The Science of Salsa: Antimicrobial Properties of Salsa Components to Learn Scientific Methodology

Abstract

Most ethnic foods and cooking practices have incorporated the use of spices and other food additives. Many common spices have crossed cultural boundaries and appear in multiple ethnic cuisines. Recent studies have demonstrated that many of these ingredients possess antimicrobial properties against common food spoilage microorganisms. We developed a laboratory exercise that promotes the use of scientific methodology to evaluate the effectiveness of salsa components at inhibiting the growth of undesirable microorganisms. Tomato, onion, garlic, cilantro, and jalapeño were tested for antimicrobial properties against a representative fungus, Saccharomyces cerevisiae, and the common food spoilage bacteria Staphylococcus aureus, Bacillus cereus, and Escherichia coli. Each component was ethanol extracted and a modification of the Kirby-Bauer method of antimicrobial sensitivity was employed. Garlic demonstrated the greatest inhibitory effects against all organisms tested. Onion demonstrated a slight inhibition of all four organisms, while cilantro showed some inhibition of all three bacteria but no effect against the fungus. Jalapeño may have slightly inhibited E. coli and S. aureus, as evidenced by a consistently measured increase in the zone of inhibition that was not statistically significant when compared to that of the control. Following the initial exercise, students were given the opportunity to repeat the exercise using other spices such as cinnamon, clove, nutmeg, and coriander. Student learning outcomes were evaluated using preliminary and secondary surveys, mainly focusing on definitions of science and hypothesis as well as the process of science. Students enjoyed this exercise and met the learning goals of understanding the process and methodology of science, as well as the interdisciplinarity inherent in the sciences. Student learning was evidenced by an increase in the number of correct responses on the secondary survey in comparison to the preliminary.

The dramatic increase in the Hispanic population of the United States coupled with America’s taste for new ethnic cuisine has led to salsa’s replacement of ketchup as the number one table condiment (2). The flavorful sauce comes in many variations, but centers on a core of tomatoes, chili peppers, and aromatic herbs. The apparent impending ubiquity of salsa raises some interesting questions regarding its popularity, but more importantly its function as a part of various culinary traditions. That is, is there more to salsa than good taste?

A number of authors have investigated the antimicrobial action of common herbs and spices, many of which are key components of common salsa preparations (5, 8). Common ingredients including garlic, cilantro, onion, and jalapeño are reported to possess antimicrobial properties (1, 3, 6, 7, 9). These plants produce the secondary metabolites allicin, linalool, thiopropanal-s-oxide, and capsaicin, respectively. The use of spices and herbs in traditional ethnic foods essentially borrows the plant’s defensive mechanism and allows humans to benefit from these metabolites. In fact, Sherman and Billing have suggested that herb and spice use in traditional recipes follows certain trends, namely that as mean annual temperature increases so does the number of spices used in traditional recipes, as well as the strength of antimicrobial activity in the spices used (9). Conversely, cooler climates tend to use fewer spices per recipe, and those additives have decreased antimicrobial activity. In salsa, it is hypothesized that a combination of aromatic secondary compounds from the vegetables and herbs and the naturally low pH help salsa to remain unspoiled for long periods, even without refrigeration. It is these antimicrobial characteristics and the probable high level of student familiarity with salsa that make it a great tool for use in the biology teaching laboratory. After introduction of the key concepts of this investigation to the students, we pointed out that fresh salsa has an unusually long shelf life, thus we prompted the students to ask the simple question: what makes salsa resistant to spoilage?

It became the students’ task to develop and test a hypothesis to determine if there were any inherent antimicrobial properties to any of the components in salsa. With this exercise we hope to meet the challenge that many educators today face in developing student-friendly yet meaningful lab activities that address National Science Education Standards (4). The objective of this project was to develop a hands-on activity that meets three specific criteria: (i) the activity directly relates to National 9–12 science content standards, (ii) the activity has clearly identifiable goals that can be assessed, and (iii) the activity is scalable to a level appropriate for college undergraduates.

