Bacteria that differentiate into highly heat-resistant endospores (HHRS strains) may survive ultrahightemperature treatment of milk and germinate in the final product. They do not noticeably spoil the milk and are nonpathogenic. The complete (>96%) 16s rRNA genes from three HHRS strains were identical, and phylogenetic analysis placed them alongside Bacillus firmus in the B. meguterium group of the genus Bacillus. Moreover, the approximately 550 nucleotides between regions U2 and U5 were invariant for seven HHRS strains. However, three cloned 16s rRNA genes from one HHRS strain, M215, showed marked size and sequence variations within the V1 and V2 regions. DNA reassociation assays confirmed the distinction between a reference HHRS strain and closely related members of the B. meguterium group, notably, B.firmus (30%), B. benzoevorans (28%), and B. circuhns (20%). Ribotyping and pyrolysis mass spectrometry both indicated that the HHRS strains belong to a homogeneous, species-ranked taxon, an exception being strain TP1248, which is slightly atypical. The HHRS strains are unusual in that they grow poorly, if at all, on nutrient agar; good growth is obtained on brain heart infusion agar. On subculture, most HHRS strains form long, filamentous rods which stain unevenly in the Gram reaction. They are strictly aerobic and do not produce acid from sugars. We propose the name Bacillus sporothermoduruns for these bacteria, which are phenotypically and phylogenetically distinct from other Bacillus species. The type strain is M215 (= DSMZ 10599).High-temperature processing of milk takes several forms, including autoclaving to produce sterilized milk and ultrahightemperature (UHT) treatment to provide a product which can be stored without refrigeration for prolonged periods, generally, several months. The typical heating regimen for autoclaving is 115 to 120°C for 15 to 20 min or 109 to 115°C for 20 to 40 rnin, and for UHT treatment the range is 135 to 142°C for a few seconds, which should result in the destruction of any vegetative cells and endospores present in the raw material. Occasionally, spoilage can occur in sterilized and UHT-treated milk, usually as a result of contamination during filling operations. Members of the genus Bacillus, notably, Bacillus badius, B. cereus, B. licheniformis, B. polymyxa, B. subtilis, and B. stearothermophilus, have been identified in such situations (10, 23, 36).In certain circumstances, exceptionally heat-resistant endospores survive the UHT or autoclaving treatment and pass into the final product. These endospores may subsequently germinate and grow in the stored milk (8, 15). These mesophilic bacteria, which differentiate into highly heat-resistant spores (HHRS), were detected first in UHT-treated milk from southern Europe in 1985 and in UHT-treated milk from Germany in 1990. Today, the problem is more widespread and has been noted in several other European countries, including the Benelux countries, France, and Spain, as well as in some nonEuropean dairies (15). HHRS bacteria multipl...

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Thirty milk samples [high-temperature short-time (HTST) milk, extended shelf life (ESL) milk (directly heated, indirectly heated, microfiltered), ultra-high temperature milk] from 17 German dairies were analysed. Total viable counts of directly or indirectly heated ESL milk were significantly lower than those in microfiltered ESL and HTST milk. Evaluation of indigenous enzyme activity revealed sufficient heat treatments in all milk samples. The manufacturing processes were differentiated by estimating furosine and acid soluble whey proteins. Sensory examinations revealed a preference for HTST heated and microfiltered ESL milk. However, a significant discrimination of drinking milk types was not possible. Vitamin losses were not detected, and concentrations of vitamins in different types of milk were comparable.Keywords Drinking milk, Microbiological status, Head load evaluation, Sensory quality, Vitamin status. I N T R O D U C T I O NExtended shelf life (ESL) milk has a shelf life of about 3 weeks under chill chain conditions and fills the gap between high-temperature short-time (HTST)-heated milk, which typically is assigned a shelf life of 10 days and ultra-high temperature (UHT)-heated milk, which can be stored for a few months without cooling (Hoffmann et al. 2006). In Germany, ESL milk has a market share of 20-25%. For comparison, UHT milk has a share of approximately 70%, and HTST milk has a market share of 5-10% (BLE 2008). The term ESL milk as well as the manufacturing process is not legally defined in the European Union. A thermal treatment or combination of heat treatment and membrane filtration are involved in the production of ESL milk. The thermal process requires direct or indirect heating at 123-127°C with a holding time of 1-5 s. Traditional HTST pasteurisation is carried out at 72-75°C for 15-30 s, and UHT milk is heated at a minimum of 135°C for a few seconds (sterilisation value F 0 ‡ 3 min) (Schwermann and Schwenzow 2008a,b;Kaufmann et al. 2009). The combined treatment process for ESL milk includes microfiltration of skim milk through ceramic membranes with an average pore diameter of 0.8-1.4 lm. As a result, a spore reduction of 3-5 log 10 steps is achieved and most other forms of microorganisms are also separated. The so enriched retentate and a specific amount of cream are heated at 123-127°C, homogenised, and mixed with the HTST heated skim milk permeate (Hoffmann et al. 2006;Schwermann and Schwenzow 2008c;Henke 2009).Microbial spoilage of heat treated drinking milk is caused by heat-resistant or recontaminating micro-organisms. The former group is represented by spore forming micro-organisms, mainly Bacillus spp. and Enterococci. The latter group consists of Gram-positive nonspore forming bacteria and Gram-negative bacteria. These organisms gain significance if they are psychrophilic or psychrotrophic and able to multiply at refrigeration temperatures (Blake et al. 1995;Mayr et al. 2004;Kress et al. 2005;Kaufmann and Kulozik 2008).Several parameters are suited for the evaluation of milk...

