<OL> <LI> To quantify the concentration of estradiol-17B in milk and dairy products and estimate the average intake of this hormone by Americans of different ages.<LI> To screen bulk tank milk samples for the presence of pathogenic strains of Staphylococcus aureus and determine the frequency of enterotoxin-producing strains of this pathogen. <LI> To optimize real-time biosensor methods for detecting bacterial pathogens in milk and dairy products and to develop gene-array methods for confirming virulence attributes and integrate these methods into HACCP systems. <LI> To develop and optimize Multi-Virulence-Gene Sequence Typing method for molecular fingerprinting of Listeria monocytogenes and use this method to track this pathogen in dairy processing plants to identify and eliminate routes of transmission. <LI> To evaluate a short pre-pasteurization heat processing step to destroy spore-forming pathogens during subsequent pasteurization of milk and identify microorganisms in pasteurized dairy products before and after filling that will lead to strategies to reduce post-pasteurization contamination. 6. To evaluate the synergistic effects of mild heat treatments and high-pressure processing on destruction of Listeria monocytogenes and other milk-borne pathogens and to understand and utilize interactions between various mold strains and intrinsic and extrinsic factors to reduce or prevent the growth of L. monocytogenes in mold-ripened soft cheeses.
NON-TECHNICAL SUMMARY: Dairy products are potential sources of harmful chemicals and biological hazards. Estrogen in milk is a potential carcinogen and microbial pathogens can enter milk and dairy products and cause foodborne illness. The purpose of this project is to detect, quantify and track the above hazards in order to prevent them from entering dairy products.<P>
APPROACH: 1. Blood plasma, urine, fresh milk, and processed milk products will be subjected to radioimmunoassay (RIA) to quantify estradiol-17B using the double antibody RIA kit from ICN Biomedical. 2. Bulk tank milk samples will be screeened for the S. aureus agr genotype using restriction fragment length polymorphism profiles. The frequency of enterotoxin-producing strains of S. aureus from bulk tank milk samples will be determined by multiplex PCR. Concordance analysis of agr genotyping and enterotoxin profiles will be conducted. 3. Real-time biosensor methods for detecting foodborne pathogens in milk and dairy products will be optimized. Gene array methods will developed for detecting virulent foodborne pathogens. HACCP protocols will be devised for integration of biosensors and gene arrays. 4. Multiplex PCR will be developed for more rapid Multi-Virulence-Sequence-Typing (MVLST) of L. monocytogenes isolates. Isolates of L. monocytogenes will be obtained from dairy and meat processors in Pennsylvania and subjected to MVLST. Data will be analyzed to determine routes of transmission and then develop effective intervention strategies to prevent post-pasteurization contamination of dairy products. 5. Bacillus spores will be subjected to mild thermal treatments to sensitize them to subsequent destruction during pasteurization. Milk will be subjected to microbioligical testing immediately after filling. Intervention strategies will be employed on those areas of the environment that appear to contribute to recontamination. 6. Cells of L. monocytogenes and other pathogens will be mixed into raw milk and subjected to high pressure processing (HPP) treatments that cause various levels of injury. Research will be conducted to determine the mechanism(s) of injury and repair following HPP. Experiments will be conducted to evaluate synergistic effects between low-level thermal treatments and HPP on destruction of foodborne pathogens in fluid milk. The effect of various intrinsic and extrinsic interactions on survival of HPP-injured L. monocytogenes will be examined during ripening and storing of mold-ripened soft-cheeses.
PROGRESS: 2003/07 TO 2005/07 <BR>
The project included 5 major parts: Identifying enzymes usable in assessing milk pasteurization; improving quality and safety of milk by non-thermal techniques; assessing effects of pasteurization and homogenization on milk components; defining molecular markers for investigating outbreaks caused by L. monocytogenes; investigating the antibacterial activity of food-grade Penicillium species. Alkaline phosphatase (ALP) and L-lactic dehydrogenase (LLD) were immobilized onto magnetic nanoparticles and their stability was investigated in developing a biosensor to assess milk pasteurization. The activity of ALP was improved after immobilization while LLD was poorly immobilized. Activity and structural changes induced to ALP and LLD by HPP at 206-620 MPa were studied. HPP up to 620 MPa slightly affected ALP without causing inactivation, while LLD activity decreased as the pressure increased, till total inactivation at 515 MPa. Changes in activity were in accordance with changes in structures, evidenced by changes in CD spectra. Non-contact ultrasound was used to decrease microbial numbers. E.coli and P. fluorescens were suspended in phosphate buffer (PB) and ultrasound was applied. A 0.05 log decrease for P. fluorescens after 1 min and 0.31 log for E. coli after 2 min were observed. Microbial injury increased to 98.6% for cells after 60 sec in PB compared with 78.4% for cells after 90 sec in peptone broth. Killing of microorganisms was barely observed although PB afforded less protection to cells than peptone buffer. To estimate daily consumption of estrogen, its levels of estrogen in milk were measured after pasteurization and homogenization. Milk from cows in later state of pregnancy has higher level of Estradiol-17B and these levels correlated with fat content. Processing decreased milk fat globule size, but E2 levels averaged 0.70 and 0.61 pg/ml in raw and processed milk, respectively. The ability of MVLST to distinguish epidemic clones and outbreak strains of L. monocytogenes was evaluated. Results showed that MVLST provided high discriminatory power (D = 0.99), epidemiological concordance (E=1.0), stability and typeability. MVLST allowed accurate identification of three known epidemic clones (I, II and III), suggesting another epidemic clone (IV) in serotype 4b of L. monocytogenes. A set of 28 single nucleotide polymorphisms (SNPs) allowed satisfactory differentiation of epidemiologically unrelated isolates. SNPs in virulence genes appear to be excellent molecular markers for investigation of outbreaks caused by L. monocytogenes. To respond to numerous outbreaks linked to consumption of soft mold-ripening and blue-veined cheeses, various Penicillium sp from soft cheeses were extracted and their antibacterial effects were investigated. Penicillium showed some degree of inhibition against Gram positive bacteria (S. aureus, L. innocua, and Bacillus sp.) but not against Gram negative ones (E. coli, and S. typhimurium). The order of inhibition was Bacillus sp. > Listeria innocua > S. aureus.
IMPACT: 2003/07 TO 2005/07 <BR>
This research will provide insight on structural changes induced on milk enzymes by pressure processing. The ascertained thermal and long term stability of ALP will be essential in utilizing this enzyme to construct a biosensor for milk pasteurization assessment. Furthermore, defining the inactivation condition for ALP will pave the way to using high pressure processing as a non-thermal mean for pathogens inactivation in milk and milk products. We also anticipate that the results of the epidemiological investigation of epidemics and outbreaks caused by L. monocytogenes will allow food processors and government regulatory agencies to track epidemic clones and outbreak strains of Listeria monocytogenes, which will allow the development of more effective intervention strategies to prevent their transmission to foods. Knowledge on the effects of pasteurization and homogenization on the concentration of extrogen in cow's milk is important in assessing the quality of milk products. This research will provide data desired by consumers and the dairy industry to quantify the mass of estrogen associated with dairy product consumption. These studies will also provide greater insight into the mechanism of secretion of estrogen into milk of the cow, and may provide alternative management strategies to reduce the concentration of estrogen in dairy products. These are questions that are important to the public. The investigation of food-grade antimicrobial in cheese has demonstrated that 7 species of Penicillium found in cheese have antimicrobial activity.