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.     

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.     

Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef

Drip samples were collected at 4-week intervals from 10 vacuum-packaged beef striploins stored for 16 weeks at −1.5 °C and assayed for populations of lactic-acid bacteria (LAB), pH and spoilage-causing fermentation products. A total of 15 LAB species were identified using pulsed-field gel electrophoresis and biochemical analysis. A pattern of succession was observed during storage between strains of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. Acetic acid production was associated with increasing LAB populations generally and butyric acid production was associated with the development of a particular strain of Leuconostoc. Changes in pH is postulated as a driver of succession.     

Influence of various preservatives on the quality of minced beef under modified atmosphere at chilled storage

The effect of preservatives on microbial quality, pH, drip-loss, roasting-loss, colour, and sensorial properties of modified atmosphere packaged (70% O₂ and 30% CO₂) minced beef (M. semimembranosus) stored at (2 ± 0.5 °C) for 12 days was investigated. Beef cubes (approx. 20 × 20 × 20 mm size) were immersed in solutions of 2% and 5% lactic acid, 2% lactic acid combined with 0.5% sodium ascorbate, 20% potassium lactate and 20% potassium sorbate before mincing. Addition of lactic acid was associated with pH drop, which increased drip-loss and roasting-loss. Application of all additives inhibited aerobic micro-organisms (10³–10⁴ CFU g⁻¹ on day 12) compared to reference sample (9 × 10⁵ CFU g⁻¹ on day 12). Lactic acid discoloured samples, while sodium ascorbate seemed to improve colour stability. Despite good visual colour characteristics, potassium sorbate treated samples were organoleptically unacceptable with massive off-flavour.     

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.     

The effect of glucose supplementation on the spoilage microflora and chemical composition of minced beef stored aerobically or under a modified atmosphere at 4 °C

Glucose was added to minced meat (pH 6.0) and stored under aerobic or a modified atmosphere (MA) composed of 80% O₂ and 20% CO₂ to assess the effects of carbohydrate on the microbial association and the chemical properties of the meat. The type of packaging affected the size and the final composition of the microflora. The microbial composition of the mince without added glucose in air or MA, given in log₁₀cfu/g respectively, were pseudomonads (9.8 and 7.3), Brochothrix thermosphacta (8.5 and 8.1), lactic acid bacteria (8.8 and 8.7), Enterobacteriaceae (7.2 and 6.1) and yeasts (4.3 and 4.2). In mince supplemented with 0.2% (w/w) glucose, similar composition and numbers were observed. Glucose, glucose 6-phosphate and lactic acid were consumed at slower rates by the flora on meat stored under MA than by the flora on meat stored in air. The addition of glucose enhanced gluconate production by the flora on meat stored in air. D-lactic and acetic acid were produced in all samples stored under the MA.     

乳酸菌直投发酵剂培养与冷干条件的优化

采用响应面法对保加利亚乳杆菌(Lactobacillusbulgaricus)和嗜热链球菌的增菌(Streptococcusther-mophilus)培养基进行了优化,并且对其冻干保护剂进行了筛选,结果表明通过流加碱控制保加利亚乳杆菌的pH值为5.8,嗜热链球菌的pH值为6.4,培养15h,菌体的浓度分别达到1.24×109cfu/mL和8.05×108cfu/mL。离心收集到的菌体,在-70℃的冰箱先预冻3h,然后在冷阱温度-51℃、真空度9Pa冻24h,最终可以得到含活菌数为1.43×1010cfu/g的高活力酸乳发酵剂。     

Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Sardinian sausage, a typical Italian dry fermented sausage. Samples were taken at different stages, and 112 strains were isolated. The isolates were characterized using the micromethod proposed by Font de Valdez et al. [Font de Valdez, G., Savoy de Giori, G., Oliver, G., & De Ruiz Holgado, A. P. (1993). Development and optimization of an expensive microsystem for the biochemical characterization of lactobacilli. Microbiologie Aliments Nutrition, 11, 215–219]. Schillinger and Lücke’s [Schillinger, U., & Lücke, F. K. (1987). Identification of lactobacilli from meat and meat products. Food Microbiology. (4), 199–208] scheme and the biochemical patterns given by Bergey’s Manual of Systematic Bacteriology [Bergey’s Manual of Systematic Bacteriology (1986). Baltimore: William and Wilkins] were used for preliminary identification. A PCR-based method was then used to confirm the results. LAB were the dominant flora during ripening. They consisted mainly of homofermentative mesophilic rods. Lactobacillus sakei (43,3%), Lactobacillus plantarum (16,6%) and Lactobacillus curvatus (13,3%) were the main isolates. The results of the biochemical identification methods agreed well with those of PCR-based identification (91% agreement).     

