Determination of the carbonation tolerance of yeasts

A simple method is described for the determination of the carbonation tolerance of yeasts. Using the method, data are presented for the carbonation tolerance of some common soft drink spoilage yeasts.     

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.     

Isolation of a hop-sensitive variant of Lactobacillus lindneri and identification of genetic markers for beer spoilage ability of lactic acid bacteria

We have isolated a hop-sensitive variant of the beer spoilage bacterium Lactobacillus lindneri DSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designated horB(L) and horC(L) that encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs of horB and horC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those of horA and ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use of horB/horC and horA is able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison of horB and horC homologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution of horB and horC homologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.     

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.     

Isolation of a Hop-Sensitive Variant of Lactobacillus lindneri and Identification of Genetic Markers for Beer Spoilage Ability of Lactic Acid Bacteria

We have isolated a hop-sensitive variant of the beer spoilage bacterium Lactobacillus lindneri DSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designated horB_L and horC_L that encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs of horB and horC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those of horA and ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use of horB/horC and horA is able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison of horB and horC homologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution of horB and horC homologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.     

Well-known quorum sensing inhibitors do not affect bacterial quorum sensing-regulated bean sprout spoilage

AIM: To investigate the potential of quorum sensing inhibitors (QSI) as food preservative agents in a food product, where bacterial spoilage is controlled by quorum sensing (QS). METHODS AND RESULTS: The effects of well-known QSI were tested on spoilage phenotypes and on QS-regulated genes of a bean sprout spoiling bacterial isolate (Pectobacterium A2JM) in laboratory substrates and in a bean sprout model system. The acylated homoserine lactones (AHL) analogues PenS-AHL and HepS-AHL decreased the specific protease activity of Pectobacterium A2JM in broth but did not reduce the expression of a QS-regulated secretion protein, and were without effect on soft rot of bean sprouts. The QSI ProS-AHL, furanone C-30, patulin, penicillic acid and 4-nitropyridine-N-oxide did not have any effect on protease activity, on gene expression or bean sprout appearance at nongrowth inhibitory concentrations. Extracts from garlic and bean sprouts induced the QS system of Pectobacterium in bean sprouts and a broth system, respectively. CONCLUSIONS: Among the several well-known QSI compounds, only PenS-AHL and HepS-AHL, inhibited QS-regulated protease activity of Pectobacterium A2JM in broth cultures, but had no effect on bean sprout spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: The QSI compounds must be selected in the specific system in which they are to function and they cannot easily be transferred from one QS system to another.     

Detection of rope spoilage in bread caused by Bacillus species

Rope spoilage of bread by eight Bacillus isolates obtained from a bakery environment was examined via direct inoculation of slices of bread with bacterial culture. The three types of loaf examined were two soft grain varieties, one containing vinegar and the other containing calcium propionate as the preservative agent, and a white variety containing calcium propionate. Differences in rope production caused by batch variation were studied by comparing seven loaves of each type of bread. Not all isolates were capable of causing extensive rope, but isolates of Bacillus subtilis , B. licheniformis , B. megaterium and B. pumilus were able to produce such spoilage. Limited rope was also caused by pre-existing Bacillus species within the loaves. The amount of rope production by an isolate was not constant on all loaf types or even between different batches of the same variety, indicating that approaches that rely on direct inoculation of loaves with culture are not applicable for assessing the rope-inducing potential of Bacillus isolates. However, it was clear from this study that vinegar in soft grain loaves was more effective than calcium propionate at inhibiting rope.     

Asaia sp., an unusual spoilage organism of fruit-flavored bottled water

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10(6) CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.     

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco

AIMS: Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. METHODS AND RESULTS: E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. CONCLUSION: E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.     

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.     

Asaia sp., an Unusual Spoilage Organism of Fruit-Flavored Bottled Water

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10⁶ CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.     

Temperature function integration and the development and metabolism of poultry spoilage bacteria

The rate of spoilage of chicken tissues, the development of spoilage bacteria, and the utilization of amino acids by spoilage bacteria as a function of temperature were more accurately described by the general spoilage curve of Olley and Ratkowsky (Food Technol. Aust. 25:66-73, 1973; Food Technol. N.Z. 8:13-17, 1973) than by the linear equation of Spencer and Baines (Food Technol. [Chicago] 18:175-179, 1964). Remaining shelf life of poultry tissues may be predicted at temperatures up to 16 degrees C by using a temperature function integrator which incorporates the general spoilage curve.     

Temperature function integration and the development and metabolism of poultry spoilage bacteria.

