Spoilage is the degradation of food such that the food becomes unfit for human consumption. Food can be spoiled by a number of means, including physical and chemical means. However, the most prevalent cause of food spoilage is microbial growth and residence in the food, which results in numerous undesirable metabolites being produced in the food that cause unwanted flavors and odors. Approximately 25% of the world’s food produced post harvest or post slaughter is lost to microbial degradation of food alone. (1)
The main culprits are microbial organisms known as specific spoilage organisms (SSOs). The concept of SSOs arises from the fact that not all bacteria cause food spoilage; indeed, the degree of food spoilage is not proportional to the amount of microbes present on the food. SSOs are solely responsible for spoilage of the food and the typical characteristics associated with that spoilage. They are typically present in very low numbers and comprise a low percentage of the microflora present on the food. (1)
Identification of SSOs is done by comparisons of the physical and chemical features of the collective spoiled products with the individual products left behind by each organism in the spoilage microflora. In particular, the qualitative ability of each organism to produce off-odors (spoilage potential) and the quantitative ability of each organism to produce spoilage metabolites (spoilage activity) are examined. This simple phenotypic identification scheme, along with a 16S rDNA gene sequencing to confirm results, allows scientists to discover which organism or organisms in the spoilage microflora are directly responsible for the spoilage. (1, 6)
Each unique environment has its own unique SSOs, because each different environment selects for particular organisms to thrive. The spoilage domain for an SSO is identified based upon the conditions (pH, temperature, water activity, and atmosphere) under which that SSO can grow and produce the metabolites that cause spoilage. (1)
Spoiled Food vs. Harmful Food
It is important to note that spoilage bacteria normally outgrow pathogenic bacteria during storage. Thus some foods may spoil before they become toxic. Spoilage bacteria and pathogenic species in spoiled food have different effects. Pathogens are responsible for the symptoms that result from eating spoiled food; SSOs may or may not have a direct harmful effect to the consumers. (2)
Spoilage in Fish
Fish spoilage manifests itself physically in numerous ways. In terms of smell, spoiled fish will generally have a fishy, sour, or ammonia-like stench. Appearance-wise, spoiled fish may appear to be dry or mushy in certain areas, and the gills may have slime. Spoiled fish will also have flesh that is soft, or does not spring back when pressed upon. Typically, spoiled fish will also have a green or yellowish discoloration; however, this arises not from spoilage metabolites, but rather oxidation of the oxygen transporters in fish blood (myoglobin to metamyoglobin) during frozen storage or from prolonged or unnecessary exposure of the fish to air. (3, 4)
Compared to other foods, fish is unique as a substrate for microbial growth. This uniqueness stems from several important factors: the poikilotherm nature of fish, a high post mortem pH in the flesh (typically greater than 6.0), the presence of non-protein-nitrogen (NPN) in large quantities, and the presence of trimethylamine oxide (TMAO). (5)
The poikilotherm nature of fish selects for bacteria that can thrive in a wide range of temperatures. For example, the microflora of temperate water fish is dominated by psychrotrophic Gram-negative, rod-shaped bacteria such as those found in the genera Pseudomonas and Moraxella, with only varying proportions of Gram-positive organisms such as Bacillus. (5)
The high post mortem pH of fish flesh is caused by the fact that fish flesh is low in carbohydrates (less than 0.5%) in the muscle tissue and that only small amounts of lactic acid are produced after death. This allows pH sensitive organisms such as Shewanella putrefaciens to grow in seafood but not in other meats. (5)
The NPN fraction of the fish flesh consists of low-molecular-weight water-soluble nitrogen contains compounds, particularly free amino acids and nucleotides, that allow it to serve as a readily available bacterial growth substrate. Decomposition of these compounds is responsible for many of the off-odors and off-flavors typically found in spoilage. For example, the breakdown of cysteine and methionine by certain microbes, both sulfur-containing amino acids, forms hydrogen sulfides and methylmercaptane respectively which causes undesirable odors to emanate from spoiled fish. (5)
The presence of TMAO in fish is well-established, and it is known to cause a high redox potential in the fish flesh, although the significance of this is not clear. The spoilage of fish is influenced most by the presence of TMAO in conditions where oxygen is not present. Some anaerobic bacteria are able to utilize TMAO as the terminal electron acceptor in an anaerobic respiration process with trimethylamine (TMA) as the primary product; TMA contributes to the characteristic ammonia-like and fishy off-flavours in spoiled fish. (1, 5)
