Elizabeth Brewster

apples on production line

© SlobodanMiljevic/E+/Getty Images

apples on production line

© SlobodanMiljevic/E+/Getty Images

Boosting Microbial Safety in Produce

Fresh produce has been the source of many outbreaks of foodborne illness that caused disease, hospitalizations, and deaths in addition to impacting growers and processors adversely. The intense media attention on fresh produce–associated outbreaks, including Escherichia coli O157:H7 in apple cider and Listeria monocytogenes in caramel apples, is a testament to the significance of food safety in consumers’ minds when they’re buying fresh products.

There is a growing need to significantly improve understanding of the ecology of foodborne pathogens and develop enhanced farm-to-fork safety approaches. Food Technology contributing editor Gülhan Ünlü recently talked with Meijun Zhu, a food science professor at Washington State University, to find out more about her innovative research on improving microbial safety for produce and low-moisture foods.

Zhu’s research group uses metagenomics and molecular and traditional microbiological techniques to study microbial safety of fresh apples produced in Washington state. The group works in a laboratory equipped with its own miniature packing line, since fresh apple packing operations are an important part of the supply chain and a possible source of microbial contamination.

"I feel that we saw a turning point in the produce industry in 2011, after the major outbreak of listeriosis associated with cantaloupes contaminated with Listeria monocytogenes."

- Gülhan Ünlü
Gülhan Ünlü

Ünlü, an associate professor in the Department of Animal, Veterinary and Food Sciences at the University of Idaho, specializes in researching microbial food safety, food biopreservation, and fermented/functional foods. Her research group has been studying bacteriocins as biopreservatives against select foodborne pathogens for several years.

Read about how these two experts assess current and potential microbial safety research involving fresh produce sanitation, plant-derived antimicrobial compounds, and the use of surrogate bacteria.

Sanitizing Advancements

Ünlü: Biofilms are an area of interest for me and many other microbiologists and food scientists, particularly biofilms of Listeria monocytogenes. What kind of work are you and your team doing to expand on our knowledge of microbial biofilms involving various food contact surfaces?

Zhu: We have done a couple of studies on biofilms, comparing different sanitizers within practical/legal concentration against Listeria biofilms on different food contact surfaces. We first compared their efficacies against different biofilms at different ages because, in industry settings, we can have existing young biofilms and some accumulated biofilms.

[Not surprisingly] we found that sanitizer is more effective against single-strain biofilms and young biofilms. So we did follow-up studies using multiple-strain, aged biofilms, looking into different sanitizers on different surfaces and trying to [determine] their efficacy. In general, we found that the efficacy with aged biofilm increased with sanitizer concentration or exposure times. Organic matter soiling, such as apple juice or diluted milk, compromised antimicrobial efficacies of sanitizer against Listeria biofilms regardless of food contact surfaces. Data highlight the importance of timely and thorough cleaning and sanitizing of food contact surfaces. We compared a handful of commonly used sanitizers in the industry, and we found that peroxyacetic acid (PAA) was the most effective against the Listeria on different surfaces, but [that] efficacy was impacted by organic matter.

In [another] follow-up study on biofilm, we are also looking into the effectiveness of steam in eliminating Listeria biofilms on different food contact surfaces. We found that short-term steam exposure caused a rapid kill of Listeria biofilm on different surfaces. Six-second saturated steam treatment can result in 2.4 to 3.2 log CFU/coupon reduction, depending on the type of surface. The efficacies of steam were minimally impacted by organic soiling. Steam treatment is more effective on stainless steel, but it’s less effective on other surfaces, like rubber surfaces.

"[Our] data indicate that low-dose ozone gas in the cold storage rooms could be a viable intervention method for the apple industry or for other fresh produce industries, depending on surface textures."

- Meijun Zhu
Meijun Zhu

Ünlü: Listeria is such an important microorganism of interest for food microbiologists. I feel that we saw a turning point in the produce industry in 2011, after the major outbreak of listeriosis associated with cantaloupes contaminated with Listeria monocytogenes. Since then, the presence of L. monocytogenes in the preharvest environment has been at the forefront of food safety concerns. What do we know now about how L. monocytogenes transfers and circulates in the farm environment?

Zhu: Fruit or produce can be contaminated by microorganisms—including pathogenic microorganisms—through the production line, and [microorganisms] can come from preharvest, processing, and transportation along the production continuum. The preharvest environment is pretty complicated. Microorganism contamination on produce in preharvest can be caused by agricultural practices such as irrigation water and soil amendments if they weren’t done properly. Pathogenic microorganisms can also be introduced by animals, whether this is domestic animals or wildlife and birds, which are more difficult or unpredictable to control. Produce can be contaminated through the environment, like run-off from agriculture farms, raindrops, wind, dust, and other fomites.

