Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni.

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.

A collaborative study to evaluate qualitatively powdered baby food validation samples artificially contaminated with Salmonella anatum.

Nineteen laboratories participated in a collaborative study to evaluate the performance of validation samples consisting of powdered infant formula and cereal-based weaning food artificially contaminated with low levels of Salmonella anatum. The Standard method BS EN ISO 6579:2002 was to be followed for the trial. Salmonella counts in each baby food were around 10 CFU/25 g (low) or 10 CFU/g (high level). Trial participants received five samples of each sample type and five blank samples. All samples contained Pseudomonas aeruginosa and Lactobacillus spp. at concentrations between 10(4) and 10(5) CFU/g as background flora. Precision data for the powdered infant formula were similar for the solid selective plating-out medium xylose-lysine-deoxycholate (XLD) and a 2nd choice of agar suitable to isolate Salmonella spp. The sensitivity was 91.3%, accordance 82.5% and concordance 83.9% for the low and 100% for the high level in all cases. For the cereal-based weaning food, the precision data for the high level were similar for XLD and the 2nd choice medium (98.8%, 97.5% and 97.5%). A slight difference was observed for the low level where XLD obtained values of 82.5%, 73.8%, 70.6% for sensitivity, accordance and concordance and the second choice medium values of 81.5%, 72.5% and 69%. The specificity, accordance and concordance of blanks for the infant formula and cereal-based weaning food were 100%.

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed.

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.

A medium for the presumptive detection of Enterobacter sakazakii in infant formula: interlaboratory study.

A standard method for the detection of Enterobacteriaceae was modified for the presumptive detection of Enterobacter sakazakii, and the modified method was validated in an interlaboratory trial with 16 laboratories from 8 European countries. The modification included a differential-elective medium for the isolation of E. sakazakii, consisting of nutrient agar (NA) supplemented with 4-methyl-umbelliferyl alpha-D-glucoside (alpha-MUG). A 25 g sample was added to 225 mL buffered peptone water. After incubation at 35 degrees or 37 degrees C for 16 or 20 h, 10 mL nonselective enrichment was transferred into 90 mL selective enrichment. The selective enrichment was streaked on violet-red bile glucose agar (VRBGA) and incubated at 37 degrees C for 24 h. It was streaked in parallel on NA plates supplemented with alpha-MUG at 50 mg/L and incubated at 25 degrees C for 16 h, and afterwards for an additional 24 h at room temperature in the dark. E. sakazakii appeared as vivid yellow colonies under normal light and showed blue/violet fluorescence under UV light on NA + alpha-MUG plates. Validation samples represented powdered infant formula without E. sakazakii (blanks) and with low (1-10 colony-forming units [CFU]/25 g) and medium (1-10 CFU/g) contamination levels. All samples contained Pseudomonas aeruginosa and Lactobacillus spp. as background flora. The specificity for blank samples was 100%. The sensitivity of the low contamination level was similar for VRBGA and NA + alpha-MUG, i.e., 66.7% (66.7% accordance, 53.9% concordance). For the medium level the sensitivities were 96.7% (93.3% accordance, 93.5% concordance) for VRBGA and 98.3% (96.9% accordance, 96.9% concordance) for NA + alpha-MUG.

Enumeration of probiotic pediococci in animal feed: interlaboratory study.

An enumeration method to be used as official control under Council Directive 70/524/EEC for probiotic pediococci used as feed additives was validated for consideration for adoption as Comitée Européen de Normalisation (CEN) and ISO standards. Seventeen laboratories in 11 European countries carried out an interlaboratory study. A spread plate method following BS ISO 15214:1998 using 4 different agars [MRS, acidified MRS, MRS with triphenyl tetrazolium chloride (TTC), and a newly developed pediococci selective medium (PSM)] was validated. Precision data in terms of repeatability (r) and reproducibility (R) of the method for each medium using different feeding stuffs with a high and a low inoculation level were determined. Pediococci were present in the samples in mixtures with other probiotics. The enumeration of pediococci on all agars showed an RSDr value of 0.4-3.1% and an RSDR of 1.3-4.8%. MRS agar was preferred, followed by acidified MRS and MRS + TTC agar. All 4 media gave similar counts. Depending on the presence and concentration of other probiotic, such as enterococci, lactobacilli, and yeast, acidified MRS or MRS + TTC agar are recommended. The PSM was selective for pediococci and can be used if this species is present at a concentration more than 10-fold lower than other species that can grow on the MRS agars. The methodology with all 4 media is not applicable to mineral feed.

