Investigation of the antioxidant properties of Persea americana seed flour altered by the fermentation process with Lactobacillus plantarum European Journal of Chemistry 15 (1) (2024) 74-78 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2024 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.15.1.74-78.2508 European Journal of Chemistry View Journal Online View Article Online Investigation of the antioxidant properties of Persea americana seed flour altered by the fermentation process with Lactobacillus plantarum Polegodage Dilushi Sureka Ruwan Kumari 1,*, Ranjani Amarakoon 1 and Madushan Dhammika Gunarathna 2 1 Department of Microbiology, Faculty of Science, University of Kelaniya, Kelaniya, 11300, Sri Lanka 2 Department of Chemistry, Faculty of Science, University of Kelaniya, Kelaniya, 11300, Sri Lanka * Corresponding author at: Department of Microbiology, Faculty of Science, University of Kelaniya, Kelaniya, 11300, Sri Lanka. e-mail: surekadilushi@gmail.com (P.D.S.R. Kumari). 10.5155/eurjchem.15.1.74-78.2508 Received: 21 January 2024 Received in revised form: 27 February 2024 Accepted: 01 March 2024 Published online: 31 March 2024 Printed: 31 March 2024 This study aims to investigate the effect of Lactobacillus plantarum fermentation on the antioxidant potential of Persea americana seed flour. The half-maximal inhibitory concentration (IC50) value of avocado seed flour after fermentation for 24, 48, and 72 h was compared with the unfermented avocado seed flour using the measurement of free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Additionally, the change in pH value was measured to confirm the continuous fermentation process within the desired period. The results for the DPPH assay for unfermented, 24, 48, and 72 h fermented avocado seed flour were 61.5±0.71, 51.18±0.10, 46.00±0.21, 43.73±0.21 µg/mL, respectively, which indicated a significant increase (p < 0.05) of IC50 values of avocado seed flour with the fermentation. Furthermore, with the fermentation period, there was a significant decrease in the pH value for the 72-hour fermented sample (4.15±0.03) compared to the unfermented sample (6.81±0.04). These results supported avocado seed as an important by-product source for the further development of health-promoting products, by confirming the increased antioxidant capacity of avocado seed flour after fermentation. DPPH Fermentation Avocado seeds Persea americana Antioxidant potential Lactobacillus plantarum Cite this: Eur. J. Chem. 2024, 15(1), 74-78 Journal website: www.eurjchem.com 1. Introduction The food industries are providing a wider range of food products to consumers. By-products and wastes generated by these industries cause many problems for the pollution of the environment [1]. At present, the development of new value- added products for commercial applications using discarded by-products and discarded materials is gaining much atten- tion. The bioactive compounds found in waste and by-products are highly nutritional and bioactive, making their recovery important for use in the development of nutraceuticals and pharmaceuticals. According to epidemiological studies, many phytonutrients found in fruits and vegetables can protect the human body against damage caused by reactive oxygen and nitrogen species [2]. Since some synthetic antioxidants could be toxic and involve high manufacturing costs but may not be as effective as natural antioxidants, it would be beneficial to identify natural and more economically viable antioxidants that could be included in foods. Many types of plants have been studied for their antioxidant properties, including oil, seeds, cereal crops, vegetables, fruits, leaves, roots, spices, and herbs [3]. Among various methods to analyze the antioxidant potential of different types of plants, the DPPH test is simple because it measures the mechanism between the antioxidant and the DPPH reagent and it is one of the most widely used methods [4]. It is based on the reduction of the DPPH molecule and causes its color change from violet to yellow-colored stable molecules via the process of transferring a hydrogen atom from the antioxidant. This test measures nonreduced violet DPPH molecules using a UV-Vis spectrometer at 515-520 nm to measure the antioxidant capacity of the sample as shown in Figure 1 [5-7]. Avocados, commonly known as Persea americana Mill., have been widely distributed in the tropical regions of the world for centuries. Nutritionally, it is a very healthy fruit. The smooth flavor of avocado has contributed to its popularity as an oleaginous fruit crop throughout the world. Aside from being fresh, it is also processed to create food, cosmetic, and pharma- ceutical products to increase commercialization and provide more added value. Due to its nutritional and bioactive composition, the avocado (P. americana) fruit has various benefits [8]. Avocados are used for their pulp, but their seeds and skins are discarded as waste. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.15.1.74-78.2508 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.15.1.74-78.2508 mailto:surekadilushi@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.15.1.74-78.2508&domain=pdf&date_stamp=2024-03-31 Kumari et al. / European Journal of Chemistry 15 (1) (2024) 74-78 75 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.1.74-78.2508 N N O2N O2N NO2 + A+ AH N H N O2N O2N NO2 Figure 1. Reaction of DPPH radical and antioxidant. AH; an antioxidant donor molecule, A; a produced free radical. The polyphenols present in these residues are powerful antioxidants and antimicrobials. In terms of avocado seeds, they represent 13-18% of the fruit and are a by-product that is not normally used. These are typically discarded along with the pulp during processing. Seed waste may pose a significant ecological problem [9]. The bioactive compounds that contain these compounds may be of interest to industry. It is reported to contain phytosterols, triterpenes, fatty acids, and two new abscisic acid glucosides. In addition to its antioxidant proper- ties, avocado seeds have also been reported to have larvicidal, fungicidal, hypolipidemic, and recently amoebicidal and giardicidal properties. Among the various applications of P. americana for ethnomedicine is the treatment of diarrhea, dysentery, toothache, and intestinal parasites, as well as skincare and beauty. In vitro studies have shown that tocopherols from the fruit inhibit the growth of prostate cancer. Seeds are also a rich source of tannins and carotenoids [10]. The use of non-edible parts of fruits, such as seeds, is becoming a trending approach to mitigate the increase in food industry waste. Many of the bioactive compounds in fruits can be inactive when the underutilized parts are discarded. As a result, fermentation can produce bioactive substances with enhanced functionality, thus improving the quality of foods. Therefore, the use of enzymes and plant microorganisms can improve the quality of microbial fermentation. There are pros and cons to each of these production approaches [11]. Therefore, fermentation can transform raw seeds into value- added products through microbiological treatment. In this study, we tested the antioxidant potential of avocado seed flour using microbial fermentation to reveal perspectives on its potential of avocado seed flour utilizing microbial fermentation to reveal perspectives on its potential use as a therapeutic agent in the future. 2. Experimental 2.1. Collection and preparation of samples For the experiment, ripe avocado fruits of a local variety were collected in June 2023 from a local supermarket in Sri Lanka. Avocado seeds can be collected by removing the fleshy part of the fruit. After cutting the seeds into 2 mm thick slices, the seeds were blanched with steam. A constant weight was obtained by drying the slices at 50 °C. The dried samples were packed in an airtight container for further analysis in the microbiology laboratory. 2.2. Preparation of the Lactobacillus plantarum starter culture According to Reference [9], starter cultures were prepared from Lactobacillus plantarum with some modifications. Here, L. plantarum stock culture was grown in sterile DeMan, Rogasa, and Sharp (MRS) broth (10.0 mL) at 37 ° C for 24 hours. This MRS broth culture was further grown in sterile medium (50.0 mL) containing avocado seed flour (2.50 g), refined sugar (1.50 g), and skim milk (1.00 g), and then incubated at 37 °C for 24 hours. 