MATERIALS AND METHODS

Student population. Elmhurst College hosts a Math and Science Summer Academy that serves as a science enrichment camp for high-school-age students. This 2-week camp enrolls approximately 40 students, who are juniors or seniors, from several Chicago-area high schools, many of which have limited resources and offer few wet-lab experiences. Student profiles typically fit into three general categories: (A) working above grade level in science and math, (B) working at grade level in science and math, and (C) working below grade level in science and math. Group A students attend the academy to gain exposure to college life and college-type academic activities. Many of these students have applied or will apply to college. Students in Groups B and C are often those who are undecided about college or do not believe that they have the ability to gain admission. The students who are not working at grade level are mainstreamed with Groups A and B and receive supplemental instruction from undergraduate tutors.

Experimental set-up. This exercise was designed to reinforce the components of scientific methodology (National Science 9–12 content standard A), whereby students actively engage in the steps during the laboratory exercise. Students are first given a preliminary survey to assess their knowledge about science and the steps in this process. This is followed by lectures on science, history, scientific methodology, and bacterial anatomy, diversity and growth (9–12 content standards F and G). As soon as the students enter the lab, they learn about aseptic technique and safety.

To demonstrate microbial diversity (9–12 content standard F) and growth, students are given the opportunity to sample a variety of sites to culture microorganisms. Students also learn to conduct a Gram stain on the cultured microorganisms (9–12 content standard E). For many participants, this is their first use of Bunsen burners and microscopes. Scientific methodology is taught using an exercise designed to test the antimicrobial properties of salsa components against the common food poisoning microbes Escherichia coli, Bacillus cereus, and Staphylococcus aureus. While not involved in food spoilage, Saccharomyces cerevisiae was included as a fungal representative which is easily cultured by students with little laboratory experience. The salsa ingredients, including tomato, onion, jalapeño, garlic, and cilantro, were subjected to ethanol extraction, whereby 5 g of fresh material was macerated in a mortar and pestle prior to the addition of 10 ml of 95% ethanol. Sterile absorbent paper discs, 7-mm in diameter, were saturated with the extract and placed on a bacterial lawn in a modification of the Kirby-Bauer technique. The nutrient agar plates were incubated at 37°C for 48 hours before students measured the zones of inhibition. Statistical analyses (analysis of variance) of the resultant halo data were conducted to demonstrate proper result analyses and interpretations using SigmaStat 3.5 (Systat Software, Inc., 2006). The exercise was repeated with the exception that students chose different herbs and spices to test for antimicrobial activity.

Assessment of student learning. Prior to developing this exercise, the biology experience in the Summer Academy was mostly lecture followed by basic microbiology education. In the laboratory this translated to culturing organisms in the environment, performing Gram stains on several isolates, and using the Kirby-Bauer method to test antimicrobial resistance of the isolates. This gave students hands-on experience in the laboratory; however, there was no way to evaluate if the students were actually learning anything about biology or microbiology. We therefore designed this progressive exercise and an assessment method to evaluate student learning. To assess the learning outcomes, students were given a preliminary and secondary survey. They were asked questions about science and scientific methodology. The following specific questions were asked: what is science, what are the steps in the scientific method, what is the purpose of developing a hypothesis, and do you need to know chemistry or math to do biology. The first three questions focused on the process of science, while the last questions dealt with the interrelatedness of math and all the hard sciences. Student responses were scored as “excellent,” “adequate,” or “poor.” For analysis purposes, “excellent” responses were given a score of +1, “adequate” responses were scored as 0, and “poor” responses were given a −1 (Table 1). To maintain consistency and objectivity, all surveys were scored by the corresponding author at the time of manuscript preparation. Furthermore, they were blind scored so that during the scoring process, it was unknown whether a preliminary or secondary survey was being examined.

TABLE 1.