Enumeration of probiotic bacilli spores in animal feed: interlaboratory study.

Fourteen out of 17 laboratories completed an interlaboratory study comparing 2 pretreatment protocols of feed samples containing authorized probiotic bacilli spores. Both methods used tryptone soy agar for enumeration. Pretreatment A involved preparation of a suspension of the feed sample in 50% ethanol. For pretreatment B, the sample was suspended in peptone salt solution and heated at 80 degrees C for 10 min. Each laboratory analyzed 12 samples (6 per pretreatment), which represented duplicates of a high (10(9) colony-forming units [CFU]/g) and low (10(5) CFU/g) level of bacilli spores or a blank that contained vegetative probiotic bacteria only. For pretreatment A, the repeatability relative standard deviation (RSD(r)) was 2.9% for the low level and 2.5% for the high. The reproducibility relative standard deviation (RSDR) values were 7.8 and 5.9%, respectively. Pretreatment B revealed RSD(r) values of 1.1 and 1.0%, and RSDR values of 5.8 and 3.4%, respectively. The heat treatment (pretreatment B) of feed samples had better precision data, resulted in higher viable bacilli counts, and was more effective in deactivating vegetative background flora. It is therefore recommended for adoption for official control purposes and for CEN and ISO standards.

Validation of an official control method for enumeration of authorised probiotic yeast in animal feed.

An official control method in the framework of Council Directive 70/524/EEC for probiotic yeast used as feed additives was validated in a collaborative study by twenty laboratories in 12 European Countries. A pour plate method following ISO 7954 using chloramphenicol glucose yeast extract (CGYE) and a plate count method using CHROMagar Candida were used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Yeast was present in the samples in mixtures with other probiotic feed additives at a lower, a higher concentration or not present. The enumeration of yeast on CGYE agar showed for the lower and higher concentration a RSD(r) of 2.4-4.9% and a RSD(R) of 7.7-8%, respectively and was preferred by the majority of labs. CHROMagar Candida had a RSD(r) of 1.9-2.8% and a RSD(R) of 1.9-5.9%. For routine analysis the use of the pour plate technique is recommended. CHROMagar Candida can be used for confirmation of the species Saccharomyces cerevisiae. The methods are not recommended for mineral feeds. The results from this study are intended for consideration for adoption as CEN and ISO standards.

Antimicrobial effects of garlic, clove and red hot chilli on Listeria monocytogenes in broth model systems and soft cheese.

Antimicrobial activity of 1% (w/v) fresh garlic, ground clove and red dried chilli on Listeria monocytogenes was tested in broth systems at 37 degrees C and at 4 degrees C for 7 h. The initial cell concentration in the broth systems was between 2 x 10(6) and 4 x 10(6) CFU/ml. At 37 degrees C, growth to viable numbers of 3 x 10(8) CFU/ml in 7 h was measured. Clove had bacteriocidal activity and reduced the count to 1 CFU/ml. Garlic displayed bacteriostatic properties, and a count of 4 x 10(6) CFU/ml was maintained. Red chilli displayed an inhibitory effect and resulted in 50% lower counts than the control. L. monocytogenes had a slow growth rate at 4 degrees C and increased from an initial value of 3 x 10(6) to 5 x 10(6) CFU/ml during 7 h. The addition of garlic resulted in 3 x 10(6) CFU/ml, and clove reduced the viable cell concentration to 1 x 10(3) CFU/ml after 7 h. Two batches of soft cheese were produced in the laboratory using milk that was supplemented with L. monocytogenes. The final cheese containing L. monocytogenes with about 1 x 10(5) CFU/g. Half of each cheese batch was supplemented with either 1% garlic or 1% clove, whereby the other half served as a control. After 7 or 11 days incubation at 4 degrees C, the cheese was incubated at abuse temperature of 25 degrees C for 7 or 3 days, respectively. No antimicrobial effects of 1% (w/w) fresh garlic or clove powder on L. monocytogenes were observed in cheese after 1 or 2 weeks at the lower or higher temperature.

A collaborative study of a method for the enumeration of probiotic bifidobacteria in animal feed.

An enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic bifidobacteria used as feed additives was validated. Seventeen laboratories in 11 European Countries carried out a collaborative study. A spread plate method following BS ISO 15214:1998 using four different agars, Man Rogosa Sharpe (MRS), acidified MRS, MRS with triphenyl tetrazolium chloride (TTC) and a selective bifidobacteria medium, was validated. Precision data in terms of repeatability (r) and reproducibility (R) of the method for each medium using different feeding stuffs with a high and a low inoculation level were determined. Bifidobacteria were present in the samples as a single component or in mixtures with other probiotics. The enumeration of bifidobacteria on all agars showed a relative standard deviation of repeatability (RSD(r)) between 1.2% and 6.3% and a relative standard deviation of reproducibility (RSD(R)) between 2.6% and 8.7%. MRS agar was preferred, followed by acidified MRS and MRS+TTC agar. The selective bifidobacteria medium gave similar counts as the MRS media. For routine analysis, the use of MRS agar with supplementation of cysteine hydrochloride (the selective bifidobacteria medium without antibiotics) is recommended. Depending on the presence and concentration of other probiotics such as enterococci, lactobacilli and pediococci, acidified MRS or MRS+TTC agar is recommended. The selective bifidobacteria medium was selective for bifidobacteria. An official control method for enumeration of probiotic bifidobacteria as a single component and in mixtures with other probiotic microorganisms in feeding stuffs was validated. The methodology is not applicable to mineral feed. The results are intended for consideration for adaptation as CEN and ISO standards.

Thermal properties of bacterial spores and biopolymers.

Differential scanning calorimetry (DSC) measurements of dormant bacterial spores is traditionally associated with an endothermic transition at around 50 degrees C. This endothermic transition was described as an indicator for two main physico-chemical states in spores. These were a glassy state in the dormant spore core as a model for spore dormancy and a heat-activated state that generally facilitates spore resuscitation. The idea of a glassy state in dormant spores is based on the observation that a similar transition as in dormant spores was observed in low moisture biopolymers that are in a glassy state. Thermal properties of spores of Bacillus subtilis and B. cereus in a dormant and germinated, resuscitated state and of an outer and an inner coatless spore mutant of B. subtilis were investigated. Biopolymers with low moisture (<15%) and high moisture (>30%) contents such as maize starch, pectin, RNA and DNA were further studied. Critical evaluation of results revealed that the low temperature transition in dormant spores has some similarities to those observed in glassy biopolymers, but also to those of fully hydrated proteins and therefore does not necessarily indicate a glassy low moisture state. Its origin can also be attributed to the outer spore coats and it occurred at a lower temperature and for a shorter duration to be of significance for thermal heat activation of spores.

Laboratory-scale preparation of soft cheese artificially contaminated with low levels of Escherichia coli O157, Listeria monocytogenes, and Salmonella enterica serovars Typhimurium, Enteritidis, and Dublin.

The production of cheese with incurred low levels of pathogenic microorganisms stressed by the production process was the aim of the study. A standard protocol for the preparation of artificially contaminated soft cheese on a laboratory scale was developed. Milk for cheese preparation was artificially contaminated with pathogenic target microorganisms at low levels, between 1 and 10 CFU/ml. Two strains of Escherichia coli OI157:H7, two strains of Listeria monocytogenes, and three Salmonella spp. (Salmonella enterica serovars Typhimurium, Enteritidis, and Dublin) were investigated. The food pathogens in the cheese were exposed to the entire production process. All three microorganism species survived the cheese production process and were detected in the final product at concentrations between 1 and 50 CFU/g. The cheese produced contains target microorganisms that have been exposed to curd formation, drainage, setting, and ripening. This cheese can be used to validate microbiological methods or to examine the target microorganisms in a natural food environment at low concentrations. It represents an alternative to artificial contamination of cheese by adding target microorganisms to a final cheese product.

Effects of hydration on molecular mobility in phase-bright Bacillus subtilis spores.

The molecular mobility of 31P and 13C in dormant Bacillus subtilis spore samples with different water concentrations was investigated by high-resolution solid-state NMR. Lowest molecular mobility was observed in freeze-dried preparations. Rehydration to a 10% weight increase resulted in increases in molecular motions and addition of excess water furthered this effect. A spore slurry which had been freeze-dried displayed after addition of excess water similar NMR spectra to native wet preparations. Dipicolinic acid (DPA), which is mainly located in the core, was detected at all hydration levels in 13C cross-polarization magic angle spinning (CPMAS) but not in single-pulse magic angle spinning (SPMAS) spectra, indicating that hydration had no effect on its mobility. The molecular mobility of 31P, present mainly in core-specific components, was strongly dependent on hydration. This result suggests reversible water migration between inner spore compartments and the environment, whereas 13C spectra of DPA indicate that it is immobilized in a water-insoluble network in the core. Scanning transmission electron microscopy revealed that freeze-dried spores were significantly longer and narrower than fully hydrated spores and had a 3% smaller volume.