Some bacteria have the ability to produce a structure called spores which allows them to survive harsh conditions, including pasteurization. In general, higher counts of these bacteria in dairy products may cause spoilage issues.

Bianchini andreia
Associate Professor / Food Science & Technology Department / University of Nebraska – Lincoln
Microbiologist & Research Associate Professor / University of Nebraska – Lincoln
Research Associate / University of Nebraska – Lincoln

Specifically in milk powder, the bacteria are associated with lower quality, affecting potential markets and prices significantly. According to the U.S. Dairy Export Council, milk powder containing spore counts higher than 1,000 colony-forming units (CFU) per gram for international markets and 500 CFU per gram for infant formula will be considered as lower quality.

Within the spore-forming bacteria group, some species have the ability to grow at lower temperatures and produce enzymes affecting the quality of fluid milk during storage and limiting its shelf life.

Since destroying or controlling this type of bacteria at later stages of milk processing is quite difficult, the identification of contamination routes is essential to design interventions that can control problematic spore-forming bacteria at early stages of the supply chain.

Tracking problematic spore-forming bacteria

Our research group at the University of Nebraska – Lincoln has studied this group of bacteria for over six years across the dairy production chain in Nebraska. We have analyzed samples from a fluid milk plant, a condensed milk plant and multiple farms within this state. These studies have allowed us to understand the prevalence and potential routes of contamination of milk with spore-forming bacteria.

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Among our findings, we’ve observed a wide variety of spore-forming bacteria that can survive pasteurization and condensation processing conditions. By analyzing the quality of the milk entering these processing plants, we observed the levels of this bacterial population were relatively constant during processing with slight or no increases during manufacturing of both types of products (fluid and condensed milk).

On-farm research

To understand where spore-forming bacteria come from, we decided to assess multiple farms in Nebraska to determine the contamination routes. First, we evaluated the farm environment (i.e. animal feed, cow teats, manure and other potential sources), finding areas that were heavily contaminated with spore formers. As Figure 1 shows, the majority of the contamination was present around the pen area (i.e., bedding, drinking water, manure and others) where cows spend most of their time.

Environmental contaminatin with sporeformers throughtout the milk chain

Lower incidences were observed in the milking parlor, and even lower levels were found at processing plants.

To better understand the source of contamination, spore formers found in pasteurized and condensed milk were identified and traced back to the farm using molecular techniques.

The identification of the bacterial species was critical to understand which spore formers are problematic for each product, and tracing back was required to determine if a single source, or multiple sources, were causing the on-farm contamination. The identification of these sources allows researchers to focus their efforts on potential interventions.

Spore-former bacteria associated with fluid milk spoilage during refrigerated storage were isolated and identified over the course of the product’s shelf life. Researchers found Bacillus and Paenibacillus species were associated with product spoilage.

The Paenibacillus group, specifically, has been shown to survive commercial pasteurization and grow under refrigerated conditions (5ºC to 6ºC or 41ºF to 43ºF). This bacterial genus can produce quality defects, such as protein coagulation and rancidity, due to their enzymatic activity.

Another research group at Cornell University has also found Paenibacillus spp. associated with spoilage of fluid milk in the northeast U.S. and observed a 63 percent prevalence of this group in raw milk. A similar contamination rate was found in our research in Nebraska.

Similar to the approach used to study fluid milk during storage, molecular tools were used to evaluate spore formers associated with condensed milk. The results of bacterial identification showed a wide variety of spore-forming bacteria that survive processing conditions, including Bacillus, Paenibacillus, Lysinobacillus, among others.

In both studies it was difficult, however, to determine a unique source of contamination due to the wide variety of species observed.

Fingerprinting bacteria

When contamination was traced back to the farm supplying raw milk to processing facilities, our Nebraska research team observed many spore formers isolated from pasteurized and condensed milk were also found in environmental samples of those farms.

This exact matching was possible by using DNA fingerprint identification. Places at the farm where those spore formers were found included cows’ teats, drinking water, manure and others. More importantly, these organisms were quite prevalent in the milking parlor area (Figure 2).

cources of sporeforming bacteria

This finding indicates environmental sources located in close proximity to the milking equipment could be the key contamination sources of this problematic group of bacteria. Therefore, researchers are currently evaluating interventions applied mainly at the parlor to see if they can reduce levels of spore-forming bacteria in raw milk, improving its overall quality.

Because some of these points of contamination could affect both processing chains (fluid and condensed milk), these interventions would benefit both sectors of the dairy industry.

Researchers have found a majority of contamination around pen areas, with lower incidneces in milking parlorResearchers have found a majority of contamination around pen areas, with lower incidences observed in the milking parlor. Photo courtesy of Andreia Bianchini.

As previously mentioned, to achieve the quality standards established for milk powder for international markets and infant formula, raw milk should have fewer than 100 CFU per milliliter or 50 CFU per milliliter, respectively. The Nebraska study, conducted over a year from at least four farms, revealed some farms delivered relatively high-quality milk having spore counts lower than 100 CFU per milliliter – but not constantly throughout the year.

On the other hand, some farms produced lower-quality milk with spore-former counts higher than the suggested parameters. This observation showed the potential effect of farm practices on the undesirable bacteria levels and the possibility of applying on-farm interventions to better control them.

Application of potential interventions

Based on our research and surveys with dairy farmers in Nebraska, we’ve chosen three potential areas of intervention to control spore formers at the farm level. These areas include changes in herd management, milking practices and sanitation procedures.

To provide guidelines regarding how to better control spore formers, it is necessary to design experiments allowing researchers to evaluate the effectiveness of the proposed interventions. That is exactly what our group is currently working on.

We are evaluating simple changes in farm practices on multiple farms in Nebraska and relating those to the levels of spore formers in the farm environment and raw milk after a period of acclimation. In this context, baseline data about the farm is collected, then changes are implemented. The farm uses the new protocol for two weeks followed by a weeklong collection of data while the protocol remains in use.

Some of the early results from one of the farms suggests some of the tested interventions are effective at reducing the spore-forming bacteria in raw milk. Among those are changes in protocol related to towels used to wipe cows’ teats, non-iodine sanitizers for teats and sanitation procedures used during clean-in-place milking lines. These interventions are now being tested on other farms to verify the results.

Because the types of spore-former bacteria found in Nebraska are similar to those found across multiple regions of the U.S. by other researchers, it is plausible potential sources of contamination are also the same.

Therefore, wider implementation of these intervention practices could improve raw milk quality in other regions. The long-term goal of our research has been to support farmers in producing raw milk of the highest quality. That will lead to dairy products of greater economic value, benefiting farmers and the overall milk industry.

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