2.3. Preparation of fermented and unfermented avocado seed flour Using dried avocado seed chips (20.0 g) and sterile distilled water (200.0 mL), three samples were prepared. After sterilizing each flask at 120 °C for five minutes, the starter plantarum cultures were transferred to three flasks for fermentation at 37 °C for 24, 48, and 72 hours. In the following steps, we washed avocado chips with sterile distilled water and soaked them for 15 minutes in a salt solution (10%, 500 mL). After that, rinse it three times with sterile distilled water to remove salt residues. Over 2-3 days, the fermented avocado seed samples were oven dried at 55 °C. Each fermented avocado seed sample was oven dried at 55 °C for 2-3 days. The dry chips were ground in a blender and sieved to an 8 mm mesh size. The unfermented avocado seed flour is prepared by the same procedure without adding the starter culture to carry out fermentation [12]. 2.4. Determination of antioxidant potential The antioxidant potential of the avocado seed flour (ASF) samples was determined using the measurement of 2,2- diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity [11]. Firstly, a suitable solvent that can be used for avocado seed extraction was found by observing the solubility of ASF in each solvent. Methanol was found to be the suitable solvent for avocado seed to perform the DPPH assay. The antioxidant activity of the methanol extract of ASF was determined using a 96-well microtiter plate according to the method described in reference [13] with slight modifications. A stock solution of the sample and the standard butylated hydroxyl-toluene (BHT) (1000 µg/mL) was prepared. A series of BHT standards with concentrations of 1000, 500, 125, 62.5, and 31.25 µg/mL were prepared. Methanolic DPPH solution (0.25 µg/mL, 40.00 µL) was added to each sample solution and the standard solution (160 µL) in the well and incubated for 30 minutes at room temperature in dark conditions. A microtiter plate reader was used to measure the absorbance of each well at 520 nm. On the basis of the equation below, the percentage inhibition was calculated. % Inhibition = (𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 − 𝐴𝐴𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑐𝑐𝑠𝑠 𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐⁄ ) × 100 (1) The calculated % inhibition corresponding to each sample in Table 1 was used to plot the graph (Figure 2) with the respective concentrations of the sample standards to obtain the IC50 2.5. Determination of the pH change The pH value of each sample (Unfermented, 24, 48, and 72 h fermented) was determined using a pH meter, calculated in triplicate and average values (Table 2). 76 Kumari et al. / European Journal of Chemistry 15 (1) (2024) 74-78 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.1.74-78.2508 Table 1. % Inhibition avocado seed flour and fermented avocado seed flour (24, 48, and 72 h). Sample concentration (µg/mL) % Inhibition Unfermented sample Fermented sample 24 h 48 h 72 h 500 68.89 92.21 89.19 89.90 250 58.00 76.56 74.67 77.67 125 55.12 65.01 60.31 62.31 62.5 49.98 52.23 53.56 52.89 31.25 47.01 37.89 41.22 40.22 Table 2. IC50 value and pH of avocado seed flour and fermented avocado seed flour (24, 48, and 72 h). Parameters Unfermented Fermented 24 h 48 h 72 h IC50 61.5±0.71 51.18±0.10 46.00±0.21 43.73±0.21 pH 6.81±0.04 5.99±0.06 4.87±0.05 4.15±0.03 Figure 2. Variation of antioxidant activity after fermentation by L. plantarum compared to the unfermented control sample (n = 3). Results are represented as mean±standard deviation. 2.6. Statistical analysis Data obtained in triplicate were carried out using the one- way analysis of variance (ANOVA) technique. Tukey’s multiple comparison test was used to identify the means that differ significantly at p < 0.05. Results were expressed as means±SEM. 3. Results and discussion Figure 2 shows the antioxidant capacity of fermented and unfermented ASF in different incubation periods. Antioxidants can be defined as reductants, and inactivation of oxidizing agents by reductants can be explained as a type of redox reaction. Avocado seeds contain considerably higher amounts of phenols, flavonoids, and paraanthocyanin, making them a good source of antioxidants obtained [13]. According to the results obtained for the antioxidant capacity of ASF corres- ponding to IC50 (the concentration of antioxidant required to give 50% inhibition of the probe in the antioxidant assay). Hence, the lower IC50 value denotes the high antioxidant activity of a given food source. IC50 values decreased in the order 0 h fermented flour (61.5±0.71) > 24 h fermented flour (51.18±0.10) > 48 h fermented flour (46.00±0.21) > 72 h fermented seed flour (43.73±0.21) (Table 2). According to the reference study [11], it has also shown an increase in the antioxidant activity of tomato seeds with L. plantarum- mediated fermentation. More