The rate of spoilage of chicken tissues, the development of spoilage bacteria, and the utilization of amino acids by spoilage bacteria as a function of temperature were more accurately described by the general spoilage curve of Olley and Ratkowsky (Food Technol. Aust. 25:66-73, 1973; Food Technol. N.Z. 8:13-17, 1973) than by the linear equation of Spencer and Baines (Food Technol. [Chicago] 18:175-179, 1964). Remaining shelf life of poultry tissues may be predicted at temperatures up to 16 degrees C by using a temperature function integrator which incorporates the general spoilage curve.     

Why fruit rots: theoretical support for Janzen's theory of microbe–macrobe competition

We present a formal model of Janzen''s influential theory that competition for resources between microbes and vertebrates causes microbes to be selected to make these resources unpalatable to vertebrates. That is, fruit rots, seeds mould and meat spoils, in part, because microbes gain a selective advantage if they can alter the properties of these resources to avoid losing the resources to vertebrate consumers. A previous model had failed to find circumstances in which such a costly spoilage trait could flourish; here, we present a simple analytic model of a general situation where costly microbial spoilage is selected and persists. We argue that the key difference between the two models lies in their treatments of microbial dispersal. If microbial dispersal is sufficiently spatially constrained that different resource items can have differing microbial communities, then spoilage will be selected; however, if microbial dispersal has a strong homogenizing effect on the microbial community then spoilage will not be selected. We suspect that both regimes will exist in the natural world, and suggest how future empirical studies could explore the influence of microbial dispersal on spoilage.     

Presence of acylated homoserine lactones (AHLs) and AHL-producing bacteria in meat and potential role of AHL in spoilage of meat

Quorum-sensing (QS) signals (N-acyl homoserine lactones [AHLs]) were extracted and detected from five commercially produced vacuum-packed meat samples. Ninety-six AHL-producing bacteria were isolated, and 92 were identified as Enterobacteriaceae. Hafnia alvei was the most commonly identified AHL-producing bacterium. Thin-layer chromatographic profiles of supernatants from six H. alvei isolates and of extracts from spoiling meat revealed that the major AHL species had an R(f) value and shape similar to N-3-oxo-hexanoyl homoserine lactone (OHHL). Liquid chromatography-mass spectrometry (MS) (high-resolution MS) analysis confirmed the presence of OHHL in pure cultures of H. alvei. Vacuum-packed meat spoiled at the same rate when inoculated with the H. alvei wild type compared to a corresponding AHL-lacking mutant. Addition of specific QS inhibitors to the AHL-producing H. alvei inoculated in meat or to naturally contaminated meat did not influence the spoilage of vacuum-packed meat. An extracellular protein of approximately 20 kDa produced by the H. alvei wild-type was not produced by the AHL-negative mutant but was restored in the mutant when complemented by OHHL, thus indicating that AHLs do have a regulatory role in H. alvei. Coinoculation of H. alvei wild-type with an AHL-deficient Serratia proteamaculans B5a, in which protease secretion is QS regulated, caused spoilage of liquid milk. By contrast, coinoculation of AHL-negative strains of H. alvei and S. proteamaculans B5a did not cause spoilage. In conclusion, AHL and AHL-producing bacteria are present in vacuum-packed meat during storage and spoilage, but AHL does not appear to influence the spoilage of this particular type of conserved meat. Our data indicate that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. H. alvei may thus influence spoilage of food products in which Enterobacteriaceae participate in the spoilage process.     

Rapid estimation of spoilage bacterial load in aerobically stored meat by a quantitative polymerase chain reaction

We report a quantitative PCR which utilizes primers from a conserved 23S rDNA sequence identified in nine different spoilage bacteria commonly present in meat. The PCR detected the spoilage bacteria by amplifying a specific 207 bp sequence from their chromosomal DNA. Quantification of PCR product by electrochemiluminescence revealed that the concentration of the amplified product was dependent on cycle number and the initial number of bacteria present in the sample. Statistical analysis of the results indicated a correlation coefficient of 0.94 (P < 0.001) between aerobic plate count and QPCR luminosity units.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

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.     

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

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.     

Clostridium novyi type B as a causative agent of bovine meat spoilage

A series of bovine meat spoilage cases in which meat from clinically healthy Belgian Blue cattle showed green discoloration are described. Histology of skeletal muscle revealed numerous spore-forming rods in the discolored areas of the meat. These organisms stained positively for Clostridium novyi by immunohistochemistry. A combination of 16S rDNA and fliC gene sequencing of bacterial DNA, isolated from the spoiled meat samples, revealed the unique presence of C. novyi type B. Although this bacterium has been implicated in clinical necrotic hepatitis in cattle, the cases described here are the first implicating C. novyi type B as a cause of bovine meat spoilage.