Evaluating Paenibacillus odorifer for its potential to reduce shelf life in reworked high-temperature, short-time fluid milk products (original) (raw)
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Spoilage potential of Paenibacillussp. in Brazilian raw milk
Ciência Rural, 2016
ABSTRACT: Bacterial spores are widespread in the environment and can contaminate milk. Spores are resistant to thermal conditions and your germination reduces milk shelf-life because the aerobic bacteria that are sporulated produce proteases and lipases. The aim of this study was identify Paenibacillus sp., the spoilage microbiota, arising from the germination of spores in raw milk and your spoilage potential. Twenty different milk samples were treated at 80°C/12min and plated to isolate spore-forming bacteria. These strains were picked in milk agar and tributyrin agar for verification of their potential proteolytic and lipolytic activities, respectively. Amplification and sequencing of the 16S rRNA gene of the strains for identification by similarity to the DNA sequences deposited in GenBank was performed. One hundred and thirty-seven isolates were obtained, of which 40 (29.2%) showed spoilage activity for milk. Of these, three (7.5%) were identified as strains of Paenibacillus sp....
International Journal of Dairy Technology, 2017
Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI-TOF-MS. Milk had spore counts <2 log 10 cfu/mL and 4 log 10 cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7°C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.
Applied and Environmental Microbiology, 2012
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.
Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product
Journal of dairy science, 2008
Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Baci...
Frontiers in microbiology, 2017
Premature spoilage and varying product quality due to microbial contamination still constitute major problems in the production of microfiltered and pasteurized extended shelf life (ESL) milk. Spoilage-associated bacteria may enter the product either as part of the raw milk microbiota or as recontaminants in the dairy plant. To identify spoilage-inducing bacteria and their routes of entry, we analyzed end products for their predominant microbiota as well as the prevalence and biodiversity of psychrotolerant spores in bulk tank milk. Process analyses were performed to determine the removal of psychrotolerant spores at each production step. To detect transmission and recontamination events, strain typing was conducted with isolates obtained from all process stages. Microbial counts in 287 ESL milk packages at the end of shelf life were highly diverse ranging from <1 to 7.9 log cfu/mL. In total, 15% of samples were spoiled. High G+C Gram-positive bacteria were the most abundant taxo...
Spoilage potential of spore-forming bacteria from refrigerated raw milk
Semina: Ciências Agrárias, 2018
Aerobic bacterial spores are an important group of microorganisms in raw milk. These microbes are thermoduric, whereas the vegetative forms are thermophilic, thermoduric and psychrotrophic and reduce the shelf life of pasteurized milk. In Brazil, there are a lack of studies on the load of aerobic spores in raw milk; thus, little is known about the spoilage activity of these organisms. The aim the present study was to quantify the aerobic spores in Brazilian refrigerated raw milk of dairy region of Castro, Paraná state, assess the potential proteolytic and/or lipolytic isolates and identify the microorganisms derived from the germination. Twenty milk samples were evaluated, and the aerobic spore count was performed after plating the samples following heat treatment at 80°C for 12 min. The activity proteolytic and lipolytic isolates were evaluated through subculture on milk agar and tributyrin agar, respectively, and these microorganisms were identified using partial 16S rRNA gene seq...
Tracking Spore-Forming Bacterial Contaminants in Fluid Milk-Processing Systems
Journal of Dairy Science, 2007
The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, shorttime pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.
International Journal of Food Microbiology, 2010
Spoilage bacteria in milk are controlled by treatments such as thermization, microfiltration and addition of carbon dioxide. However, little information is known about the changes in microbial communities during subsequent cold storage of treated milk. Culture-dependent methods and a direct molecular approach combining 16S rRNA gene clone libraries and quantitative PCR (Q-PCR) were applied to obtain a better overview of the structure and the dynamics of milk microbiota. Raw milk samples were treated by the addition of carbon dioxide (CO 2 ), thermization (TH) or microfiltration (MF) and stored at 4°C or 8°C up to 7 d. Untreated milk (UT) was used as a control. Psychrotrophic and staphylococci bacteria were enumerated in the milk samples by culture methods. For the molecular approach, DNA was extracted from milk samples and 16S rRNA gene was amplified by PCR with universal primers prior to cloning. The Q-PCR method was used to evaluate the dynamics of dominant bacterial species revealed by clone library analysis of 16S rRNA gene. Comparison of the 16S rRNA gene sequence indicated that the two most abundant operational taxonomic units (OTU), determined at 97% identity, belonged to the class Gammaproteobacteria (40.3% of the 1415 sequences) and Bacilli (40%). Dominant bacterial species in UT, CO 2 and TH milk samples at day 3 were affiliated with Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acinetobacter and Trichococcus. Dominant bacterial species detected in MF milk were Stenotrophomonas, Pseudomonas and Delftia, while Pseudomonas species dominated the bacterial population of UT, CO 2 and MF milk samples at day 7. Staphylococcus and Delftia were the dominant bacterial species in thermized milk. Q-PCR results showed that populations of S. aureus, A. viridans, A. calcoaceticus, C. variabile and S. uberis were stable during 7 d of storage at 4°C. Populations of P. fluorescens, S. uberis and total bacteria increased in UT and CO 2 milk samples during 7 d of storage at 8°C and were noticeable from day 3. This study shows new microbial species which can develop during cold storage after milk treatment and contributes to identifying causes of reduced shelf life and deterioration of technological properties of milk during storage.
2017
Dairy processing facilities need to control spoilage microorganisms in fluid milk in order to reach markets and increase their profits. The major contributors for early spoilage on fluid milk are sporeforming bacteria capable of growing under refrigeration temperature and Post- Pasteurization Contamination (PPC) organism. Since these two groups of organisms have different survival characteristics, strategies to control them may require different approaches. Therefore, identification of bacteria contributing to spoilage becomes essential. The objective of this research was to identify the spoilage bacteria, mainly sporeformers, associated with laboratory heat-treated milk (80°C for 12 min) and commercially-pasteurized fluid milk, throughout a production chain in Nebraska. Identification was done using the rpoB gene and/or partial 16S rDNA sequences. Raw and pasteurized milk samples were collected from different locations (farm, trucks, processing plant, and packaged products) during ...
Dairy Science & Technology, 2016
The ingredients used for the development and production of milk-based products pose the risk of the introduction of new, emerging spore-formers producing highly thermoresistant spores. Therefore, the aim of this study was to examine the heat resistance of spores isolated from dehydrated ingredients, intermediate and final products. Furthermore, the influence of the heating medium (milk or phosphate buffer) on the heat resistance was determined in order to assess which medium is best to use in the context of dairy processing. Sixteen spore-forming strains from seven different species (Bacillus amyloliquefaciens, Bacillus flexus, Bacillus subtilis, Bacillus thermoamylovorans, Bacillus smithii, Geobacillus pallidus, Geobacillus stearothermophilus) producing thermoresistant spores were selected after their isolation from diverse food products obtained from local dairies such as cocoa powder, milk powder, spices, and dessert products. Spores produced from the chosen strains were tested regarding their heat resistance at 110, 120, and 125°C for 30 min. Highly thermoresistant spores surviving a heat treatment for 30 min at 125°C in milk were produced by B. amyloliquefaciens and G. stearothermophilus. The inactivation of the spores was higher in milk than in phosphate buffer. This study highlights that not only raw milk but also dehydrated ingredients are important sources of thermoresistant spores in dairy processing. Since