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Undergraduate Laboratory Exercises Specific to Food Spoilage Microbiology Abigail B. Snyder, Randy W. Worobo, and Alicia Orta-Ramirez

Abstract: Food spoilage has an enormous economic impact, and microbial food spoilage plays a significant role in food waste and loss; subsequently, an equally significant portion of undergraduate food microbiology instruction should be dedicated to spoilage microbiology. Here, we describe a set of undergraduate microbiology laboratory exercises that focus specifically on food spoilage which were taught in 2 lab periods as part of the undergraduate food microbiology lab curriculum at Cornell University. The lab was broken down into 3 exercises. Two exercises lead students to determine the likely source of contamination in a canned salsa through (exercise 1a) plating and observation of colony morphology and (exercise 1b) determination of the thermal resistance for those isolates. The final exercise (2) involved detection of the spoilage bacterium Alicyclobacillus in apple juice. Spoiled juice demonstrations were also prepared in this exercise for students to observe sensorial changes resulting from spoilage, emphasizing that spoilage is not always visually detectable. Students were able to successfully determine the source of contamination based on the results of their laboratory findings, which they used to make recommendations for production to reduce microbial food spoilage in the canned salsa product. Based on student answers to discussion questions provided following lab exercises, participants were able to (a) identify the significance of microbial spoilage and how spoilage is principally different from food safety, (b) describe varying sensorial changes associated with microbial spoilage, and (c) employ methods and analysis to evaluate sources and type of contamination. Downloadable handouts and stepwise instructions are available as supporting information.

Introduction

programs in place (Snyder and Worobo 2015). While most undergraduate curriculum revolves around general techniques geared toward foodborne pathogens, spoilage microorganisms represent an additional and importantly different aspect of food microbiology (FAO 2012). In order to increase undergraduate exposure to analytical methods and problem-solving approaches geared toward the control of food spoilage microbes, there is need to develop and implement spoilage-related laboratory exercises in undergraduate curricula. Based primarily on a few key principles, analytic approaches for dealing with microbial food spoilage differ from analytical approaches for dealing with foodborne pathogens. These differences create a need for laboratory exercises that are specific to spoilage microbes (Gram and others 2002). The most obvious difference is the identity of the pertinent microbes. Foodborne pathogens are primarily bacterial, while spoilage organisms include bacteria, yeast, and molds (Wu and Kim 2007). Importantly, even the relevant spoilage bacteria include different genera and species with different characteristics when compared to pertinent foodborne bacterial pathogens. For example, the spoilage bacterium Alicyclobacillus has different growth conditions (media, incubation temperature and oxygen levels, and time requirements), environmental sources, and potential commercial consequences MS 20151452 Submitted 24/8/2015, Accepted 27/3/2016. All authors are with Dept. of Food Science, Cornell Univ., 411 Tower Rd., Ithaca, NY 14853, U.S.A. than does, as a counter example, pathogenic E. coli (Zhao and others 2015). A 2nd striking difference between spoilage and Direct inquiries to author Orta-Ramirez (E-mail: [email protected]).

Undergraduate food microbiology courses are intended to provide students with a basic background in the microbial analysis of food (Jay and others 2006). This includes general laboratory techniques for enumeration and detection of pertinent foodborne microbes (Yousef and Carlstrom 2002). Curriculum focuses primarily on foodborne pathogens (pathogenic Escherichia coli, Listeria moncytogenes, Salmonella spp., and so on) and some fermentative organisms, which may be limited to lactic acid bacteria. Typically, these foci would then only include the relevant analytical methods that are organism-specific. Even less specific analyses, like total plate count and coliform detection, which are used to determine general sanitation, are often utilized to indicate the potential for pathogen contamination (Yousef and Carlstrom 2002). Approximately 48 million cases of foodborne illness and 3000 deaths result from pathogen-contaminated product annually in the United States (Scallan and others 2011a, 2011b), making foodborne pathogens a crucial part of undergraduate food science curriculum. However, spoilage also plays an important role in food production and many companies have stringent microbial quality

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78 Journal of Food Science Education • Vol. 15, 2016

doi: 10.1111/1541-4329.12089

Spoilage microbiology laboratory exercises . . .

