Use of conductance methods to predict bacterial counts in fish

Conductance methods to measure bacterial growth are more rapid than conventional methods for assessing the load of spoilage bacteria in fish. With the correct choice of medium, an estimate of the count can be obtained within 24 h which shows a very good correlation with the conventional methods. Moreover, the conductance changes correlate better with counts of those organisms thought to be responsible for spoilage. The Malthus conductance instrument provides an automated system capable of the simultaneous monitoring of 128 different samples, resulting in considerable savings of time and effort over traditional plate counting techniques.     

Isolation and characterization of Pseudomonas from processed chicken in Jamaica

Inflation and changes in the poultry industry in Jamaica have necessitated an increased understanding of spoilage flora and the examination of methods for increasing the shelf life of chilled eviscerated poultry. The efficacy of five antibiotics was determined. Despite the widespread use of neooxytetracycline in the treatment of poultry infections, tetracycline concentrations of < 10 μg ml^-1 were found to be highly effective against isolates of the main spoilage organism Pseudomonas.     

Theoretical Considerations when Estimating the Mesophyll Conductance to CO(2) Flux by Analysis of the Response of Photosynthesis to CO(2)

The conductance for CO₂ diffusion in the mesophyll of leaves can limit photosynthesis. We have studied two methods for determining the mesophyll conductance to CO₂ diffusion in leaves. We generated an ideal set of photosynthesis rates over a range of partial pressures of CO₂ in the stroma and studied the effect of altering the mesophyll diffusion conductance on the measured response of photosynthesis to intercellular CO₂ partial pressure. We used the ideal data set to test the sensitivity of the two methods to small errors in the parameters used to determine mesophyll conductance. The two methods were also used to determine mesophyll conductance of several leaves using measured rather than ideal data sets. It is concluded that both methods can be used to determine mesophyll conductance and each method has particular strengths. We believe both methods will prove useful in the future.     

A note on shelf-life extension of British fresh sausage by vacuum packing

Vacuum packing of British fresh sausage in a low oxygen permeability film (Diolon) extended the product shelf-life at 6 degrees C to more than 20 d compared with 9-14 d in conventional packs. After 10 d storage, counts of key spoilage organisms such as yeasts and Brochothrix thermosphacta were generally 2 log cycles lower in vacuum packs. Vacuum-packed sausages also displayed a slower rate of loss of free sulphite. Variations in pack permeability to SO2 were not responsible for this. Losses of free SO2 in stored sausages are largely due to the production of sulphite-binding agents by yeasts. Selective enumeration of these yeasts showed them to be inhibited by conditions of vacuum packing. The extension of shelf-life observed is ascribed to the reduction in growth rate of the spoilage flora in vacuum packs coupled with the consequent maintenance of inhibitory levels of sulphite for a longer period.     

Multi-Loci Sequence Typing (MLST) for Two Lacto-Acid Bacteria (LAB) Species: <Emphasis Type="Italic">Pediococcus parvulus</Emphasis> and <Emphasis Type="Italic">P. damnosus</Emphasis>

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.     

The Detection of Microbial Spoilage in Canned Foods Using Thin-layer Chromatography

Thin-layer chromatography was investigated as an inexpensive alternative to traditional microbiological techniques for the detection of microbial growth in canned vegetables. It was compared with culture methods, pH determination and direct microscopy in batches of canned vegetables with a known incidence of spoilage.     

THE PREVENTION OF MICROBIAL SPOILAGE IN WHOLE SHELL EGGS BY HEAT TREATMENT METHODS

SUMMARY: The prevention of microbial spoilage in whole shell eggs by heat treatment methods has been investigated. After shell eggs had been immersed for 3 min in water at 144·5 ± 0·5°F the incidence of microbial rotting was reduced by over 90%. Consistent reductions in spoilage losses were obtained with farm steeped eggs, eggs produced under commercial conditions, and eggs held in cold storage for 6 months. Heat treatment efficiently controlled green, sour and black rots in stored eggs, but although reductions were noted in the number of mould rots, it cannot be guaranteed that this type of spoilage can be controlled with similar certainty.
Internal quality in heat treated eggs was satisfactory. During protracted holding in cold storage, heat treatment appeared to result in a better internal egg quality and a more stable keeping quality, than when untreated eggs were held under similar conditions.     