SPOILAGE OF FRESH FISH
The niche on spoiling fresh fish and the microbes that live there
Raw fish spoilage is part of the 25% of food products that is lost yearly due to microbial activity. Microbes are found on the outer body covering and the inner surfaces of fresh fish, such as the skin, gills, and GI tract. The poikilotherm nature of fresh fish allows a wide variety of bacteria to grow, including the Gram-negative, rod-shaped bacteria which belong to the genera Pseudomonas, Moraxella, Acinetobacter, Shewanella, Flavobacterium, Aeroemonadaceae, and Vibrionaceae, and Gram-positive bacteria such as Bacillus, Micrococcus, Clostridium, Lactobacillus, and Corynebacterium. Psychrotrophs are bacteria that can tolerate cold temperature and grow at 0 degree Celsius but grow optimally around 25 degrees Celsius.(1,5)
At the time of being captured, fresh fish contain on their bodies a wide variety of microorganisms, also known as the microflora of the fish. Depending on the region of water from which the fish are caught, the microflora of the fish can have different degrees of complexity. For example, fish that are caught in very cold and clean waters have a lower number of psychrotrophic and psychrophilic microbes whereas fish caught in warmer waters have somewhat higher counts of mesophilic microbes. In addition, fish captured from polluted warm water have a selection of unique microbes due to the presence of a large number of Enterobacteriaceae. However, regardless of where the fish has been caught, only a number of the microbes is able to proliferate on the post-mortem fish. Of these surviving microbes, only a small portion can generate metabolites that create the off-flavors, off-odors and discolorations that humans find unsuitable for consumption. In fresh fish, the specific spoilage bacteria include Shewanella putrefaciens and Pseudomonas spp.(5,6)
The immune system of alive or newly caught fish can be deadly to the spoilage bacteria S. putrefaciens and Pseudomonas spp. since the immune system of the fish is still functional and keeps the flesh of the fish sterile by suppressing the growth of bacteria in that location. However, post-mortem, the immune system deteriorates. Without the immune system barrier, some bacteria invade the flesh by entering between the muscle fibers while others establish colonies on the flesh. As the fish body breaks down, some metabolic biomaterials make their way to the surface and become available to the microbes S. putrefaciens and Pseudomonas spp.(6)
Since fish are typically put on ice immediately post-harvest, S. putrefaciens and Pseudomonas spp. often pass through a lag phase of approximately one to two weeks in order to adjust to the new environment. How long the lag phase lasts depends on how long the bacteria need to make the appropriate biosynthetic materials and prepare for growth. After these processes have been complete, S. putrefaciens and Pseudomonas spp. enter the exponential phase, in which it grows at an exponential rate. Over time, communities of these microbes are established that produce various metabolites that are associated with spoilage.(6)
In dead fresh fish, enzymes can bring about the destruction of cells via autolytic changes. Both S. putrefaciens and Pseudomonas spp. can produce hypoxanthine from inosine or inosine monophosphate which come from the autolytic changes in dead fish and use them as biosynthetic materials to grow.(6)
For fresh chilled fish stored in air, Pseudomonas spp. typically produce biogenic amines, ketones, aldehydes, esters, sulfur compounds. S. putrefaciens typically produce TMA, H2S, acetic acid, and other sulfur compounds. S. putrefaciens can use trimethylamine oxide (TMAO) as the terminal electron acceptor to generate TMA (7,8). This is an anaerobic respiration process that helps S. putrefaciens generate energy in the form of ATPs via the Kreb’s cycle(5). Due to the very small amount of carbohydrate that most fish contain (less than 0.5% of body composition), very small amounts of lactic acid are produced, making the pH on the body of post-mortem fresh fish usually above 6.0 which is appropriate for the growth of the pH-sensitive bacteria S. putrefaciens (5). Furthermore, breakdown of certain amino acids on fresh fish also establishes an appropriate environment for bacteria to proliferate. For example, the breakdown of amino acids such as glycine, serine, and leucine help the bacteria gain esters, ketones, and aldehydes for its metabolism.(7,9)
In general, without further preservation techniques, the environment of a fresh fish provides an abundance of biosynthetic materials that the spoilage organisms such as Shewanella putrefaciens and Pseudomonas spp. can readily use to survive and proliferate. The products of metabolic processes of these microbes include biological compounds that signal spoilage and render the fish unsuitable for human consumption.