Scoring of preliminary and secondary surveys with sample responses for individual questions

Question	Score	Example
What is science?	Excellent (+1)	“A way of knowing through a reliable, yet not infallible, set of collected real data”
	Adequate (0)	“A way of knowing through observation and experimentation”
	Poor (−1)	“The study of all living things” (study of life) or “The study of everything that makes up our world”
What are the steps in the scientific method?	Excellent (+1)	“Observe, problem, hypothesis, materials, procedure, results, conclusion, share results with scientific community”
	Adequate (0)	“Hypothesis, procedure, materials, test hypothesis, record results”
	Poor (−1)	“Develop hypothesis, write down info, state if hypothesis was correct or not”
What is the purpose of developing a hypothesis?	Excellent (+1)	“To help you determine what results you get. Predicting can help you understand the outcome.”
	Adequate (0)	“To see if what you predict will actually happen”
	Poor (−1)	“To find out if you were right or not about an experiment or even a question”
Why would you need biology, chemistry, and math to answer questions about bacterial growth?	Excellent (+1)	“All three were necessary because if not for our research in the lab, our knowledge of living organisms, and the mathematical equations and numbers used to record, average, and total our data, we would have not been able to come to the same conclusion”
	Adequate (0)	“You need biology because bacteria are living things. You need chemistry to speed up the growth through enzymes. You need math to calculate the growth, which will probably be exponential.”
	Poor (−1)	“You need biology to know about bacterial growth…and you need chemistry to test and know how to handle the equipment…and you need math to solve equations.”

When answering the first question, what is science, excellent answers were those that explained that science was a process which involved data collection and could only be used to study things in the real world (Table 1). Adequate answers were missing one of these three components, and poor answers were missing more than one component. Students who gave excellent responses for the steps in the scientific method were able to explain that it included a hypothesis, testing of the hypothesis, analysis of the results, and reporting the results to colleagues in the field (Table 1). Adequate answers were those that included hypothesis and testing of hypothesis but which might not include reporting the findings to the scientific community. Poor answers were missing three of the four components of excellent answers. In the third question, students were asked to explain the purpose of developing a hypothesis. Excellent answers were those that stated that a hypothesis was a predicted outcome based on prior knowledge and that forming a hypothesis prior to beginning work would help to keep the research focused on one specific question (Table 1). Poor answers stated that a hypothesis was “an educated guess” with little or no rationale for the importance of developing this prior to beginning work. Adequate responses were judged based on the student clearly indicating that a hypothesis was a prediction of experimental outcome with some explanation as to how this would be important during experimentation. Finally, students were asked whether or not it was important to know chemistry or math to conduct biological experiments. Excellent answers were responses that stated that all of these fields were connected and therefore necessary (Table 1). Adequate answers stated that knowing one or the other was necessary and poor answers indicated that one did not need to know chemistry or math to conduct biological experiments. All four questions were subjected to chi-square analyses and the multivariate analysis of variance using SPSS version 15.0 (Statistical Package for the Social Sciences, 2006).

Science teaching standards. The National Science Education Content standards addressed are 9–12 content standard A, science as inquiry; 9–12 content standard E, science and technology; 9–12 content standard F, personal and social perspectives; and 9–12 content standard G, history and nature of science.

RESULTS

Experimental results. Students found that garlic consistently exhibited inhibitory activity against all four of the test organisms, while each of the remaining components had variable inhibition, depending on the organism against which it was tested (Table 2; Fig. 1). For example, Bacillus cereus growth was greatly inhibited by garlic, with a mean zone of inhibition size of 29 mm, and slightly inhibited by onion and cilantro, with mean zone sizes of 9.56 mm and 8.42 mm, respectively, relative to the ethanol control. Escherichia coli growth was inhibited only by garlic (32.67 mm), and Staphylococcus aureus was inhibited by garlic and cilantro only, with zone sizes of 32.42 mm and 17.89 mm, respectively. The fungal representative Saccharomyces cerevisiae was inhibited by garlic, although not to the same degree as the prokaryotes, as the mean zone of inhibition was only 22 mm in diameter. In addition, there may have been a very slight inhibitory effect of onion on S. cerevisiae growth (6.06 mm compared to 5.92 mm for ethanol control). This effect was not significantly different from the control but was consistently present in each trial. Analysis of variance of student results consistently showed garlic to have significantly larger zones of inhibition.

TABLE 2.