Shelf life and stability traits of traditionally and modified atmosphere packaged ground beef patties treated with lactic acid bacteria, rosemary oleoresin, or both prior to retail display

Previous research indicates that lactic acid bacteria (LAB) can inhibit pathogenic bacteria. This research evaluated effects of LAB inclusion on the shelf life of traditionally packaged ground beef patties; as well as the effects and possible interaction of LAB and rosemary oleoresin (RO) on the stability of high oxygen MAP ground beef during display. In both package types, trained and consumer evaluations indicated no effect (P > 0.05) of LAB on lean color and off-odor. Display affected trained and consumer sensory evaluations and indicated declined stability over time. Thiobarbituric acid values were lower for traditionally packaged ground beef with LAB (P < 0.05) and MAP ground beef with RO or RO and LAB (P < 0.05). Overall, LAB had no effect on the shelf life and stability of traditionally or high-oxygen MAP packaged ground beef patties. Therefore, utilization of LAB in ground beef to reduce pathogenic bacteria is viable without alteration of spoilage indicators.     

真空冷冻干燥过程中保护剂对乳酸菌的保护机理

通过综述乳酸菌发酵剂生产过程中真空冷冻干燥前期不同保护剂的添加对其保护机理,提出合理的保护措施,为工业化生产较高稳定性的乳酸菌发酵剂提供理论依据.     

乳酸菌对玉米加工特性的影响及基于均匀设计优化玉米发酵条件

本实验选择东北地区大面积推广种植具有代表性的8个普通玉米品种和2个糯玉米品种进行研究,通过乳酸菌发酵,分析玉米保水力、膨胀势、溶解度和糊化特性的变化趋势;并以膨胀势作指标,采用均匀实验设计,优化适于发酵的玉米品种及发酵条件。实验结果表明:经乳酸菌发酵,玉米品种间加工特性变化差异显著(p<0.05),糯玉米的保水力、溶解度显著高于普通品种玉米(p<0.05);而膨胀势、回生值显著低于普通品种玉米(p<0.05);普通品种玉米的峰值粘度和衰减值经发酵作用有所降低,而糯玉米品种的峰值粘度和衰减值经发酵后升高。在所选品种中郑单958最适于发酵,料液比1∶2(g/m L),37℃发酵5 d,所得玉米粉膨胀势高达12.93 g/g,其加工特性得到有效改善。      

DVS乳酸菌种菌株保存过程中遗传稳定性分析

对从某直投式酸奶发酵剂中分离出的乳酸菌菌株在保存过程中的遗传稳定性进行了分析。观察各种保存情况下各菌株复壮时的形态．用RAPD方法分析不同保存方法保存不同时间的乳酸菌基因组的变化，并用SDS-PAGE对比不同保存方法保存不同时间乳酸菌的全细胞蛋白质表达的差异。结果表明，乳球菌在4℃保存和-80℃保存过程中基因组有变化，蛋白质表达无变化；冻干保存前后基因组和蛋白质表达均无变化。杆菌1在4℃保存过程中基因组基本无变化．蛋白质表达有变化；-80℃保存过程中基因组有变化，蛋白质表达无明显变化；冻干保存前后基因组和蛋白质表达均无变化。杆菌2在4℃保存过程中基因组和蛋白质表达均有变化；-80℃保存过程中基因组和蛋白质表达均有变化：冻干前后基因组和蛋白质表达均无变化。采用RAPD方法可以有效地对乳酸菌种菌株保存过程中的遗传稳定性进行分析。     

Fingerprint of lactic acid bacteria population in beef carpaccio is influenced by storage process and seasonal changes

We have investigated the population structure of lactic acid bacteria (LAB) for several beef carpaccio available on the market with the purpose of comparing the effect of storage process (modified-atmosphere packaging and vacuum-packaging) and of seasonal changes on this microbial population. Out of 60 samples we have characterised 214 isolates accounting for 10 LAB species and 35 isolates accounting for 11 non-LAB species. Lactobacillus sakei, Leuconostoc carnosum and Leuconostoc mesenteroides were the most prevailing LAB species with a frequency of identification within 66%, 62% and 52% of the samples respectively. These 3 species were also characterised by a phenotypic intra-species diversity of isolates based on colony morphology. We showed that the prevalence was increased 1.5 fold for L. sakei and L. mesenteroides during the summer sampling in comparison to the spring or the fall sampling suggesting an environmental origin of these two species. Seasonal variations were also observed for the prevalence of Lactobacillus fuchuensis and L. carnosum in spring (2- and 1.5-fold increase, respectively) and of Brochothrix thermosphacta in fall (6-fold increase). Finally, we demonstrated that the growth potential after the sell-by-date was favourable of 1.25 log₁₀ cfu g⁻¹ to Leuconostoc spp. in modified-atmosphere packaging and of 1.38 log₁₀ cfu g⁻¹ to Lactobacillus spp. in vacuum-packaging. In conclusion, we show that important and unsuspected traits in bacterial population dynamics can be unravelled by large sampling strategies. We discuss about the need to take this assessment into account for further studies on bacterial ecosystems of meat.     