SPOILAGE OF PROCESSED FISH
The spoilage activity of lightly preserved fish and fish products may develop in spite of the inhibitory strength of the processing and storage conditions. The microorganisms living off the food products have evolved and managed to endure the physical and chemical processing techniques, including CO2 and vacuum packing, salting, heating or pasteurization, and addition of preservatives (1,5,10). Numerous studies have identified several SSOs including Photobacterium phosphoreum and lactic acid bacteria (Lactobacillus and Carnobacterium) which are largely responsible for spoilage of lightly preserved products (1,5,10,11,12,13)
Niches and their microbes
CO2 and vacuum packing
CO2 and vacuum packing are used to create an anaerobic environment by which to eliminate aerobic SSOs and extend the life of the fish products. However, these processing techniques do not guarantee the sterility and safety of the fish products due to the presence of microbes that are able to utilize alternative mechanisms of survival. In such an oxygen-deprived living niche, Gram-negative and CO2-resistant microbes generate their energy from fermentative processes and respiratory mechanisms involving electron acceptors other than oxygen (1,5,11).
Photobacterium phosphoreum is a Gram-negative, psychrotolerant, bioluminescent large cell microbe that thrives in the CO2 and vacuum packing anaerobic environment by a respiratory mechanism that uses TMAO as the electron acceptor. TMAO is reduced to trimethylamine (TMA), giving the product spoiled flavors. However, Photobacterium phosphoreum does not survive well when it encounters freezing conditions or spices that are designed to extend shelf life (1,5,10,12).
Another group of bacteria that has the ability to adapt to the anaerobic packing environment is lactic acid bacteria (LAB). These are Gram-positive, fermentative bacteria that produce lactic acid and antibacterial peptides called bacteriocins to survive in a resource-restricted niche. The low pH resulting from the production of lactic acid in combination with bacteriocins help LAB gain a competitive advantage to neighboring bacteria by attacking them (1,5,11).
Many bacteria are only able to survive within a certain range of salt concentration (0.2 - 5% NaCl concentration) (5,14). Therefore, salting in fish is used to bring about extreme salt concentrations such that bacterial growth is dramatically reduced. However, even in this extreme living environment, salt-loving bacteria called halophiles can survive in salt concentrations of up to 10-20% NaCl (14). The ways that halophiles deal with the highly soluted media are to exchange Na+ with K+ to keep a low intracellular concentration of Na+ and increase the intracellular glycerol in such way that the water flow is balanced between the inside and outside of cell (14).
Some fish products undergo a mild heat treatment like pasteurization at a specific temperature in a sufficient period of time to kill heat-sensitive bacteria. However, Gram-positive bacteria such as Clostridium sp. and Bacillus anthracis are specific microbes that are able to perpetuate in the face of pasteurization by producing spores (1,5). In response to environmental stresses such as pasteurization, these microbes go through sporulation to produce spores, which are specialized, dormant cells that are resistant to heat, desiccation, radiation and antibiotics (14). The spores will germinate and grow again if the surrounding conditions are favorable.
Addition of Preservatives
Many semi-preserved seafood are subjected to the addition of preservatives like sorbate and benzoate with an attempt to extend the storage shell life of those products. Sorbate and benzoate are natural or synthetic organic compounds that reduce the pH and increase the NaCl concentration, which eliminate many Gram-negative bacteria. However, microbes such as acid-tolerant LAB (lactobacillus) and yeasts are able to remain active in this environment and become part of the surviving spoilage domain (1,11).
1. Employment of 16S rDNA gene sequencing techniques to identify phenotypically difficult-to-identify culturable eubacteria from food and water (15)
In the past, SSOs could only be detected by their phenotypic expressions on spoiled seafood. However, current research utilizing 16S rDNA gene sequencing has been shown as a successful method for more exact identification of phenotypically difficult-to-identify SSOs in foods.
