SUMMER 2024

Rebecca Shenk

Carob (Ceratonia siliqua L.) is an agricultural product prevalent around the Mediterranean. The fruit of these trees, in the form of seed pods, are commonly used to produce flour/powder, syrup, or chocolate alternatives. More than 135,000 tons are produced each year (Tzatzani and Ouzounidou, 2023). However, the main use of carob industrially are its seeds, used to make locust bean gum. 80% of the fruit, the pulp, is wasted during the production of this product, and thus research has been needed to encourage the human consumption of carob fruit. This summer, I joined a group of researchers at Universitat Politècnica de València to find benefits of the carob plant and its seed pods. We wanted to determine antimicrobial and antioxidant properties.

Primarily, we needed to determine the best way to extract the polyphenolic compounds from the carob. Polyphenols are well known for their antioxidant properties and health benefits, such as preventing heart disease and cancer (Stavrou et al., 2018). This was the main task I assisted with. We used the Trolox
equivalency test to determine antioxidant capacity, and Gallic Acid equivalent to determine total polyphenol content, but to run these tests we needed to be able to get the extracts to fall within the standard curve. We tested the germ and the peel, as well as the whole pod. The pods arrived weekly for our testing. We received pods from Mallorca, Valencia, and Portugal, with 5 pods each from 10 trees at each location. We also received these at a variety of stages of growth, because when the pods stay on the trees longer, they begin to turn brown, and their polyphenolic content increases. For the germ and the peel, we weighed out 2 grams each, dehydrated them, and then ground them up into powder. For the whole pod, we weighed out 2 grams, mixed it with 15 ml of our solvent, and then used the ultra-turrax homogenizer to mix it. For each of the three categories of testing, we mixed it with 15ml of the solvent initially, then mixed it with a vortex, then placed it in an ultrasonic bath, and then placed it in a centrifuge.

We repeated this several times to extract all of the soluble compounds as possible. We adjusted our solvents, the number of repetitions, and the temperature of the ultrasonic bath and the centrifuge. We used water, salt water, acetone, ethanol, or methanol to extract the compounds. We determined that a temperature of 20-21°C was best, because some of the soluble compounds began to degrade at higher temperatures. We also settled on a mixture of acetone, ethanol, and water as our solvent.

We created a mixture of 30% ethanol and 70% deionized water, and a mixture of 50% ethanol and 50% DI water. We mixed, centrifuged, and decanted twice with the ethanol solution and once with the acetone solution, and then filled each sample to 50mL using a mixture of 10% acetone, 57% DI water, and 33%
ethanol, to maintain the solvent ratio and create a consistent volume. After this, we were able to use our spectrophotometer tests to determine antioxidant capacity and polyphenol content. Trolox reacts with DPPH to create a standard curve, and the DPPH solution is purple at first, and turns lighter when reacting with Trolox or the antioxidant compounds in the extracts. We used different dilutions for each of the 3 extractions due to their different antioxidant capacities. We needed them to stay within the Trolox line. Using the spectrophotometer and this equivalency method, we could see the higher antioxidant capacities based on the lowest absorptions. Similarly, Gallic Acid reacts with Folin to create a standard curve for polyphenolic content. Folin starts off yellow and turns grayish clear when reacting with polyphenols.

We also tested antimicrobial potential. We wanted to see if the compounds extracted from the carob would combat Listeria monocytogenes or Escherichia coli. We inoculated our extracts with the bacteria at different concentrations, and then placed a few drops on a petri dish which we then filled with tryptic soy agar. We did not see any differences in the E. coli plates, but we did see a significant reduction in the number of colonies in our Listeria plates.

Beyond learning an immense amount of lab skills, I learned many analytical skills. In the laboratory, there are no correct answers found in a textbook, you must find them yourself. The scientists I had the honor of working with taught me to keep very detailed records and notes, and I learned the importance of a very organized lab notebook. We planned out our procedures in extreme detail, which made it easy to spot problems. I also read a lot of scientific literature this summer, which allowed me to help create methodology and adjust it knowledgably. Additionally, while working in Spain, I learned a vast amount of Spanish. I had already taken several years in school, but nothing can compare to the immersion. While most of my research was conducted in English, there were many lab associates that I needed to work with who spoke very little or no English. I thus needed to speak only Spanish, and learned how to communicate with a limited vocabulary. This lab project is still ongoing, and I am excited to see the final results and its applications. I hope to return to this lab group potentially for graduate school as this experience was truly invaluable to me.