Sept. 4, 2014
LINCOLN, Neb. — When perishable foods near the end of their shelf life, we usually attribute their unpleasant color or odor to some bad species of bacteria or fungi that finally has blossomed, giving a whole new sensory experience to what was once a mouth-watering commodity.
However, while the direct connection between spoilage microbes and that "ewww factor" was pretty well established in the 1800s by Louis Pasteur, some foods, such as fresh pork sausage, break all of the rules when it comes to spoilage, said University of Nebraska-Lincoln food microbiologist Andy Benson.
In pork sausage, which has an extraordinarily long shelf life (60-80 days at refrigeration temps), the microbes generally have been found innocent of causing spoilage. Sausage's long shelf life stems partly from the antimicrobial nature of the spices blended into the product, but dismissal of microbes as the culprit came about because historically, microbiological testing always showed that microbial growth on product ceases after 15-30 days, a full 30-45 days before onset of the ewww factor.
This disconnect between microbe growth and the onset of sensory failure led food scientists to attribute spoilage of sausage to some unique combination of long-lived microbial products, remaining enzymatic activity from the meat and some unknown environmental factors.
Recent research by a team led by Benson and in collaboration with ConAgra Foods has shed new light on this mystery, and it turns out microbes aren't off the hook after all.
The issue was brought to UNL by ConAgra scientists Jairus David, Gordon Smith and Stefanie Gilbreth. Benson and his team (Rohita Sinha, Junjie Ma and Joseph Nietfeldt) used metagenomics to dig deeper into the causes of sausage spoilage.
"What resulted was several amazing discoveries," Benson said of findings recently published in the journal Applied and Environmental Microbiology.
First, Benson's group showed that multiple waves of growth and death occur among members of the complex microbial community in sausage during cold storage.
"Traditional microbiological methods, which measure only the total population abundance or the abundances of select groups of organisms, all show that population growth ceases after 15-30 days, leaving an apparently static population for the remainder of the shelf life," said Benson, a professor in UNL's Department of Food Science and Technology.
However, using metagenomics, Benson's team was able to quantify nearly all of the organisms in the food, finding that the population is far from static. Indeed, waves of different microbial species could be seen growing, displacing other members of the population.
"Instead of a static population, you have a remarkably dynamic system with multiple waves, known as successions in ecological terms, occurring over time," Benson said. "And it's likely that each wave sets the stage for emergence of the next wave, as well as its own demise."
Working with ConAgra scientists Jairus David and Indarpal Singh, who measured different chemical properties of the sausage during the shelf life, Benson's team identified a small set of organisms that are the most likely culprits in the chemical changes in the sausage that ultimately bring about spoilage.
This type of association study, which is commonly used to study relationships between the human microbiome and disease, was essentially unprecedented in food, Benson said.
Benson's group also found that the many of the organisms that bring about sensory displeasure actually originate from the spice blends used to make the sausage, not from cross-contamination from the pork processing.
"This unparalleled application of metagenomics to 'source-tracking' in foods is yet another demonstration of how understanding fundamental questions about the ecosystem (such as the assemblage of organisms in the sausage and their origins) can ultimately translate into useful information for the corporate partner (such as how to screen ingredients)," he said.
In addition to source-tracking, Benson's team also observed a remarkable event occurring only in sausage treated with the antimicrobial compound lactate-diacetate. Among the different treatments being tested by ConAgra as shelf-life extenders, lactate-diacetate completely eliminated the waves or successions of microbes in the sausage, and instead caused only a limited amount of growth of a single species, known as Lactobacillus graminis.
However, what happens to the microbial population at day 30 of storage in treated samples was "totally unexpected," Benson said. After remaining static for 30 days, the entire population in LD-treated sausage takes an abrupt nosedive.
"In metagenomic terms, this means the DNA from the static population simply disappears," he said. "Such an abrupt and across-the-board disappearance of DNA from a population reeks of a massive, interspecies programmed cell death.
"We normally think of PCD in higher organisms such as plants and mammals where selective death of specific cells and cell types is often programmed into developmental pathways," Benson said. In bacteria, programmed cell death is not well understood, though "most taxa do appear to have components of a rudimentary PCD system.
"If it is indeed a massive PCD that occurred, we want to know how it happened and what signaled it. While the fundamental biology of PCD in bacterial populations is truly fascinating, the potential applications are far-reaching," Benson said.
Benson said the collaboration between UNL and ConAgra "is a good example of the outcomes that can happen when UNL researchers with remarkable tools for discovery partner with private entities that have unique problems, a model that many would agree is the Holy Grail for Innovation Campus."I have to admit, studying the microbiome of fresh pork sausage probably doesn't sound like the sexiest of scientific adventures. We won't make the cover of Science or Nature, but it holds as much potential for fundamental discovery as any other microbial ecosystem. It's all in your perception, sensory and otherwise," he said.
Andrew Benson, Ph.D.
Food Science and Technology
IANR News Service