The laboratory technique of using 16S rDNA gene sequencing to identify various organisms is explained in-depth. First, PCR amplification of the DNA preparations of various isolates was performed in order to generate amplicons upon which sequence analysis could be performed. From subsequent sequencing and analysis of these amplicons, researchers concluded that reliable identifications could be made using this technique.
2. Development of a smart packaging for the monitoring of fish spoilage (16)
The reasoning behind this method of monitoring fish spoilage lies in the fact that when fish spoils, a variety of basic volatile amines are released into the headspace of the container, which is detectable with the appropriate pH indicators (a pH sensitive dye that changes colors appropriately). Researchers concluded from laboratory trials that the sensor accurately tracks amine concentration in the packaging headspace and thus the degree of spoilage of the fish; ongoing trials are being done to test the validity of this method in refrigeration temperatures as well as in other common packaging scenarios.
3. Soft tissue infection and bacteremia caused by Shewanella putrefaciens (17)
Due to a recent discovery of a patient who was identified as infected with Shewanella putrefaciens, scientists are currently investigating and paying attention to the ability of bacteria that are not usual pathogens in clinical cases to cause diseases in humans. In this study, scientists regarded Shewanella putrefaciens as some of the unusual and opportunistic pathogens that could infect and endanger the health of immuno-compromised patients.
4. Bacterial membranes: the effects of chill storage and food processing (18)
Currently, a combination of novel preservation techniques with refrigeration is being investigated to assess whether this approach can prolong the shelf life of fresh fish and fish products. New techniques such as ultrasound, high hydrostatic pressure, and pulse electric field have the potential to disrupt the cold-adapted proteins and membrane lipids of spoilage bacteria such as Pseudomonas spp., shunning their metabolic processes and thus reducing the spoilage potential of these bacteria.
5. Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl) homoserine Lactone (12)
Photobacterium phosphoreum is a known spoilage specific organism that produces a type of chemical communication signals called N-acylated homoserine lactones (AHLs). In this study, AHL was discovered in Photobacterium phosphoreum strains that did not express bioluminescence. Therefore, it was suggested that AHLs may serve as a negative regulatory component of bioluminescence. Furthermore, even though the role of AHLs in causing food spoilage remains unclear, the activity of AHLs has been linked to the production of degradative enzymes which could be involved in the spoilage process.
Fish spoilage is not a negligible portion of the approximate 25% of the world’s food produced post harvest or post slaughter that is lost to microbial activity. In fish and fish products, some microbes, called specific spoilage organisms, generate chemical compounds that are characteristic of spoilage, including the unsuitable food flavors, odors, discolorations, and textures. The various ways microbes are able to survive and proliferate despite radical changes in the environment all serve to highlight the ability of microbes to evolve and adapt to the resources available in a particular niche.
However, not all of the details concerning microbial interactions that lead to fish spoilage have been elucidated and thus further research is needed. This knowledge will help extend the shelf life of fish products by assisting the invention of new preservation techniques that prevent microbial spoilage and protect the consumers from food-borne illnesses.