Lactic acid bacteria community dynamics and metabolite production of rye sourdough fermentations share characteristics of wheat and spelt sourdough fermentations

Four spontaneous rye sourdough fermentations were performed over a period of ten days with daily back-slopping. Samples taken at all refreshment steps were used for culture-dependent and culture-independent characterization of the microbiota present. Furthermore, an extensive metabolite target analysis was performed through a combination of various chromatographic methods, including liquid chromatography coupled to mass spectrometry (LC/MS) and gas chromatography coupled to mass spectrometry (GC/MS). Spearman’s rank correlation coefficients were calculated and a principal component analysis (PCA) was performed on the data obtained in this study combined with data obtained previously for wheat and spelt sourdoughs. In general, the establishment of a stable microbial ecosystem occurred through a three-phase evolution, with mainly Lactobacillus plantarum and Lactobacillus fermentum dominating the rye sourdough ecosystems. PCA revealed that ornithine and mannitol were positively correlated with rye sourdoughs, contributing to bacterial competitiveness at the onset of sourdough production. Wheat and spelt sourdoughs showed a high degree of similarity, although certain compounds (e.g. indolelactic acid) appeared to be specific for spelt sourdoughs. The production of amino acid metabolites, mainly hydroxy acids (e.g. phenyllactic acid) and alcohols (e.g. 3-methyl-1-butanol), contributed to the equilibration of the redox balance and further enhanced the competitiveness of dominant species in stable sourdoughs.     

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 °C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.     

Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions

The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied in fermented milk under non-refrigerated conditions (30 °C) with a view to develop ambient-stable fermented milk with live LAB. Five yeasts tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowia lipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but not Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by ~ 10² to 10⁵-fold. Seven yeasts examined (Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membranifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus in fermented milk by ~ 10³ to 10⁶-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10⁶-fold, but the same yeast failed to improve the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermented milk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and that the specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecular mechanism of such interaction of yeasts with LAB remains to be found.     

Effect of storage temperature and gas permeability of packaging film on the growth of lactic acid bacteria and Brochothrix thermosphacta in cooked meat emulsions

The effect of gas permeability of packaging film on the growth of lactic acid bacteria and Brochothrix thermosphacta in cooked meat emulsions stored at 0, 8 and 15 °C was investigated. The estimated parameters from Gompertz equation for the assayed temperature–oxygen permeability combinations showed LAB development to be significantly greater than those of B. thermosphacta. The influence of the two sources of variation (oxygen permeability of packaging film and temperature) on the growth parameters of LAB and B. thermosphacta was analysed showing a significant effect (P<0.001) of the temperature on both bacterial population while the film permeability had only a significant influence (P<0.001) on B. thermosphacta growth. Under the conditions of this study the packaging film influenced the maximum counts and growth rates of both organisms. Since the inhibition of B. thermosphacta occurred when the meat product was vacuum-packaged in films possessing high oxygen permeability and the effect of pH was found not to be associated with the growth inhibition, accumulation of hydrogen peroxide produced by LAB may possibly be one of the main factors responsible for B. thermosphacta inhibition. Shelf-life of vacuum-packaged cooked meat emulsions in high oxygen transmission rate films will be guarantied and a temperature abuse will not result in an increase of spoilage by LAB.     

Treatment of Escherichia coli O157:H7 with lactic acid, neutralized electrolyzed oxidizing water and chlorine dioxide followed by growth under sub-optimal conditions of temperature, pH and modified atmosphere

The utilization of sub-lethal decontamination treatments gains more and more interest due to the increased consumers' demand for fresh, minimally processed and convenient food products. These products rely on cold chain and hurdle (combination) technology to provide microbiological safety and quality during their shelf life. To investigate the ability of surviving cells to resuscitate and grow in a food simulating environment, sub-lethal decontamination treatments were coupled with subsequent storage under sub-optimal growth conditions. For this purpose chlorine dioxide (ClO₂) and neutralized electrolyzed oxidizing water (NEW)-treated cultures of Escherichia coli O157:H7 were inoculated in TSB-YE of pH 5.8 and a_w 0.99, and stored at 10 °C, 12.5 °C and 15 °C, under four different atmospheres (0%, 30% and 60% CO₂ balanced with N₂, and air). Due to the severity of injury, lactic acid-treated cells were inoculated in TSB-YE pH 7.0. Data obtained reveal that the fraction of sub-lethally injured E. coli O157:H7 undergoes an additional inhibitory effect during the storage period under of sub-optimal conditions. Observed extension in the lag growth phase was a direct consequence prior sub-lethal injury. The effects of liquid ClO₂ and NEW were less pronounced in comparison to lactic acid. The current study signifies the potential utilization of appropriate combination of different extrinsic and intrinsic factors in the elimination or growth inhibition of food-borne pathogens.