Figure 1–Heat-treated product and the morphologies for the isolated spoilage organisms for (L to R): Incipiently spoiled salsa, postprocessing contamination, and underprocessing.

pathogenic microbes is that for spoilage organisms, outgrowth is required for a product to be deemed unfit (Dainty 1996). For pathogens, however, presence alone is often enough to precipitate an intervention (Mandal and others 2011). This requirement for outgrowth means that monitoring techniques and laboratory procedures may differ, or that different processing and storage conditions may be relevant to prevent outgrowth in a given product. It also means that producers’ response may vary. Additionally, and unlike food safety, for which stringent standards are in place, food quality is much more subjective (USDA 2015). Companies could employ a range of control limits and corrective actions depending on their given stringency for microbial food quality. These 3 issues, microbe identity, requirement for outgrowth, and individualized quality standards, make analytical approaches for food spoilage microbes unique from those for pathogens, and emphasize the need for content geared toward addressing these specific challenges. In order to increase undergraduate student exposure to methods for addressing spoilage, we included these laboratory exercises in our food microbiology lab course. These activities revolve around diverse spoilage organisms and utilize media specific to their detection and enumeration. Food quality loss as dictated by deleterious changes in sensory qualities is also a concept including a range of different product-specific faults that are related to spoilage (Phillips 1996; Ercolini and others 2006; Pateras 2007); therefore, sensorial observations of spoiled products were also included and the identity of organisms isolated from spoilage products used to help determine where in the production line spoilage occurred. These exercises can be implemented quickly with supplies and expertise available in the majority of food microbiology labs.

Exercise 1a

Figure 2–Sample student data for thermal destruction curves of 3 different microbes isolated from spoiled salsa. Data were collected at 60 °C.

Available on-line through ift.org

Colony morphology of spoilage organisms isolated from shelf stable salsa Thermal processing of foods, when performed correctly, significantly reduces the population of a target pathogenic organism. As a consequence, the treatment also reduces the population of other heat-sensitive spoilage organisms as well. However, some spoilage organisms may survive more extreme environmental conditions, Vol. 15, 2016 • Journal of Food Science Education 79

Spoilage microbiology laboratory exercises . . . such that controls such as water activity reduction, acidification, and heat treatments, designed to inhibit the growth of bacterial pathogens, are not severe enough to restrict the growth of all spoilage organisms. The types of spoilage organisms present may indicate a specific failure mode during food production. Spoilage of ingredients prior to thermal processing is referred to as “incipient spoilage.” After the heat treatment, the microbial population itself may be drastically reduced by the application of a processing step, but the physical properties of the food product will have already been degraded. On the other hand, the presences of multiple heat-sensitive organisms that are often associated with the environment are indicators of postprocessing contamination. Finally, underprocessed products may spoil as a result of thermal stable organisms that were able to survive the administered heat treatment. Plating of an underprocessed product would result in enumeration of a more homogenous microbial population. In this exercise, students cultured the organisms found on heat-treated salsa that was contaminated 1 of 3 possible ways and were expected to hypothesize about how the spoilage had occurred based on the number and morphology of their resulting colonies (Figure 1). Expected learning outcomes—At the completion of this activity, students will be able to:

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How does the thermal process play a role in the elimination of microbiota and what would we guess about the thermal resistance of the one isolate observed? Why would postprocessing contamination result in the presence of a variety of different organisms compared to contamination before processing?

