Identification and Characterization of Psychrotolerant Sporeformers Associated with Fluid Milk Production and Processing

Ivy, Reid A.; Ranieri, M.L.; Martin, N.H.; Bakker, Henk C. den; Xavier, Bruno M.; Wiedmann, Martin; Boor, Kathryn J.

doi:10.1128/aem.06536-11

Cited by 140 publications

(173 citation statements)

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“…rpoB and 16S rRNA gene alignments were performed in the MegAlign program (DNAStar, Inc. Madison, WI). rpoB sequences (632 bp) from a total of 1,288 isolates representing the Paenibacillus genus (n ϭ 737), the Bacillus genus (n ϭ 467), and genera formerly classified as Bacillus (e.g., Viridibacillus; n ϭ 84) collected from farm environments, raw milk, fluid milk-processing plants, and HTST fluid milk products were analyzed to identify unique subtypes (21). rpoB sequences lacked sufficient conservation for design of TaqMan primers and probes that could detect all 737 Paenibacillus sequences represented in this collection.…”

Section: Methodsmentioning

confidence: 99%

“…Plating onto BHI agar supplemented with X-Gal allowed simultaneous APC determination and identification of ␤-galactosidase (␤-Gal)-positive spore-forming bacteria, which, in milk, have generally been found to be Paenibacillus spp. (21). From APC plates, both X-Gal-positive and -negative colony counts were recorded.…”

Section: Methodsmentioning

confidence: 99%

“…To validate the primers and probe, Paenibacillus and closely related Bacillus strains (n ϭ 9 for each genus) were selected to represent the rpoB allelic types (ATs; i.e., those isolated Ն10 times) most frequently found among a collection of over 1,200 isolates collected from dairy farms, processing plants, raw milk, and pasteurized fluid milk (21). An additional 8 Bacillus strains (or strains of the closely related genera Lysinibacillus, Oceanobacillus, and Viridibacillus) and 7 Paenibacillus strains were included to represent genetic diversity (Table 2).…”

Section: Methodsmentioning

confidence: 99%

“…Currently, only limited phenotypic methods are available to differentiate between Bacillus spp. and closely related Paenibacillus spp., including cold growth, which requires 7 to 10 days of incubation, and lactose utilization, which can be difficult to interpret and is not a consistent indicator of sporeformer genus (21).…”

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Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Ranieri

Ivy

Mitchell

et al. 2012

Appl Environ Microbiol

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Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C T ) of 19.14 ؎ 0.54. While 14/17 Bacillus isolates showed no signal (C T > 40), 3 Bacillus isolates showed very weak positive signals (C T ‫؍‬ 38.66 ؎ 0.65). The assay provided a detection limit of approximately 3.25 ؋ 10 1 CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (>1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product. Despite advances in food preservation techniques, bacterial spoilage remains a leading cause of global food loss (14). Nearly one-third of all food produced worldwide is estimated to be lost postharvest, and much of this loss can be attributed to microbial spoilage (16). Dairy products constitute one of the leading sectors impacted by food loss in the United States, as nearly 20% of conventionally pasteurized (high temperature, short time [HTST]) fluid milk is discarded prior to consumption each year (23). In the United States, the shelf life of fluid milk ranges from approximately 1 to 3 weeks. Most consumer complaints result from the growth of psychrotolerant bacteria, typically, either nonspore-forming Gram-negative rods or Gram-positive sporeforming bacteria (12,18,19,28,34,39). The presence of psychrotolerant, non-spore-forming bacteria (e.g., Pseudomonas) in pasteurized milk indicates either inadequate heating of the milk or, more commonly, postpasteurization contamination (6). Therefore, pasteurized milk contamination with Pseudomonas and other non-spore-forming bacteria can be controlled or eliminated by adhering to pasteurization specifications for minimum time and temperature combinations (8) and by adhering to proper sanitation and equipment maintenance protocols, particularly with respect to milk filler sites (33). Conversely, Grampositive psychrotolerant sporeformers can survive pasteurization as spores, germinate, and then grow ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Ranieri

Ivy

Mitchell

et al. 2012

Appl Environ Microbiol

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“…Milk pasteurization is a common practice for safeguarding consumers from food-borne pathogenic bacteria (Ranieri et al 2009). This practice is used worldwide in order to increase the shelf-life of this highly perishable food product (Ivy et al 2012). After pasteurization, cleaning in place (CIP) processes are designed in order to maintain a clean and hygienic environment, including piping and fitting systems (Bayoumi et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Diversity and biofilm-forming capability of bacteria recovered from stainless steel pipes of a milk-processing dairy plant

Cherif-Antar

Moussa–Boudjemâa

Didouh

et al. 2015

Dairy Sci. & Technol.

104

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Bacteria may adhere to and develop biofilm structures onto dairy surfaces trying to protect themselves from adverse conditions such as pasteurization and CIP processes. Thus, biofilms are considered common sources of food contamination with undesirable bacteria. The purpose of this study was to evaluate the diversity of the microbiota attached to stainless steel surfaces in pre-and post-pasteurization pipe lines of a milk-processing plant. Seventy Gram-positive isolates were identified as Enterococcus faecalis (33), Bacillus cereus (26), Staphylococcus hominis (8), Staphylococcus saprophyticus (2), and Staphylococcus epidermidis-Staphylococcus aureus (1) species. Fifty-five Gram-negative isolates were identified to the species Escherichia coli (18), Klebsiella pneumoniae (13), Acinetobacter calcoaceticus (6), Serratia marcescens (6), Enterobacter spp. (5), Pseudomonas aeruginosa (4), Escherichia vulneris (2), and Proteus mirabilis (1). Fifty-five different strains were detected by the RAPD technique. These were subjected to an in vitro assay to evaluate their biofilm-forming capability. E. faecalis (7), A. calcoaceticus (4), K. pneumoniae (3), S. hominis (3), and P. aeruginosa (2) were the species in which more biofilm producer strains were encountered. The adhered microbiota was also assessed by the PCR-DGGE culture-independent technique. This analysis revealed a greater bacterial diversity than that revealed by culturing methods. In this way, in addition to the bacteria detected by culturing, DNA bands belonging to the genera Chrysobacterium and Streptomyces were also identified. This study emphasizes that knowledge of attached Dairy Sci. & Technol. (2016) 96:27-38 DOI 10.1007

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The GenusBacillus

Ehling‐Schulz

Messelhäußer²

2014

DNA Methods in Food Safety

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Identification and Characterization of Psychrotolerant Sporeformers Associated with Fluid Milk Production and Processing

Cited by 140 publications

References 95 publications

Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Diversity and biofilm-forming capability of bacteria recovered from stainless steel pipes of a milk-processing dairy plant

The GenusBacillus

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