Abigail B. Snyder

Chemical and genetic characterization of bacteriocins: antimicrobial peptides for food safety.

Antimicrobial peptides are produced across all domains of life. Among these diverse compounds, those produced by bacteria have been most successfully applied as agents of biocontrol in food and agriculture. Bacteriocins are ribosomally synthesized, proteinaceous compounds that inhibit the growth of closely related bacteria. Even within the subcategory of bacteriocins, the peptides vary significantly in terms of the gene cluster responsible for expression, and chemical and structural composition. The polycistronic gene cluster generally includes a structural gene and various combinations of immunity, secretion, and regulatory genes and modifying enzymes. Chemical variation can exist in amino acid identity, chain length, secondary and tertiary structural features, as well as specificity of active sites. This diversity posits bacteriocins as potential antimicrobial agents with a range of functions and applications. Those produced by food-grade bacteria and applied in normally occurring concentrations can be used as GRAS-status food additives. However, successful application requires thorough characterization.

Rapid authentication of concord juice concentration in a grape juice blend using Fourier-Transform infrared spectroscopy and chemometric analysis

Concord grape juice is associated with many health benefits, and so it can be sold at a premium price. However, there is currently no method to verify the percent composition of Concord grape juice in grape juice blends. In order to guard against potential adulteration, a rapid method for authentication is required. Fourier Transform infrared (FT-IR) spectroscopy was used to develop a model which predicts the percent composition of Concord grape juice. The model was based on a training set of 64 samples with Concord concentrations ranging from 50% to 100%. Data was collected on an external validation set with a standard error of prediction of 5.6% using 7 factors. The results suggest the feasibility of using FT-IR coupled with chemometrics as a production-scale tool for authentication claims of Concord in grape juice blends, protecting consumers and businesses against deceptive labelling.

Characterization and control of Mucor circinelloides spoilage in yogurt.

Consumer confidence in the food industry is severely affected by large-scale spoilage incidents. However, relatively little research exists on spoilage potential of members of the fungal subphylum Mucormycotina (e.g. Mucor), which includes dimorphic spoilage organisms that can switch between a yeast-like and hyphal phase depending on environmental conditions. The presence of Mucor circinelloides in yogurt may not cause spoilage, but growth and subsequent changes in quality (e.g. container bloating) can cause spoilage if not controlled. The purpose of this study was to evaluate the effects on M. circinelloides of pasteurization regimen, natamycin concentrations, and storage temperature in yogurt production, as measured by fungal proliferation and carbon dioxide production. A strain of M. circinelloides isolated from commercially spoiled yogurt showed greater yogurt-spoilage potential than clinical isolates and other industrial strains. D-values and z-values were determined for the spoilage isolate in milk as an evaluation of the fungus ability to survive pasteurization. Natamycin was added to yogurt at 0, 5, 10, 15, and 20ppm (μg/ml) to determine its ability to inhibit M. circinelloides over the course of month-long challenge studies at 4°C, 15°C, and 25°C. Survivors were recovered on acidified PDA and carbon dioxide levels were recorded. The D-values at 54°C, 56°C, and 58°C for hyphae/sporangiospores were (in min) 38.31±0.02, 10.17±0.28, and 1.94±0.53, respectively, which yielded a z-value of 3.09°C. The D-values at 51°C, 53°C, and 55°C for yeast-like cells were (in min) 14.25±0.12, 6.87±1.19, and 2.44±0.35, respectively, which yielded a z-value of 0.34°C. These results indicated that M. circinelloides would not survive fluid milk pasteurization if contamination occurred prior to thermal treatment. CO2 production was only observed when M. circinelloides was incubated under low-oxygen conditions, and occurred only at temperatures above 4°C. Addition of 10ppm and greater of natamycin inhibited the growth and CO2 production of M. circinelloides under moderate temperature abuse when compared to the untreated control. These data suggest that yogurt spoilage (container bloating) caused by anaerobic growth of M. circinelloides is due to post-pasteurization contamination. Temperature abuse facilitated spoilage as CO2 production was observed in yogurt incubated at 15°C and 25°C, but not at 4°C. The addition of at least 10ppm of natamycin prevented M. circinelloides growth in both hyphal and yeast-like phases, as well as CO2 production in temperatures of up to 15°C for 30days.