Several outbreaks of Cronobacter spp. (Enterobacter sakazakii) have been described as food-borne illness in neonates and infants. Powdered infant formula has been identified as a source of infection, especially in hospital nurseries, where a bulk of formula nutrient is prepared for the whole day and instructions for preparation are not always followed correctly. Neonates who are underweight or immunosuppressed are especially at risk for an E. sakazakii infection. Considering that milk powder is the main ingredient of powdered infant formula, we analyzed the incidence and distribution of E. sakazakii in a milk powder-producing plant. We looked specifically at the spray-drying towers and roller dryers. Selected isolates from samples taken from the environment and final product were typed by pulsed-field gel electrophoresis to investigate the epidemiology of the organism within the production area of the plant. Seven pulsed-field gel electrophoresis types were detected in the spray-drying area, which presumably entered the plant through an aperture for process air and an improperly controlled roller shutter. Furthermore, textile filters for exhaust air of both the spray-drying towers were identified as internal reservoirs of the pathogen. For economic reasons, powder from the textile filters is reintroduced into the product flow; this can contaminate the final product. For the production of milk powder to be used as an ingredient of powdered infant formula, it was suggested to terminate the process of reintroducing the filtered powder into the product flow. A second transmission route was identified in the roller dryer section of the factory. It could be shown that contaminated milk concentrate could pass the process unheated, thus leading to a contamination of the product with E. sakazakii.

Forty-seven Acinetobacter spp. isolates from milk powder obtained from a powdered milk producer in Germany were investigated for their antibiotic resistance susceptibilities, in order to assess whether strains from food harbor multiple antibiotic resistances and whether the food route is important for dissemination of resistance genes. The strains were identified by 16S rRNA and rpoB gene sequencing, as well as by whole genome sequencing of selected isolates and their in silico DNA-DNA hybridization (DDH). Furthermore, they were genotyped by rep-PCR together with reference strains of pan-European groups I, II, and III strains of Acinetobacter baumannii. Of the 47 strains, 42 were identified as A. baumannii, 4 as Acinetobacter Pittii, and 1 as Acinetobacter calcoaceticus based on 16S rRNA gene sequencing. In silico DDH with the genome sequence data of selected strains and rpoB gene sequencing data suggested that the five non-A. baumannii strains all belonged to A. pittii, suggesting that the rpoB gene is more reliable than the 16S rRNA gene for species level identification in this genus. Rep-PCR genotyping of the A. baumannii strains showed that these could be grouped into four groups, and that some strains clustered together with reference strains of pan-European clinical group II and III strains. All strains in this study were intrinsically resistant toward chloramphenicol and oxacillin, but susceptible toward tetracycline, tobramycin, erythromycin, and ciprofloxacin. For cefotaxime, 43 strains (91.5%) were intermediate and 3 strains (6.4%) resistant, while 3 (6.4%) and 21 (44.7%) strains exhibited resistance to cefepime and streptomycin, respectively. Forty-six (97.9%) strains were susceptible to amikacin and ampicillin-sulbactam. Therefore, the strains in this study were generally not resistant to the clinically relevant antibiotics, especially tobramycin, ciprofloxacin, cefepime, and meropenem, suggesting that the food route probably poses only a low risk for multidrug resistant Acinetobacter strains or resistance genes.