Ünlü: In 2015, there was an unusual multistate outbreak associated with prepackaged caramel apples, resulting in at least 35 cases of illness, 34 hospitalizations, and seven deaths. I know that your research group has worked on microbial safety of fresh apples—can you give us more details about how some specific sanitizers are working?

Zhu: During the U.S. Food and Drug Administration (FDA) investigation of this listeriosis outbreak in apples, L. monocytogenes was found on multiple food contact surfaces, including polishing brush, drying brush, conveyor, and wooden bin. This indicates that the postharvest handling and packing environment can be a significant source of contamination.

Various presumptive intervention steps have long been employed to reduce microbial risks on apples in the apple packing facilities. One such intervention is spray bar sanitizer intervention; however, there is limited information available about the practical efficacy of this antimicrobial intervention. With the help of the Washington Tree Fruit Research Commission and the Washington apple industry, we have been evaluating and validating practical efficacies for commercially used sanitizer(s) against L. monocytogenes on apple surfaces in lab and pilot scales, and then verifying their efficacy on multiple commercial apple packing lines.


©SlobodanMiljevic/iStock/Getty Images Plus


©SlobodanMiljevic/iStock/Getty Images Plus

In the Washington apple industry, PAA is currently the most commonly used sanitizer in spray bar intervention. Our study indicated that PAA at 80 ppm—the FDA allowed limit without further rinse requirement—is more effective compared with 100 ppm free chlorine applications. But their reduction is still limited, with spray wash of 80 ppm PAA at 2 min causing about 1.7 (lab scale) or around 1.0 (in-plant testing) log reduction, indicating the need to improve the effectiveness of PAA. We have been evaluating different ways to improve this efficacy. We found that PAA applied at the elevated temperatures—about 43°C–46°C—or in combination with lauric arginate can significantly boost anti-Listeria efficacies of PAA, but it’s [still] not enough.

So we need additional intervention methods to ensure the safety of fresh apples. After harvest, apples are typically stored in a cold commercial storage room for up to 12 months, [which] provides an ideal condition for us to apply antimicrobial gas interventions. Thus, we further examined the efficacy of low-dose continuous gaseous ozone application in Listeria controls on fresh apples. Our studies found that low-dose ozone gas at 50–87 ppb continuously in commercial CA [controlled atmosphere] cold storage rooms resulted in more than 5 log reduction of Listeria on apples, regardless of apple variety, after six to nine months. These data indicate that low-dose ozone gas in the cold storage rooms could be a viable intervention method for the apple industry or for other fresh produce industries, depending on surface textures.

Ünlü: I am pretty excited to hear that! Similar to the use of hurdle technologies in other food processing applications, your work indicates that combination treatments can be successful in the fresh produce industry as well.

Plant-Derived Antimicrobial Compounds

Ünlü: I’m seeing spices and their essential oils gaining popularity as antimicrobial compounds, especially in light of the continuing consumer demand for clean labels. Your research group has used cinnamon oil and grape seed extract as antimicrobials against select foodborne pathogens. Do you think there’s a future for these kinds of antimicrobials?

Zhu: Consumers want more natural food products, so that’s why we’re looking into different options for plant-derived natural food additives. We are using cinnamon oil to see if there is antimicrobial efficacy or how effective [it is] against Shiga toxin-producing E. coli (STEC), including O157 and non-O157. We found that at lower concentrations like 0.025%, cinnamon oil can inhibit [both] non-O157 and O157 E. coli growth. The minimum bactericidal concentrations varied between 0.05% and 0.1%, depending on STEC strains. Cinnamon oil at 0.1% could effectively introduce a bactericidal effect against all strains we tested within 15 minutes.

We are also looking at the molecular level to see how this cinnamon oil can interfere with virulence factor or stress response. We’re finding that cinnamon oil at subinhibitory concentrations can significantly reduce the level of Shiga toxin production and inhibit stress response gene and quorum sensing signaling molecules. [Beyond that], we are looking at cinnamon oil against Salmonella [in] low-moisture foods. We have found that the efficacy of cinnamon oil is reduced on low-moisture food like almonds. This highlights [the fact that] once bacteria adapt to low-moisture food, it is more difficult to kill them. We have not moved forward to [study] this with Listeria yet, but these data indicate that cinnamon oil [has] potential for controlling foodborne pathogens in one way.

I want to point out that one of the limitations on cinnamon is . . . sensory characteristics. Cinnamon oils have strong flavors, so . . . we need to work out [a balance] between concentration and antimicrobial activities.

Ünlü: Perhaps one avenue would be taking this cinnamon oil and incorporating it into packaging material to deal with flavor-related issues.

Zhu: Yes, that’s definitely a very good idea.

Ünlü: I think this is really great news regarding inhibition of enterohemorrhagic E. coli as well as Salmonella. I also really appreciate that with this particular project, you were able to use the tools of general microbiology as well as molecular biology.