Bactericidal thurincin H causes unique morphological changes in Bacillus cereus F4552 without affecting membrane permeability

Thurincin H is an antilisterial bacteriocin produced by Bacillus thuringiensis SF361. It exhibits inhibitory activity against a wide range of Gram-positive foodborne pathogens and spoilage bacteria including Listeria monocytogenes, B. cereus, and B. subtilis. This hydrophobic, anionic bacteriocin folds into a hairpin structure maintained by four pairs of unique sulfur to α-carbon thioether bonds. As its hydrophobicity and structure are quite different from most archived bacteriocins, this study aimed to elucidate its mode of action and compare it with the mechanisms of other well-characterized bacteriocins. The results indicated that, although bactericidal to B. cereus F4552, thurincin H did not lead to optical density reduction or detectable changes in cell membrane permeability. B. cereus F4552 imaged by scanning electron microscopy after treatment with thurincin H at 32 × MIC showed regular rod-shaped cells, while only cells treated with thurincin H at the elevated levels of 256 × MIC showed loss of cell integrity and rigidity. Both concentrations caused greater than 99% of cell viability reduction. In contrast, nisin caused significant cell membrane permeability at concentration as low as 2 × MIC. These results indicated a difference in the mode of action for thurincin H compared with the generalized pore-forming mechanism of many lantibiotics, such as nisin.

Developing and optimizing bacteriophage treatment to control enterohemorrhagic Escherichia coli on fresh produce.

Bacteriophages are potentially useful in controlling foodborne pathogens on minimally processed products since phage application is a non-destructive treatment. The purpose of this study was to evaluate the efficacy of a newly isolated environmental bacteriophage against enterohemorrhagic Escherichia coli on fresh produce, and optimize the treatment with consideration for potential application. Seven anti E. coli O157:H7 EDL933 bacteriophages were isolated from various sources; the most promising was isolated from municipal wastewater. This isolate (designated as E. coli phage OSY-SP) was propagated with the host, in a growth medium, to a titer of 10(8) PFU/ml. Before inoculation into fresh produce, E. coli phage OSY-SP was incubated with the host bacterium, spent medium was filter-sterilized, and the resulting crude lysate was used as a source of phage inocula for preliminary experiments. For optimized testing, phage in the crude lysate was purified by ultra-centrifugation and resuspension in phosphate-buffered saline. Efficacy of phage treatments was determined as a function of fresh produce type (cut green pepper or spinach leaves), treatment time (2 or 5min rinsing), and temperature of holding treated produce (4°C, 25°, or a combination of both temperatures). Cut green pepper was treated with UV light, to eliminate background microbiota, then spot-inoculated with E. coli O157:H7 EDL933 on cut edges, and the inoculum was allowed to dry. Because of its susceptibility to damage, baby spinach leaves were not subjected to a decontamination treatment. These leaves were inoculated with the green fluorescent protein-labeled E. coli O157:H7 B6-914 to facilitate inoculum enumeration in the presence of background microbiota. Phage suspension was applied to the inoculated fresh produce that was subsequently held for three days under variable storage conditions. The optimized phage treatment decreased the populations of pathogenic E. coli by 2.4-3.0logCFU/g on cut green pepper (5-min rinse) and 3.4-3.5logCFU/g on spinach leaves (2-min rinse), during 72h storage. The majority of this decline was caused by the antimicrobial action of the phage. These findings suggest the utility of bacteriophage to selectively control pathogens on fresh produce.

Undergraduate Laboratory Exercises Specific to Food Spoilage Microbiology

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.

Risk Mitigation for Immunocompromised Consumers of Mucormycete Spoiled and Fermented Foods: Germain Guidance and Remaining Needs

Mucoralean invasive fungal infections, while unusual among the general population, have a high mortality rate among immunocompromised individuals who become infected. They are also common spoilage organisms in cultured dairy products, some fresh produce, and baked goods. Additionally, Mucor and Rhizopus spp. are utilized in the production of traditional fermented foods including mold ripened cheeses and fermented soy products. The risk that consumption of these foods poses to immunocompromised consumers has been previously identified. However, actionable guidance on implementation of appropriate dietary restrictions and microbial specification targets for food manufacturers serving these populations is scarce and is limited by insufficient data regarding traceback analysis in cases of invasive fungal infections where food is the suspected transmission vector. Culture-dependent and molecular subtyping methods, including whole genome sequencing, will improve identification of the point source. In turn, the empirically determined information on root-cause can best direct the development of appropriate food safety policies and programs.

