1. Isolate and identify predominant spore-forming bacteria in the milk processing. </P>
2. Adapt the terbium chloride (TbCl3) method for spore detection in milk. </P>
3. Measure spore levels during milk processing.
This project will provide milk powder processors with tools to help them rapidly detect bacterial spores in real time. Endospores are specialized bacterial structures that survive pasteurization, intense heat, and chemicals. Under appropriate conditions, endospores can germinate into vegetative cells, multiply, release more spores and eventually form biofilms that contaminate dairy products and ingredients. Bacillus and Clostridium are two common genera endospore-producing bacteria found in soil and water. Standard methods for quantifying bacterial endospores generally require at least 48 hours and rely on spore germination, which can vary under different growth conditions and by bacterial species. Direct microscopic counts of bacterial endospores cannot be used to determine if the spores are viable. Bacterial spores can also be quantified fluorescently by binding the dipicolinate (DPA) component of the spore coat with terbium chloride (TbCl3). When bound to TbCl3, DPA fluoresces in proportion to its concentration. This method is not species or strain specific and has the advantage of rapidly detecting DPA from any spore. TbCl3 has been used to rapidly detect spores released during a bioterrorism event, but it has not been used in milk or dairy products. </P> The goal of this study is to adapt the TbCl3 method to detect and quantify bacterial spores during milk processing and in the final product. Quantifying endospores before and after milk pasteurization, and at specific points during processing will help identify locations for intervention strategies.