Fish spoilage bacteria--problems and solutions

Microorganisms are the major cause of spoilage of most seafood products. However, only a few members of the microbial community, the specific spoilage organisms (SSOs), give rise to the offensive off-flavours associated with seafood spoilage. Combining microbial ecology, molecular techniques, analytical chemistry, sensory analysis and mathematical modelling allows us to characterise the SSOs and to develop methods to determine, predict and extend the shelf life of products.     

Microbial interaction in cooked cured meat products under vacuum or modified atmosphere at 4 degrees C

AIMS: To investigate the antagonistic activity of two lactic acid strains against the spoilage microflora in cooked cured meat products, vacuum or modified atmosphere packed at 4 degrees C and to determine the inhibitory capacity of their bacteriocins. METHODS AND RESULTS: Frankfurter-type sausages and sliced cooked cured pork shoulder were inoculated with Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 or with their bacteriocins. The microbial, physico-chemical (pH, L- and D-lactate, acetate and ammonia) and colour changes were studied. Results under vacuum packaging showed that in the uninoculated samples of the pork product the spoilage microflora grew but in the inoculated ones the spoilage microorganisms (e.g. Brochothrix thermosphacta and enterococci) reduced during the storage. This observation was more pronounced in the samples with the addition of bacteriocins. In the frankfurter-type sausages the spoilage microflora did not grow in the uninoculated and inoculated samples. In the modified atmosphere enriched in CO2 the population of spoilage microflora remained at low levels in both products, indicating that CO2 has an effect on the spoilage microorganisms' growth. In the pork product the concentrations of acetate and d-lactate increased while L-lactate decreased, but in the frankfurter-type sausages increase of acetate and D-lactate was not observed. CONCLUSIONS: Lactic acid strains had an effect on the spoilage microflora growth but did not affect, negatively, the organoleptic properties of the products. These strains may be used as biopreservative cultures or their bacteriocins could be an important contribution to microbiological quality of meat products. SIGNIFICANCE AND IMPACT OF STUDY: Establishment of biopreservation as a method for extension of shelf life of meat products.     

Quorum sensing in the context of food microbiology

Food spoilage may be defined as a process that renders a product undesirable or unacceptable for consumption and is the outcome of the biochemical activity of a microbial community that eventually dominates according to the prevailing ecological determinants. Although limited information are reported, this activity has been attributed to quorum sensing (QS). Consequently, the potential role of cell-to-cell communication in food spoilage and food safety should be more extensively elucidated. Such information would be helpful in designing approaches for manipulating these communication systems, thereby reducing or preventing, for instance, spoilage reactions or even controlling the expression of virulence factors. Due to the many reports in the literature on the fundamental features of QS, e.g., chemistry and definitions of QS compounds, in this minireview, we only allude to the types and chemistry of QS signaling molecules per se and to the (bioassay-based) methods of their detection and quantification, avoiding extensive documentation. Conversely, we attempt to provide insights into (i) the role of QS in food spoilage, (ii) the factors that may quench the activity of QS in foods and review the potential QS inhibitors that might "mislead" the bacterial coordination of spoilage activities and thus may be used as biopreservatives, and (iii) the future experimental approaches that need to be undertaken in order to explore the "gray" or "black" areas of QS, increase our understanding of how QS affects microbial behavior in foods, and assist in finding answers as to how we can exploit QS for the benefit of food preservation and food safety.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Estimation of radiation resistance values of microorganisms in food products