ASIDE: The Interaction between Listeria monocytogenes and Spoiled Shrimp
The ocean is full of bacteria. The ocean is an environment that allows many bacteria to thrive. In turn, those that inhabit the oceans are also susceptible to becoming hosts and carriers of these bacteria. On type of bacteria that is found on the inhabitants of the ocean is Listeria monocytogenes. Listeria monocytogenes is a Gram-positive bacterium that is motile via flagella and when eaten by pregnant women, it causes meningitis in their newborns. This organism is found more on chitinous seafood such as shrimp, crab and lobster . In an attempt to find the difference between chitin-containing and chitin-free waters, the two were tested for L. monocytogenes. The results found that the addition of chitin stimulated the growth of Listeria monocytogenes. Outside of the water, shrimp is still a carrier of this bacterium. In a report, it was found that L. monocytogenes was found on the exoskeleton but not in the digestive tract of shrimp that were exposed to high levels of that bacterium in aquaculture tanks. This bacterium is considered a foodborne pathogen. Listeria monocytogenes is aerobic, microaerophilic, facultative anaerobic, catalase positive, oxidase negative and esculin hydrolysis positive. It is a ubiquitous microbe that has the ability to grow and survive under a variety of different conditions and grows from temperatures of about 0 to 44°C. Not only is it able to grow in various temperatures, it is tolerant of higher ionic concentrations. L. monocytogenes is able to grow in 2 to 4% salt concentrations. This means that not only will L. monocytogenes grow on spoiled shrimp, but it will also grow on lightly preserved shrimp. L. monocytogenes has a minimum pH value of 4.1 which is an acidic value. Generally, less acidic products such as shrimp, are usually more susceptible to spoilage by bacteria and pathogenic growth. The approximate pH of the shrimp is 6.8-7.0 making it susceptible to bacterial spoilage, specifically in this case, by L. monocytogenes. Acidity has a couple of effects on respiring microbial cells: it renders the food less optimal as an environment for key enzymatic reactions, and it influences the transport of nutrients into the cell. Other important functions of the microbe such as synthesizing and utilizing DNA and ATP require the microbe’s environment to have a neutral pH. Lovett et. al. showed in 1988 that L. monocytogenes grew readily in inoculated samples of raw shrimp with the pathogen attaining maximum populations greater than 108 CFU/g in the shrimp following 14 days of storage at 7°C. The minimum growth temperature of L. monocytogenes is 32°F. Since the minimum growth temperature is slightly cold, the relative humidity of the environment will be higher, because the two are inversely proportional. Higher humidity causes surface spoilage of the shrimp, and also an accommodating environment for the proliferation of L. monocytogenes. An energy source, nitrogen source, vitamins (such as the B vitamins) and minerals are essential to have for the bacterium to grow. Some of the energy sources include simple sugars, alcohols and amino acids, the primary nitrogen source is amino acids, and since L. monocytogenes is Gram-negative, it synthesizes its own B vitamins. But under large concentrations of CO2, the growth of L. monocytogenes is slowed down. L. monocytogenes are not the only bacteria you can find on the surface of the spoiling shrimp. There are also other bacteria such as spoilage microorganism, other pathogens and innocuous microorganisms as well as desirable microorganisms that aid in the preservation of the shrimp. Some of the bacteria are competitors to L. monocytogenes and others are helpful to it. The competitors of L. monocytogenes are usually the spoilage bacteria because it needs the resources that the spoiling shrimp offers. This competition can inhibit the growth of L. monocytogenes. Some other bacteria actually stimulate the growth of L. monocytogenes. Pseudomonas species have been show to stimulate L. monocytogenes (Marshall and Schmidt, 1988) by providing more available substrates for their growth through proteolysis and lipolysis. The presence of the essentials for the bacteria to grow along with other helping factors allows L. monocytogenes to thrive on the spoiling shrimp. It also helps that L. monocytogenes is already a bacterium that can grow in a range of temperatures and ionic concentrations. L. monocytogenes continues to grow, while using up the resources from the spoiling shrimp, depleting the resources available in the shrimp.
(1) Gram and Dalgaard, 2002 L. Gram and P. Dalgaard, Fish spoilage bacteria-problems and solutions, Current Opinion in Biotechnology 13 (2002), pp. 262–266.
(2) F. Feldhusen, The role of seafood in bacterial foodborne diseases, Microbe Infection 2 (2000), pp. 1651–1660.
(3) Fresh and Frozen Seafood: Selecting and Preserving It Safely. 2 Oct. 2006. U.S. Food and Drug Administration. 25 Aug. 2008. <http://www.cfsan.fda.gov/~lrd/seafsafe.html#store>
(4) Jeremiah, Lester E., ed. Freezing Effects on Food Quality, pp. 113-114. Boca Raton: CRC Press, 1996.
(5) Gram and Huss, 1996 L. Gram and H.H. Huss, Microbiological spoilage of fish and fish products, International Journal of Food Microbiology 33 (1) (1996), pp. 121–137.
(6) Huss, 1995. H.H. Huss, Editor, Quality and Quality Changes in Fresh FishFAO Fish. Tech. Pap. 348, FAO, Rome, Italy (1995)
(7) Man, C.M.D. and Jones, A.A. 1999. Shelf Life Evaluation of Foods, pp. 110-133. Aspen Publisher Inc., New York, NY.