Exercise 1b Thermal resistance of spoilage organisms isolated from canned salsa This exercise is a continuation of the morphological examination presented above. The thermal tolerance of isolated organisms from a heat-treated product gives an indication of whether contamination occurred before or after processing, or, alternatively, if the product was underprocessed. Students determined D-values and z-values for isolates from a heat-treated salsa product that was spoiled 1 of 3 possible ways. Coupled with the enumeration and examination performed in the previous exercise, students were expected to conclude whether the product was underprocessed, contaminated postprocessing, or was subject to incipient spoilage. Expected learning outcomes—At the completion of this exercise, students will be able to:

Distinguish among 3 different types of microbial spoilage (in- r Perform the necessary laboratory procedures to generate thermal destruction curves. cipient, postprocessing contamination, and underprocessing). r Apply information regarding the number of microbial survivors r Calculate D-values and z-values for microbial food isolates. plated from heat-treated salsa to determine the source of con- r Interpret the results of the thermal resistance exercise and the morphological exercise to determine where the contamination tamination. r Apply information regarding the variability in colony mor- of the heat-treated salsa occurred. phologies observed from plating the microbial survivors on Experiment overview heat-treated salsa to determine the source of contamination. Teams were provided with overnight broth culture tubes (5 mL tubes) of 1 isolate taken from their spoiled product (6 in total). Experiment overview Pseudomonas, E. coli, and Bacillus subtilis were provided to represent Prior to class, 3 types of spoiled heat-treated salsa products were the incipient, postprocessing, and underprocessing contamination, prepared: (a) The incipient spoiled salsa was prepared by allowing respectively. Groups were not told the identity of the organism, the salsa to spoil at room temperature before the hot water bath only that it had been previously isolated from their product. For treatment. (b) The underprocessed product was prepared by the each organism, groups performed thermal destruction tests at 55, addition of a thermal resistant spore-former, such as a Bacillus spp. 60, and 65 °C. Every 5 min (for time points 0, 5, 10, 15, 20, and These jars were removed after only 1 to 2 min in the water bath. 25 min) one of their 5 mL tubes was removed from the water baths (c) The postprocessing contaminated jar was processed along with and used for dilution and plating. Because students had no prior the rest of the jars of salsa, but after the water bath treatment, these knowledge of the heat resistance of their organism, they plated jars were left open and a variety of acid tolerant organisms were dilutions of the homogenized salsa from 100 up to 10−6 . After added, including yeast and molds as well as bacteria such as E. coli. 24 h incubation at room temperature, students enumerated the Spoiled products were randomly distributed among student bacterial population at each time point, constructed kill curves, teams. Each team was responsible for stomaching and plating di- and calculated the D-values and z-values. lutions from their respective salsa on trypticase soy agar. After 48 h incubation at 25 °C, students calculated total plate counts Discussion points and noted the number of different colony morphologies. Students shared the data among all the groups so that differences in results could be observed and contrasted. Teams shared the raw data of population counts at each time point per temperature Discussion points and individuals performed calculations. Based on these results, Students shared the data among all the groups so that differences students were asked to draw conclusions about the relative thermal in results could be observed. Teams shared total plate counts and tolerance of these organisms and whether or not their respective number of unique morphologies. Based on these results, students thermal tolerances indicated they would or would not be able to made hypotheses about the point in processing at which contamsurvive a thermal process. ination occurred. Specific questions that were discussed were:

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Why does the incipiently spoiled product appear spoiled (that is, noticeable quality changes), but no/few microbes were able to be isolated? Why would only 1 morphology appear in the underprocessed product?


Exercise 2 Detection and sensory observations of Alicyclobacillus-spoiled apple juice The presence of particular organisms alone is not always an indication of spoilage, although the detection of certain organisms Available on-line through ift.org

Spoilage microbiology laboratory exercises . . .