Several statistical methods, including the conventional technique of Schmidt and Nank, were evaluated for estimating radiation resistance values of various strains of Clostridium botulinum by the use of partial spoilage data from an inoculated ham pack study. Procedures based on quantal response were preferred. The tedious but rigorous probit maximum likelihood determination was used as a standard of comparison. Weibull's graphical treatment was the method of choice because it is simple to utilize, it is mathematically sound, and its ld(50) values agreed closely with the reference standard. In addition, it offers a means for analyzing the type of microbial death kinetics that occur in the pack (exponential, normal, log normal, or mixed distributions), and it predicts the probability of microbial death with any radiation dose used, as well as the dose needed to destroy any given number of organisms, without the need to assume the death pattern of the partial spoilage data. The Weibull analysis indicated a normal type kinetics of death for C. botulinum spores in irradiated cured ham rather than an exponential order of death, as assumed by the Schmidt-Nank formula. The Weibull 12D equivalent of a radiation process, or the minimal radiation dose (MRD), for cured ham was consistently higher than both the experimental sterilizing dose (ESD) and the Schmidt-Nank average MRD. The latter calculation was lower than the ESD in three of the five instances examined, which seems unrealistic. The Spearman-K?rber estimate was favored as the arithmetic technique on the bases of ease of computation, close agreement with the reference method, and providing confidence limits for the ld(50) values.     

A note on shelf-life extension of British fresh sausage by vacuum packing

Vacuum packing of British fresh sausage in a low oxygen permeability film (Diolon) extended the product shelf-life at 6°C to more than 20 d compared with 9–14 d in conventional packs. After 10 d storage, counts of key spoilage organisms such as yeasts and Brochothrix thermosphacta were generally 2 log cycles lower in vacuum packs. Vacuum-packed sausages also displayed a slower rate of loss of free sulphite. Variations in pack permeability to SO₂ were not responsible for this. Losses of free SO₂ in stored sausages are largely due to the production of sulphite-binding agents by yeasts. Selective enumeration of these yeasts showed them to be inhibited by conditions of vacuum packing. The extension of shelf-life observed is ascribed to the reduction in growth rate of the spoilage flora in vacuum packs coupled with the consequent maintenance of inhibitory levels of sulphite for a longer period.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

黄酒贮存酸败关键微生物的分离鉴定

【背景】贮存陈酿是黄酒生产的重要工艺环节,但由于黄酒中营养物质含量丰富,贮存陈酿过程中时常出现酸败变质的现象,尤其是在大罐的陈酿过程中酸败的发生对黄酒行业造成较大的经济损失。【目的】解析黄酒贮存陈酿过程中造成黄酒酸败的关键微生物,为黄酒贮存酸败微生物的控制提供依据。【方法】采用高通量测序技术分析不同来源酸败黄酒中的主要微生物种类;设计针对性培养基分离培养酸败黄酒中的难培养微生物;设计微生物特异性引物,对16S r RNA基因高度相似的酸败微生物进行区分鉴定;将分离的黄酒酸败微生物接入到未发生酸败的黄酒中验证其生酸能力。【结果】高通量测序技术分析结果显示,酸败黄酒中污染微生物主要为乳酸杆菌属(丰度95%)微生物,在种水平上分析比对结果显示两种难培养微生物[耐酸乳杆菌(Lactobacillus acetotolerans)和食果糖乳杆菌(Lactobacillus fructivorans)]的相对丰度达到了82%以上;采用改进的分离培养基分离出酸败黄酒中的难培养污染微生物36株;利用耐酸乳杆菌rec A基因和食果糖乳杆菌tuf基因的特异性引物准确鉴定出这些微生物菌株为28株耐酸乳杆菌和8株食果糖乳杆菌;将两种主要酸败微生物接入到未发生酸败的黄酒中,培养2周后能够显著提高黄酒酸度。【结论】基于高通量测序的未培养技术可作为食品中难培养污染微生物快速分析的有效方法。基于未培养技术和可培养技术相结合,首次解析并验证黄酒贮存过程中造成黄酒酸败的主要微生物为耐酸乳杆菌和食果糖乳杆菌。     

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef

A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min.     

Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 degrees C

AIMS: To evaluate the microbial spoilage, formation of biogenic amines and shelf life of chilled fresh and frozen/thawed salmon packed in a modified atmosphere and stored at 2 degrees C. METHODS AND RESULTS: The dominating microflora, formation of biogenic amines and shelf life were studied in two series of storage trials with naturally contaminated fresh and thawed modified atmosphere-packed (MAP) salmon at 2 degrees C. Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon at more than 10(6) cfu g(-1) and the activity of this specific spoilage organism (SSO) limited the shelf life of the product to ca 14 and 21 d in the two experiments. Despite the high levels of P. phosphoreum, less than 20 mg kg(-1) histamine was observed in fresh MAP salmon prior to sensory spoilage. Freezing eliminated P. phosphoreum and extended the shelf life of MAP salmon at 2 degrees C by 1-2 weeks. Carnobacterium piscicola dominated the spoilage microflora of thawed MAP salmon and probably produced the ca 40 mg kg(-1) tyramine detected in this product at the end of its shelf life. CONCLUSIONS: Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon but produced only small amounts of biogenic amines in this product. The elimination of P. phosphoreum by freezing allowed this bacteria to be identified as the SSO in fresh MAP salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of P. phosphoreum as the SSO in fresh MAP salmon facilitates the development of methods to determine and predict the shelf life of this product, as previously shown with fresh MAP cod.     

A rapid chemical spot test for the detection of lactic acid as an indicator of microbial spoilage in preserved foods

A ckland , M.R. & R eeder , J.E. 1984. A rapid chemical spot test for the detection of lactic acid as an indicator of microbial spoilage in preserved foods. Journal of Applied Bacteriology 56 , 415–419.
A rapid method for the detection of lactic acid in preserved foods has been developed, based upon a chemical spot test which does not require solvent extraction or derivatisation of the lactic acid in the sample. The test can be completed within 5 min and was shown to confirm microbial spoilage detected by cultural techniques in a range of preserved foods. In addition the test was able to indicate microbial spoilage in samples where cultural techniques failed because the spoilage organisms were dead. The test may not be appropriate for products which have a naturally high lactic acid content.     

Genome sequence of a food spoilage lactic acid bacterium, Leuconostoc gasicomitatum LMG 18811T, in association with specific spoilage reactions

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium causing spoilage of cold-stored, modified-atmosphere-packaged (MAP), nutrient-rich foods. Its role has been verified by challenge tests in gas and slime formation, development of pungent acidic and buttery off odors, and greening of beef. MAP meats have especially been prone to L. gasicomitatum spoilage. In addition, spoilage of vacuum-packaged vegetable sausages and marinated herring has been reported. The genomic sequencing project of L. gasicomitatum LMG 18811T was prompted by a need to understand the growth and spoilage potentials of L. gasicomitatum, to study its phylogeny, and to be able to knock out and overexpress the genes. Comparative genomic analysis was done within L. gasicomitatum LMG 18811T and the three fully assembled Leuconostoc genomes (those of Leuconostoc mesenteroides, Leuconostoc citreum, and Leuconostoc kimchii) available. The genome of L. gasicomitatum LMG 18811T is plasmid-free and contains a 1,954,080-bp circular chromosome with an average GC content of 36.7%. It includes genes for the phosphoketolase pathway and alternative pathways for pyruvate utilization. As interesting features associated with the growth and spoilage potential, LMG 18811T possesses utilization strategies for ribose, external nucleotides, nucleosides, and nucleobases and it has a functional electron transport chain requiring only externally supplied heme for respiration. In respect of the documented specific spoilage reactions, the pathways/genes associated with a buttery off odor, meat greening, and slime formation were recognized. Unexpectedly, genes associated with platelet binding and collagen adhesion were detected, but their functionality and role in food spoilage and processing environment contamination need further study.     

Stress tolerance in fungi -- to kill a spoilage yeast

The fungal spoilage of ingredients of food manufacture is an economic problem, often causes product loss and may constitute a health hazard. To effectively combat fungal food spoilage, a mechanistic understanding of tolerance for, and adaptation to, the preservation method used is crucial. Both are dependent on the genetic make-up and growth history of the organism. In the post-genomic era we are arriving at a situation in which, in the model organism Saccharomyces cerevisiae, physiological data, classical molecular biology and whole-genome responses can be combined to obtain explanatory and predictive models for growth. For food spoilage fungi we have not yet reached such a level of understanding, but we may use the knowledge gained for S. cerevisiae for the prevention of spoilage.