importantly, a significant increase in antioxidant potential was observed for fermented seed flours for 72 h. Therefore, 72 h fermented ASF has the highest antioxidant potential. These results show a significant effect of microbial fermentation on the antioxidant potential of avocado seed flour. According to the previous study [12] on the application of microbial fermentation using L. plantarum on jackfruit seed (Artocarpus heterophyllus) seeds, it has clearly proven the positive impact of microbial fermentation on the antioxidant potential of A. heterophyllus seeds, and how the antioxidant potential of A. heterophyllus seeds has increased significantly with fermentation time. Phytochemicals in plant materials are mainly responsible for their antioxidant properties. As phytonutrients are important for human health, they are functionally essential for human consumption [14]. The increase in antioxidant potential is possibly due to the synthesis of bioactive peptides during fermentation by L. plantarum. Bioactive peptides are often inactive in the parent proteins and can be generated through the hydrolysis of different protein sources using a wide variety of approaches, mainly via fermentation using proteolytic microbes such as lactic acid bacteria (LAB) [14]. Figure 3 shows how the pH value of each sample changed with fermentation time. According to the results obtained, there is a significant decrease in the pH value of all fermented samples (24, 48, and 72 h) compared to the unfermented sample. When comparing all fermented samples, pH values of 24, 48, and 72 h fermented samples were 5.99±0.12, 4.87±0.05, and 4.15±0.13, respectively (Table 2). By the significant decline in pH value, it can be confirmed that the production of acids by the microbial fermentation process confirms the continuous process of microbial fermentation during the period of 72 h. This result also agrees with almost all lactic acid bacteria-mediated and also natural fermentation according to previous studies [15]. This may be due to the production of lactic acid by L. plantarum in the process of synthesis of energy for their metabolism [15]. Figure 3 shows how the calculated IC50 values decrease in order 0 h fermented flour (61.5±0.71) > 24 h fermented flour (51.18±0.10) > 48 h fermented flour (46.00±0.21) > 72 h fermented seed flour (43.73±0.21). 0 10 20 30 40 50 60 70 80 90 100 31.25 62.5 125 250 500 In hi bi tio n (% ) Sample concentration (µg/mL) Unfermented 24h Fermented 48h Fermented 72h Fermented Kumari et al. / European Journal of Chemistry 15 (1) (2024) 74-78 77 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.1.74-78.2508 Figure 3. Variation of the pH value after fermentation by L. plantarum compared to the unfermented control sample (n = 3). Results are represented as mean ± standard deviation. Figure 4. Variation in IC50 values after fermentation by L. plantarum compared to the unfermented control sample (n = 3). Results are represented as mean±standard deviation. 4. Conclusions In conclusion, this study has indicated how microbial fermentation affects the potential health beneficial effects of avocado seed flour, such as antioxidant activities. In particular, the potential of avocado seed flour to effectively scavenge various free radicals increased with the fermentation period compared to the unfermented sample. These promising pharmaceutical properties indicate the value of avocado seeds. However, further studies that reveal these promising results can be translated into clinically or nutritionally useful agents are necessary. Acknowledgments The authors thank the University of Kelaniya for providing all laboratory facilities and chemicals needed for this study. The authors thank all academic and non-academic staff of the Department of Chemistry and the Department of Microbiology at the University of Kelaniya for the immense support given during this study. Disclosure statement Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Dilushi Sureka Ruwan Kumari Polegodage, Methodology: Dilushi Sureka Ruwan Kumari Polegodage, Investigation: Dilushi Sureka Ruwan Kumari Polegodage, Data Curation: Madushan Dhammika Gunarathna, Writing - Original Draft: Dilushi Sureka Ruwan Kumari Polegodage, Writing - Review and Editing: Madushan Dhammika Gunarathna, Supervision: Ranjani Amarakoon. ORCID and Email Polegodage Dilushi Sureka Ruwan Kumari surekadilushi@gmail.com https://orcid.org/0000-0001-6632-9034 Ranjani Amarakoon ranjaniam@kln.ac.lk https://orcid.org/0009-0006-9398-0248 Madushan Dhammika Gunarathna dgdhammika@gmail.com https://orcid.org/0009-0007-0262-0405 References [1]. Sagar, N. A.; Pareek, S.; Sharma, S.; Yahia, E. M.; Lobo, M. G. Fruit and vegetable waste: Bioactive compounds, their extraction, and possible utilization. Compr. Rev. Food Sci. Food Saf. 2018, 17, 512–531. [2]. Halliwell, B. Antioxidants and human disease: A general introduction. Nutr. Rev. 2009, 55, S44–S49. [3]. Ramarathnam, N.; Osawa, T.; Ochi, H.; Kawakishi, S. The contribution of plant food antioxidants to human health. Trends Food Sci. Technol. 