"I’m seeing spices and their essential oils gaining popularity as antimicrobial compounds, especially in light of the continuing consumer demand for clean labels."

- Gülhan Ünlü

Sending in Surrogate Bacteria

Ünlü: Nonpathogenic surrogate bacteria are commonly used in a variety of food challenge studies in place of foodborne pathogens. I know that you have identified and used surrogate bacteria in your research on microbial safety of produce and low-moisture foods. What exactly are surrogate bacteria, and how would someone select and validate potential surrogate bacteria when verifying microbial inactivation processes?

Zhu: Based on FDA guidelines, surrogate bacteria are defined as nonpathogenic species or strains used in place of pathogenic organisms in a particular treatment or intervention.

When choosing surrogates for a study, the No. 1 thing to keep in mind is that the surrogate strain [cannot be] pathogenic. We also want bacteria that are genetically stable all the time. We want those identified surrogates to have equivalent characteristics [of] adhesion to our food or food surface. And we definitely want to have surrogates with similar or slightly higher resistance when exposed to a particular treatment or particular intervention method, compared with target strains.

Ideally, a surrogate strain should have a slightly higher resistance than the target pathogen, providing additional marginal protection against the target pathogen. However, it should not have overly higher resistance than the target strains. Otherwise, this could potentially cause overprocessing of food products, resulting in food quality downgrading or deterioration.

Additional traits to consider are surrogates that are convenient, handy, easy to prepare, easy to enumerate, and easy to grow with consistent growth potential.

Ünlü: Just looking at your research, I see you’re trying to inactivate, intervene [in], and detect foodborne pathogens in food and food production environments. Your use of surrogate organisms in your research makes a huge difference in cost, safety, and legal liability, I think.

Zhu: We try to avoid using pathogenic bacteria in industry settings. It’s risky, No. 1, and No. 2, very costly, so that is a waste.

Listeria innocua, by nature, is a well-known surrogate for L. monocytogenes because it’s not pathogenic and is genetically very relevant, but Listeria innocua is not commonly recommended [for use as] a surrogate at this moment in the industry. Right now, screen protocol in the food processing industry is generally targeting genetically related Listeria. So that means when we introduce Listeria innocua, we cannot know if this will lead to persistent Listeria species in that packing facility, [which will require] additional cleaning and may lead to product holding. This highlights the need for an alternative nonpathogenic, non-Listeria surrogate that can be used in industry settings.

"When choosing surrogate [bacteria] for a study, the No. 1 thing to keep in mind is that the surrogate strain [cannot be] pathogenic. We also want bacteria that are genetically stable all the time."

- Meijun Zhu

Ünlü: Well, that makes a lot of sense. To my knowledge, the difference between Listeria monocytogenes and Listeria innocua is only 5% at the genomic level. So it would be very difficult to make that differentiation when needed.

Zhu: Based on different side-by-side testing, we found that Enterococcus faecium NRRL B-2354 is a suitable surrogate of L. monocytogenes during fresh apple sanitizer interventions. We are using this in our fresh apple in-plant testing and validation, and we also compared E. faecium NRRL B-2354 for our low-moisture food safety studies.

E. faecium NRRL B-2354 being used as a surrogate for Salmonella has a history starting with the Almond Board of California trying to identify this as a surrogate for Salmonella during almond thermal processing [in 2007]. There is research interest [now] in trying to use this Enterococcus as a surrogate for Salmonella in different low-moisture foods. Even though, in general, E. faecium NRRL B-2354 is a presumptive surrogate for Salmonella in low-moisture food storage and subsequent thermal interventions, its behavior is also impacted by food matrix, water activity treatment, treatment temperature, and other factors. Thus, it is recommended to conduct side-by-side testing along with the target pathogen under an identical condition to validate the suitability of a surrogate candidate before using.

Looking to the Future

Ünlü: We have a lot of recent produce-associated outbreaks here in the United States and all over the world, and these outbreaks demonstrate the critical need for increased research in multiple areas to ensure the safety of fresh produce. Unquestionably, the food safety challenges facing growers, packers, processors, retailers, and definitely consumers of fresh and fresh-cut produce are quite complex and multifaceted. Established and ongoing research projects have provided insights on produce contamination at multiple steps in the supply chain, and future research activities will develop science-based intervention technologies that minimize potential contamination risk and strengthen produce safety. With that being said, I’m really curious about what your future research on produce safety will deal with.

Zhu: One of the directions we are moving forward on is biocontrol. We are trying to control foodborne pathogens using biological agents—such as bacteriophages or some isolated bacteria from produce surfaces—as antagonists to control foodborne pathogens. [We think] this is our future.

Ünlü: That is wonderful to hear, especially in a time when consumers are more and more interested in using fewer chemicals. I think this is really the way to go.

About the Author

Elizabeth Brewster is a freelance writer based in Evanston, Ill. ([email protected]).