Evaluation of anhydrous processing and storage methods of the temperate bacteriophage ɸV10 for integration into foodborne pathogen detection methodologies.

Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage ɸV10 at ambient temperature without aqueous storage solutions while limiting loss of viability. ɸV10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of ɸV10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23°C. Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for ɸV10 to form lysogens in an E. coli O157:H7 host was not negatively affected by lyophilization. Drying ɸV10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing ɸV10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies.

The effect of a novel low temperature-short time (LTST) process to extend the shelf-life of fluid milk.

Pasteurization has long been the standard method to extend the shelf-life of dairy products, as well as a means to reduce microbial load and the risk of food-borne pathogens. However, the process has limitations, which include cost effectiveness, high energy input, and reduction of product quality/organoleptic characteristics. In an effort to reduce these limitations and extend shelf-life, this study examined a novel low temperature, short time (LTST) method in which dispersed milk in the form of droplets was treated with low heat/pressure variation over a short treatment time, in conjunction with pasteurization. Lactobacillus fermentum and Pseudomonas fluorescens Migula were exposed to conventional pasteurization treatments with and without LTST. Using these organisms, the LTST addition was able to reduce microbial load below detection limits; 1.0 × 10(1) cfu/mL, from approximately 1.2 × 10(8) and 1.0 × 10(7) cfu/mL for L. fermentum and P. fluorescens Migula, respectively. In addition, the shelf-life of the treated, raw, and uninoculated product was prolonged from 14 to 35 days, compared with standard pasteurization, to as long as 63 days with the LTST amendment. Sensory analysis of samples also demonstrated equal or greater preference for LTST + pasteurization treated milk when compared to pasteurization alone (α = 0.05). Conventional pasteurization was effective at reducing the above mentioned microorganisms by as much as 5.0 log10 cfu/mL. However, LTST was able to achieve 7.0-8.0 log10 cfu/mL reduction of the same microorganisms. In addition, BActerial Rapid Detection using Optical scattering Technology detected and identified microorganisms isolated both pre- and post-treatment, of which the only organisms surviving LTST were Bacillus spp. Increased lethality, improved shelf-life, and equal or better organoleptic characteristics without increased energy consumption demonstrate the effectiveness of the incorporation of LTST. The improved shelf-life may potentially have major impacts in the dairy industry in terms of shipping and overall sustainability.