Inhibitory effects of salsa components on growth of Bacillus cereus, Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae

Salsa ingredient	Mean ± standard deviation of the diameters of zones of inhibition (mm)a
	Bacillus cereus	Escherichia coli	Staphylococcus aureus	Saccharomyces cerevisiae
Ethanol control	6.42 ± 5.2	8.59 ± 3.8	10.50 ± 4.9	5.92 ± 3.8
Garlic	29.09 ± 2.5	32.67 ± 3.0	32.42 ± 7.3	22.25 ± 4.2
Tomato	2.09 ± 2.3	1.34 ± 3.3	4.92 ± 2.1	0
Onion	9.56 ± 2.0	4.33 ± 4.1	4.42 ± 0.9	6.06 ± 1.2
Jalapeño	0	4.42 ± 0.9	4.50 ± 2.5	0
Cilantro	8.42 ± 5.0	8.02 ± 4.8	17.89 ± 9.7	0

^aValues shown represent the data collected during summers of 2005 and 2006.

FIG. 1.

(A.) Representative plates showing inhibition of Bacillus cereus by extracts of garlic (G), onion (O), and cilantro (CI), compared to ethanol control (C). (B). Representative plate of Escherichia coli inhibited by garlic extract.

Assessment results. Initially, we pooled the responses to the assessments from 2005–2007 and performed a chi-square analysis for each question, testing for differences in response distribution (Fig. 2). Student responses to the assessment questions showed significant improvement on all questions. These questions dealt specifically with the nature of science and scientific methodology (see above). Frequency data illustrate a trend in improvement of answer quality reflected in fewer poor answers and an increase in excellent and adequate responses (Fig. 2). For example, the number of poor responses for question 1 [X²(3, n = 108) = 29.56, P < 0.001] dropped from 76 to 39, adequate answers increased from 36 to 54, and the number of excellent responses increased from 1 to 15. When asked to give the steps in the scientific method, student responses also showed significant improvement [X²(3, n = 108) = 11.22, P = 0.011], with the greatest improvement being the increase in excellent responses from 42 to 63. Adequate responses decreased from 47 to 32, and poor responses decreased from 9 to 4. In defining a hypothesis, we saw a major improvement in student response between adequate and excellent answers on the pre- and post-tests, with adequate answers decreasing from 65 to 44, and excellent answers increasing from 14 to 33 [X²(3, n =108) = 22.39, P < 0.001]. Question 4 dealt with the interrelatedness of math and science fields. Individual student responses to this assessment instrument indicated that students had some understanding of this concept prior to attending the academy. However, major improvements were seen in the decrease of poor and adequate answers on the pre- and posttests, with poor answers decreasing from 36 to 17 and adequate answers decreasing from 64 to 43. These decreases corresponded with a concomitant increase in excellent responses from 12 to 47 [X²(3, n = 108) = 32.51, P < 0.001].

FIG. 2.

Means of tracked pre- and posttest responses. Symbols: ▪, question 1 (What is science?); □, question 2 (What are the steps in the scientific method?); ▴, question 3 (What is the purpose of developing a hypothesis?); •, question 4 (Why would you need biology, chemistry, and math to answer questions about bacterial growth?).

Tracked pre- and posttests were analyzed in an effort to look for improvement in individual student performance on questions 1 through 4. Our hypothesis was that subjects would demonstrate improved performance on posttest questions after completing the laboratory activities. In all cases it was determined that there was significant improvement in student responses to all questions (Fig. 2). Trends in the tracked student responses were analyzed with the multivariate analysis of variance (SPSS 15.0). Student responses showed significant improvement over all questions from the pretest to the posttest regardless of year (P value, 0.048). The individual questions were then analyzed for differences in response means from pretest versus posttest. In all cases the differences in means were significant, and posttest means were greater than pretest means, which is consistent with the stated hypothesis (Table 3).

TABLE 3.

Paired t test (pretest versus posttest) for subject responses to individual questions

Question	Number of responses	Paired samples test value	Degrees of freedom	P valuea
What is science?	108	−5.705	107	<0.001
What are the steps in the scientific method?	108	−4.509	107	<0.001
What is the purpose of developing a hypothesis?	108	−.2095	107	0.039
Why would you need biology, chemistry and math to answer questions about bacterial growth?	108	−5.972	107	<0.001

^aAll P values were statistically significant.