Figure 3–Filtration systems used by students to detect Alicyclobacillus in apple juice. Shown beside a positive result, colony growth on plated filter following 5 days of incubation at 45 °C.

may be used as an indication of a product’s capacity to spoil. This is in contrast to many foodborne pathogens in which detection is a sufficient violation of food safety. Food quality is based on a host of sensory attributes, and the metabolism of various organisms has different effects. Occasionally, visible growth of the organism renders a product unacceptable to consumers, for example, mold spoilage is often characterized by apparent mycelia, and some yeast and bacteria produce haze or turbidity in beverages. In other cases, the spoilage organisms may have no visible hallmark, and only flavor or aroma is impacted because of the degradation of nutrients and/or production of off-flavors. Similarly, proteolysis and lipolysis from the metabolic action of spoilage microbes may cause the production of off-aromas and flavors, slime, decrease in firmness, discoloration, or gas production (Jay and others 2006). In this exercise, students detected the presence of Alicyclobacillus, a bacterium with high spoilage potential, in apple juice. Demonstrations of Alicyclobacillus-spoiled juice were also prepared, and students were expected to note what sensory attributes were altered as a result of this particular spoilage and what attributes were not (notably, no visual indication of spoilage). Expected learning outcomes—At the completion of this exercise, students will be able to:

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Describe the varying changes in organoleptic properties that occur as a result of microbial spoilage. Apply the appropriate methods (vacuum filtration and selective media) to detect the presence of high spoilage potential microorganisms in apple juice.

Experiment overview Whole bottles of filtered apple juice were purchased and 1/3 of the bottles were inoculated with Alicyclobacillus. Previously spoiled juice was provided as a demonstration and students were asked to observe the sensorial changes to aroma and visual appearance. Each team was provided with a bottle of apple juice and a sterile vacuum-driven filtration unit. Students filtered the entire bottle of juice and placed the spent filter onto a plate of acidified potato dextrose agar, pH 3.5, and incubated at 45 °C for 5 days. After Available on-line through ift.org

5 days, teams observed their plated filters for colony growth and putative detection of Alicyclobacillus (Figure 3).

Discussion points Students were asked to consider the range of organoleptic changes they had observed in spoiled food from personal experience. Then, in the context of the experiment, from the range of possible sensory changes, students were asked to describe their observations of the Alicyclobacillus-contaminated apple juice. Students discussed the significance of a lack of visual change due to spoilage, and the challenge that it poses to both the detection and consumer experience. Students were additionally led in a discussion about the challenges of detection when Alicyclobacillus is present at such low levels. The discussion was extended for advanced student groups, and instructors asked students to evaluate the use of detection alone for assessing and limiting microbial spoilage in all food products or for all organisms. The students determined why certain products are most associated with Alicyclobacillus and why its detection in these products is a useful laboratory exercise.

Adaptations These exercises can be adapted depending on the instructor’s resources and needs. It can be expanded and contextualized by the addition of more spoiled food demos. This would facilitate additional discussion about the sensory changes due to spoilage, how various spoilage organisms affect food quality differently, and address the obvious differences between microbial pathogens, spoilage organisms, and fermenters. If laboratory space or equipment is limiting, the data can be provided to students who can construct thermal destruction curves and calculate D-values and z-values. Alternatively, exercises 1a and 1b could be performed consecutively instead of concurrently, so that students may actually isolate their target organism themselves.

Student Results At the end of each semester in which this set of activities was presented, students were asked to evaluate their skills regarding different components of the course. Students were asked to evaluate themselves based on knowledge, confidence in the subject, Vol. 15, 2016 • Journal of Food Science Education 81

Spoilage microbiology laboratory exercises . . . and experience with the subject. Students were asked to rank themselves on a scale of 1 to 5 in terms of these issues where 1 is low and 5 is high knowledge/confidence/experience. The average student response from 2 semesters worth of presenting this material is as follows. Regarding “food spoilage” students rated their knowledge obtained as 3.4, their confidence with the subject as 3.3, and their level of experience as 3.2. These values were on par with or slightly above scores received in other topic areas presented (for example, media, Gram reactions, and so on). It should be noted that these self-reports were issued as part of a final exam at the end of the semester, not part of student feedback immediately following the presentation of the materials. These above average scores suggest that students retained the information over the course of the semester. Additionally, follow-up with graduates 1+ years after completing the course, students report retaining much of the content presented through this set of laboratory exercises.

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