Outgraded produce variably retains surface inoculated Escherichia coli through washing

The use of secondary quality produce has gained attention as a solution to food waste in both the U.S. and Europe. The purpose of this study was to evaluate the impact of using secondary quality or outgraded produce on the retention of surface inoculated E. coli following a rinse treatment on four model fresh produce systems (apple, tomato, carrot, lettuce). A three-strain cocktail of rifampicin-resistant generic E. coli, with a concentration of 9.0u202flogu202fCFU/mL, was spot-inoculated on the intact surfaces of U.S. No.1 grade produce items and damaged or decayed areas of outgraded produce items. Generally, outgraded produce of all four kinds retained higher levels of inoculated E. coli following two postharvest treatments, chlorinated (150u202fppm) or water only. However, physical damage, those defects which compromised the integrity of the produce surface, lead to significantly greater E. coli levels following rinsing than did physiological defects. Compared to U.S. No.1 quality apples, outgraded apples retained 4.3u202f±u202f1.4u202flogu202fCFU/g more E. coli following water only treatment, and 3.6u202f±u202f1.7u202flogu202fCFU/g more following chlorine treatment. Outgraded tomatoes retained significantly more (3.5u202f±u202f1.1u202flogu202fCFU/g) inoculated E. coli following water only rinse and 3.0u202f±u202f1.4u202flogu202fCFU/g more inoculated E. coli following chlorine treatment than U.S. No.1 quality tomatoes did under the same treatment conditions. Outgraded carrots retained 1u202f±u202f1.1 log more CFU/g inoculated E. coli following water only treatment and 0.5u202f±u202f0.8 log more CFU/g inoculated E. coli following chlorine treatment, compared to U.S. No.1 carrots. Outgraded lettuce leaves retained 1.6u202f±u202f0.5u202flogu202fCFU/g more inoculated E. coli following water only treatment and 4.1u202f±u202f0.4u202flogu202fCFU/g more inoculated E. coli following chlorine treatment than did U.S. No.1 quality lettuce leaves under the same treatment conditions. Treating with 150u202fppm chlorine was not sufficient to eliminate the increased microbial retention associated with secondary quality or outgraded produce, and the efficacy of disinfection was greatly affected by type of defect. Apples with physical damage retained significantly higher E. coli loads than did those with physiological defects, an additional 2.6u202flogu202fCFU/g under chlorine treatment and 0.8u202flogu202fCFU/g more under was water only treatment. Tomatoes with physical damage had a 1.3-log CFU/g and 0.6-log CFU/g average increase of retained E. coli counts compared to those with physiological defects following a chlorine and water only treatment, respectively. Although a chlorine dip provided only a modest reduction in pathogens, generally, outgraded produce with physiological defects may present less food safety risks if introduced into the fresh market than does produce with physical damage due to their enhanced retention of bacterial cells. Therefore, as industry considers how to minimize its food waste problem, preferentially directing physically damaged produce away from the fresh market will help to minimize risk while maximizing food resources.

The Science of a Sundae: Using the Principle of Colligative Properties in Food Science Outreach Activities for Middle and High School Students.

The opportunities for outreach activities for professionals and academics in food science are extensive, as too are the range of participants’ experience levels and platforms for delivery. Here, we present a set of activities that are readily adaptable for a range of students (ages 10 to 18) in multiple platforms (demonstration table and hands-on workshop). Our activity, collectively called “The Science of a Sundae,” has three units, one for each of the three parts of a sundae: the caramel sauce, the cherry, and the ice cream. In each unit we use these familiar food items to illustrate how colligative properties (or, simply, “solutions” for younger students) impact the chemical, microbiological, and sensorial properties of food. We have used these activities to present to over 1000 students and their parents/chaperones. Grade levels of student participants have ranged from 5th grade through high school, and these activities have been presented in the form of a demonstration table at science events as well as a set of three 45-minute workshops in a classroom setting. Educational impact of these activities was evaluated with 7th grade students (n = 77) who participated in the 3-phase workshop. On average, students who took the posttest (after participation in the workshop) scored 36% higher than students who took the pretest (prior to participation in the workshop). These results and instructor observations suggest the merit of this lesson and its adaptability among ages and platforms.