₂) (MA)). The ageing method had no effect on a beef lipid oxidation intensity marker (malondialdehyde (MDA)), whereas packaging systems containing O₂ (A and MA) significantly increased lipid oxidation intensity (5 and 13 times higher than under V, respectively). Adding antioxidants to diets of animals given a PUFA-rich diet significantly improved lipid stability in steaks; the combination of vitamin E and PERP was more efficient than vitamin E alone for the most deleterious beef packaging.     

Effects of L- or D-lactate-enhancement on the internal cooked colour development and biochemical characteristics of beef steaks in high-oxygen modified atmosphere

The effects of L- or D-lactate on internal cooked colour development of steaks packaged in high-oxygen (80% O₂/20% CO₂) modified atmosphere packaging (MAP) was investigated. Ten USDA Select beef strip loins were divided individually into 4 equal-width sections, and one of four treatments (control, 0.3% sodium tripolyphosphate, 2.5% L-lactate + 0.3% sodium tripolyphosphate, and 2.5% D-lactate + 0.3% sodium tripolyphosphate) was assigned randomly to the loin sections. Loin sections were injected to approximately 10% of their raw weight. Steaks packaged in high-oxygen MAP were stored in the dark at 1 °C for 10 days. Instrumental internal colour of raw and cooked steaks (70 °C), total reducing activity (TRA), NADH concentration, and percent myoglobin denaturation (PMD) were measured. Cooked steaks enhanced with 2.5% L-lactate/phosphate maintained higher a*/b* ratios, lower hue values, higher TRA and NADH concentration, and lower PMD than the control and D-lactate-injected steaks, whereas enhancement with 2.5% D-lactate did not affect cooked colour, TRA, NADH, or PMD. Thus, inclusion of an L-lactate/alkaline phosphate blend increased the reducing activity of muscle tissues by replenishing NADH and subsequently decreased the thermal denaturation of myoglobin by maintaining the reduced state of myoglobin in the high-oxygen package.     

The spoilage characteristics of Brochothrix thermosphacta and two psychrotolerant Enterobacteriacae in vacuum packed lamb and the comparison between high and low pH cuts

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n = 306) inoculated with ~ 100 CFU of individual bacteria were stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (− 1.5 to 7 °C) for up to 84 days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7 °C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.     

Modified atmosphere packaging improved quality of kohlrabi stems

To improve the keeping quality of the kohlrabi stems and avoiding wilting of the leaves, modified atmosphere packaging (MAP) with oriented polypropylene and amide-polyethylene copolymer bags was applied. Avoiding wilting of the kohlrabi leaves is crucial because European consumers consider the freshness of kohlrabi leaves as a key quality parameter. Whole kohlrabies were stored within, above-mentioned sealed bags, for 14 days at 0 °C and 95% RH, followed by 3 days at 10 °C. In the MA packages gas compositions of about 5 kPa O₂ and 10-15 kPa CO₂ were generated. The respiratory activity, C₂H₄ production, firmness (of stems and leaves), sugars, organic acids contents, as well as chemical and sensory quality attributes were monitored. The respiratory rate of the kohlrabi stems in air at 0 °C was 10-11 mg CO₂/kg/h with a C₂H₄ production lower than 0.05 μl/kg/h (traces). However, when the temperature was increased at 10 °C the respiration rate raised 3.5 folds. Acidity, pH, soluble solids, sugars and organic acids content and firmness did not show significant changes at the end of both cold storage and retail sale periods. MAP was very effective improving keeping quality of the stems and retarding wilting of the leaves. During the retail sale period at 10 °C, the bags must to be perforated to avoid anaerobic conditions.     

Chemical composition, cytotoxicity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat

The present study describes the phytochemical profile and the protective effects of Ceratonia siliqua pods essential oil (CsEO), a food and medicinal plant widely distributed in Tunisia. Twenty five different components were identified in the CsEO. Among them, the major detected components were: Nonadecane, Heneicosane , Naphthalene, 1,2-Benzenedicarboxylic acid dibutylester, Heptadecane, Hexadecanoic acid, Octadecanoic acid, 1,2-Benzenedicarboxylic acid, Phenyl ethyl tiglate, Eicosene, Farnesol 3, Camphor, Nerolidol and n-Eicosane. The antimicrobial activity of CsEO was evaluated against a panel of 13 bacteria and 8 fungal strains using agar diffusion and broth microdilution methods. Results have shown that CsEO exhibited moderate to strong antimicrobial activity against the tested species. In addition, the inhibitory effect of this CsEO was evaluated in vivo against a foodborne pathogens Listeria monocytogenes, experimentally inoculated in minced beef meat (2 × 10² CFU/g of meat) amended with different concentrations of the CsEO and stored at 7 °C for 10 days. The antibacterial activity of CsEO in minced beef meat was clearly evident and its presence led to a strong inhibitory effect against the pathogens at 7 °C. On the other hand, the cytotoxic effects of the essential oil against two tumoral human cell lines HeLa and MCF-7 were examined by MTT assay. The CsEO showed an inhibition of both cell lines with significantly stronger activity against HeLa cells. The IC₅₀ values were 210 and 800 μg/ml for HeLa and MCF-7 cells, respectively. Overall, results presented here suggest that the EO of C. siliqua possesses antimicrobial and cytotoxic properties, and is therefore a potential source of active ingredients for food and pharmaceutical industry.     

Effect of thyme essential oil and packaging treatments on fresh Mediterranean swordfish fillets during storage at 4 °C

The present study evaluated the effect of thyme essential oil and packaging on fresh Mediterranean swordfish fillets during storage at 4 °C. Treatments in the present study included the following: air (A), modified atmosphere packaging (M), air with thyme oil (AT) and MAP with thyme oil (MT). Of the physicochemical parameters examined, TBA values for A and M swordfish samples were variable, indicative of no specific oxidative rancidity trend, whereas MT treatment inhibited lipid oxidation in swordfish samples during storage. On the basis of microbiological and sensory data, TMA-N and TVB-N limit values of acceptability for Mediterranean swordfish, of ca. 3.72 and 24.5 mg N/100 g, for the initiation of fresh Mediterranean swordfish spoilage, may be proposed. Of the treatments used in the present study, MT and M were the most effective for the inhibition of pseudomonads and H₂S-producing bacteria in swordfish. Lactic acid bacteria and Enterobacteriaceae (to a lesser extent) were also found to be part of the natural microbial flora of swordfish, irrespective of packaging treatment. Based primarily on sensory data, the shelf-lives of fresh refrigerated Mediterranean swordfish were 8 and 13 days under aerobic and MAP conditions, respectively. Addition of 0.1% thyme essential oil extended the product’s shelf-life under aerobic conditions by 5 days, whereas the combination of MAP and thyme oil resulted in a significant shelf-life extension of the swordfish fillets, i.e. by approximately 7½ days, according to sensory data, as compared to the control sample.