DISCUSSION

Experimental findings. This exercise offers an effective inquiry-based method to deliver and reinforce the process of scientific methodology. Students formulate a hypothesis as to which salsa ingredient(s) will demonstrate antimicrobial activity against Escherichia coli, Bacillus cereus, Staphylococcus aureus, or Saccharomyces cerevisiae. Many students hypothesize that based on their pungency, onion and jalapeño will exhibit the greatest degree of inhibition. After testing their hypothesis, students observe that garlic consistently inhibits all four of the organisms against which it is tested, while the other salsa components demonstrate limited inhibition, if any, against selected organisms. These results provide an excellent opportunity for students to discuss hypothesis acceptance or rejection, as well as a setting for sharing individual results with their peers. Many students are surprised by the lack of efficacy of jalapeño extracts. This generates a discussion of the shortcomings of the procedure, specifically that only compounds which are both ethanol extractable and water soluble can be tested for antimicrobial properties. Capsaicin, produced by all chili peppers to some degree, is one example of a chemical reported to possess strong antimicrobial activity against Bacillus subtilis, a retarding effect on growth of E. coli, and growth enhancement of S. cerevisiae(3). Due to the insoluble nature of capsaicin, student results did not follow those in the literature. Other results were quite consistent with published studies. For example, cilantro has been reported to have antimicrobial activity against gram-positive bacteria and S. cerevisiae, but not against gram-negative bacteria (1). The results obtained by our students are consistent with these findings. Interestingly, repeated trials of this procedure clearly indicate that antimicrobial activity is greatly diminished or absent in extracts that are not freshly prepared prior to use or if dried herbs are utilized.

The exercise described here can also be used in higher-level courses. With little modification, the exercise is useful to demonstrate the scientific method in nonmajors biology courses. In this type of course, students would be expected to have more of an understanding of the process involved in conducting science. In addition, this exercise has been used in a senior-level microbiology course for biology majors. Students are required to examine primary literature to determine which herbs, spices, or food additives may have antimicrobial properties and against which specific microorganisms. Furthermore, students must also choose their procedures based on those reported in the literature. After performing their assays, students must prepare a formal laboratory report. Essentially, when used in a senior-level course, students must independently incorporate previous knowledge and current literature to conduct an inquiry-based exercise of their own design.

Learning outcomes. This teaching method addresses National Science Education standards 9–12 content standards A, E, F, and G—science as inquiry, science and technology, personal and social perspectives, and history and nature of science (4). In addition, this method meets the first four of the five science teaching standards; (A) incorporate an inquiry-based program, (B) facilitate learning by supporting inquiry, discussion, curiosity, and responsibility in science education, (C) conduct ongoing assessment of student learning and teaching effectiveness, and (D) provide a learning environment which includes the space, time, and resources necessary for learning science (4). The most significant finding, however, is that the activity actually works as intended and accomplishes the articulated goals. Students showed significant improvement in answering questions concerning the definition of science, the steps in the scientific process and the meaning of each of these components. The student attitude survey also shows that students enjoyed the activity and gained an improved attitude about science after involvement in the experiment. A few example responses to this exercise follow:

“Loved it. Was hands on and learned a lot. Kept me alert and focused. Keep it.”

“Fun and educating. I learned a lot.”

“It was fun because I got to experiment with things I barely knew about.”

“Best class ever.”

“Very interesting and informative with Dr. Arriola’s introduction to science and Dr. Marsh’s introduction to experiments with real results. Insightful.”

“It was fun and an experience I never had.”

“I thought it was very interesting. I loved doing the experiments.”

“I learned something new.”

“[It] was very interesting. I learned a lot of things in that class.”

Given the success of this activity, we will continue to use it to expand our data set in Summer Academy and nonmajors classes. We are in the process of training other faculty who teach nonmajors courses so that they may utilize this exercise in their classes. Our plan is to integrate the activity into the Science Methods course so secondary education majors can incorporate this exercise as a lesson plan in their portfolio.