(8) Gram et al., 1990. L. Gram, C. Wedell-Neergaard and H.H. Huss, The bacteriology of fresh and spoiling Lake Victorian Nile perch (Lates niloticus). Int. J. Food Microbiol. 10 (1990), pp. 303–316.
(9) Herbert and Shewan, 1976. R.A. Herbert and J.M. Shewan, Roles played by bacterial and autolytic enzymes in the production of volatile sulphides in spoiling North Sea cod (Gadus morhua). J. Sci. Food Agric. 27 (1976), pp. 89–94.
(10) P. Dalgaard , Qualitative and quantitative characterization of spoilage bacteria from packed fish. Int J Food Microbiol 26 (1995), pp. 319–333.
(11) Gram L, Ravn L, Rasch M, Bruhn JB, Christensen AB, Givskov M: Food spoilage – interactions between food spoilage bacteria. Int J Food Microbiol 2002.
(12) Flodgaard, L.R., Dalgaard, P., Andersen, J.B., Nielsen, K.F., Givskov, M., and Gram, L. (2005) Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-hydroxyoctanoyl) homoserine lactone. Appl Environ Microbiol 71: 2113–2120.
(13) Joffraud J-J, Cardinal M, Cornet J, Chasles J-S, Léon S, Gigout F & Leroi F (2006) Effect of bacterial interactions on the spoilage of cold-smoked salmon. Int J Food Microbiol 112: 51–61.
(14) Slonczewski, Joan L., John W. Foster, and Kathy M. Gillen. Microbiology : An Evolving Science. Boston: W. W. Norton & Company, Incorporated, 2008.
(15) Xu, J., N. Heaney, S. A. Marshall, B. C. Millar, D. A. McDowell, A. McMahon, I. S. Blair, P. J. Rooney, and J. E. Moore, 2005: Employment of 16S rDNA gene sequencing techniques to identify phenotypically difficult-to-identify culturable eubacteria from foods and waters. Int. J. Food Sci. Technol. 40, 229–233.
(16) A. Pacquit, J. Frisby, Danny Diamond, K.T. Lau, A. Farrell, B. Quilty and Dermot Diamond, Development of a smart packaging for the monitoring of fish spoilage, Food Chemistry 102 (2007), pp. 466–470.
(17) Pagani, L., A. Lang, C. Vedovelli, O. Moling, G. Rimenti, R. Pristera, and P. Mian. 2003. Soft tissue infection and bacteremia caused by Shewanella putrefaciens. J. Clin. Microbiol. 41:2240-2241.
(18) Russell, 2002 N.J. Russell, Bacterial membranes: the effects of chill storage and food processing. An overview, Int. J. Food Microbiol. 79 (2002), pp. 27–34.
REFERENCES FOR THE ASIDE
 Ryser, Elliot T., and Elmer H. Marth, eds. Listeria, Listeriosis, and Food Safety. 3RD ed. New York: Taylor & Francis Group, 2007. 636-40. Roger. UCSD, La Jolla. Keyword: Listeria.
 Schmidt, Ronald H., and Gary E. Rodrick. Food Safety Handbood. John Wiley & Sons, 2003. 137-50. Roger. Knovel. UCSD, La Jolla. Keyword: Food and Saftey Handbook.
 Rutherford, Thomas J., Douglas L. Marshall, Linda S. Andrews, Patti C. Coggins, M. W. Schilling, and Patrick Gerard. "Combined effect of packaging atmosphere and storage teperature on groth of Listeria monocytogenes on ready-to-eat shrimp." Food Microbiology 24 (2007): 703-10. Science Direct. <http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6wfp-4nfr54h-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=b8073b8717b2017b7d4167a2a5ede4bc>.
 Novak, John S. Microbial Safety of Minimally Processed Foods. New York: C R C P LLC, 2002. Ch.3. Roger. UCSD, La Jolla. Keyword: Listeria monocytogenes.
 "Listeria monocytogenes." Wikipedia. <http://en.wikipedia.org/wiki/listeria_monocytogenes#cite_ref-dworaczek_kubo_dykes_2002_17-0>.
 Van Wagner, L.R. 1989. FDA takes action to combat seafood contamination. Food Process. 50: 8–12.
Edited by Tal Do, Phillip Lai, Duy Nguyen, and Tania Yaser, students of Rachel Larsen