The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to reduce cost and loss of productivity due to bacteriophage (phage) contamination in fermentation of dairy and other products. Bacteriophage contamination is the leading cause of failure in fermentation processes used in the food and pharmaceutical industries. The risk of phage contamination in food fermentation processes, particularly in the dairy industry, is significantly enhanced due to a large diversity of suppliers of both the raw ingredient (milk) and the necessary microbial cultures. The current and emerging analytical technologies cannot rapidly detect phages in food materials such as milk. Thus, the combination of the risk of contamination, and the lack of rapid detection methods, results in a significant economic loss for the dairy industry. Early detection of phage contamination will permit early intervention in the fermentation process, which will lead to significant cost savings. In addition, the test could be extended to other food areas, pharmaceutical production, and biochemical production.
The SBIR project addresses the need for rapid detection of phage contamination in a fermentation process through a novel photonic platform technology. Specifically, a platform will be developed for detecting phage in cheese making processes. The platform combines nanophotonics, microfluidics, and electrophoresis, and is based on discoveries made at University of California - Davis. The system is focused on the manipulation of charged phages with electrophoresis, and their accelerated amplification in bacteria. In addition, the system uses electrophoretic capture of phages into a nanophotonic crystal to achieve highly sensitive detection of the target phages. The salient features of the platform are: a) High-throughput, fast trapping of phages; b) High sensitivity and large dynamic range for quantification of phage concentration; and c) Scalable design that can be produced at low cost.
In dairy fermentation, a quick turnaround time for detecting phages that attack the bacterial fermentation process is desired. One of the major outcome of the project was the ability to rapidly isolate phages from relevant matrix. We had proposed that the isolation of phages would take about ~1 hr. The proposed approach involved multiple steps. During our Phase I work, we came up with a novel approach that reduced the time required for the isolation of phages from 1 hr to less than 20 min. The isolation is a single step approach with an ability to concentrate phages. We had proposed to demonstrate the feasibility with one industrially relevant phage. In this proposal period, we have shown the isolation and concentration of phages for two industrially relevant phages. We have developed methods for isolating and concentrating bacteriophages of lactic acid bacteria from both whey protein solution and growth medium. The strains of bacteriophage and their respective hosts used were phage PL-1 and its host L. paracasei subspecies paracasei, and phage J-1 and its host L. casei subspecies casei. Samples of bacteriophage at known concentrations ranging from 102 PFU/mL to 108PFU/mL were isolated using special filters. PFU here stands for plaque forming unit and is a measure of number of phages. The isolated phages were plated with their respective bacterial hosts via the double layer agar method to establish the recovery of the phages from the sample. Following isolation, we demonstrated detection of phages in about half an hour. Our lowest fluorescence signal above negative control corresponded to1000 PFU/mL. This translated to 100 phages, as 10ul of sample was used for detection. The tests were performed on nanophotonic test chips. The test chips consisted of nanostructures imprinted onto poly methyl methacrylate coated on indium tin oxide - glass slides.