1995, 6, 75–82. [4]. Sirivibulkovit, K.; Nouanthavong, S.; Sameenoi, Y. Paper-based DPPH assay for antioxidant activity analysis. Anal. Sci. 2018, 34, 795–800. [5]. Garcia, E. J.; Oldoni, T. L. C.; Alencar, S. M. de; Reis, A.; Loguercio, A. D.; Grande, R. H. M. Antioxidant activity by DPPH assay of potential solutions to be applied on bleached teeth. Braz. Dent. J. 2012, 23, 22– 27. [6]. Cheng, Z.; Moore, J.; Yu, L. (lucy) High-throughput relative DPPH radical scavenging capacity assay. J. Agric. Food Chem. 2006, 54, 7429– 7436. 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Unfermented 24h Fermented 48h Fermented 72h Fermented pH Sample 40 45 50 55 60 65 Unfermented 24h Fermented 48h Fermented 72h Fermented IC 50 va lu e Sample mailto:surekadilushi@gmail.com https://orcid.org/0000-0001-6632-9034 mailto:ranjaniam@kln.ac.lk https://orcid.org/0009-0006-9398-0248 mailto:dgdhammika@gmail.com https://orcid.org/0009-0007-0262-0405 78 Kumari et al. / European Journal of Chemistry 15 (1) (2024) 74-78 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.1.74-78.2508 [7]. Wei, A.; Shibamoto, T. Antioxidant/lipoxygenase inhibitory activities and chemical compositions of selected essential oils. J. Agric. Food Chem. 2010, 58, 7218–7225. [8]. Duarte, P. F.; Chaves, M. A.; Borges, C. D.; Mendonça, C. R. B. Avocado: characteristics, health benefits and uses. Cienc. Rural 2016, 46, 747– 754. [9]. Ortiz, M. A.; Dorantes, A. L.; Gallndez, M. J.; Cardenas, S. E. Effect of a novel oil extraction method on avocado (Persea americana mill) pulp microstructure. Plant Foods Hum. Nutr. 2004, 59, 11–14. [10]. Lu, Q.-Y.; Arteaga, J. R.; Zhang, Q.; Huerta, S.; Go, V. L. W.; Heber, D. Inhibition of prostate cancer cell growth by an avocado extract: role of lipid-soluble bioactive substances. J. Nutr. Biochem. 2005, 16, 23–30. [11]. Mechmeche, M.; Kachouri, F.; Ksontini, H.; Hamdi, M. Production of bioactive peptides from tomato seed isolate by Lactobacillus plantarum fermentation and enhancement of antioxidant activity. Food Biotechnol. 2017, 31, 94–113. [12]. Jayus; Setiawan, D.; Giyarto Physical and chemical characteristics of jackfruit (Artocarpus heterophyllus lamk.) seeds flour produced under fermentation process by Lactobacillus plantarum. Agric. Agric. Sci. Procedia 2016, 9, 342–347. [13]. Folasade, O. A.; Olaide, R. A.; Olufemi, T. A. Antioxidant properties of Persea Americana m. Seed as affected by different extraction solvent. Journal of Advances in Food Science Technology 2016, 3(2), 101–106. https://ikprress.org/index.php/JAFSAT/article/view/3501 (accessed January 1, 2024). [14]. Nkhata, S. G.; Ayua, E.; Kamau, E. H.; Shingiro, J.-B. Fermentation and germination improve nutritional value of cereals and legumes through activation of endogenous enzymes. Food Sci. Nutr. 2018, 6, 2446– 2458. [15]. Obadina, A. O.; Akinola, O. J.; Shittu, T. A.; Bakare, H. A. Effect of natural fermentation on the chemical and nutritional composition of fermented soymilk Nono. Niger. Food J. 2013, 31, 91–97. Copyright © 2024 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at https://www.eurjchem.com/index.php/eurjchem/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (https://www.eurjchem.com/index.php/eurjchem/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). https://ikprress.org/index.php/JAFSAT/article/view/3501 https://www.eurjchem.com/index.php/eurjchem/terms http://creativecommons.org/licenses/by-nc/4.0 https://www.eurjchem.com/index.php/eurjchem/terms 1. Introduction 2. Experimental 2.1. Collection and preparation of samples 2.2. Preparation of the Lactobacillus plantarum starter culture 2.3. Preparation of fermented and unfermented avocado seed flour 2.4. Determination of antioxidant potential 2.5. Determination of the pH change 2.6. Statistical analysis 3. Results and discussion 4. Conclusions Acknowledgments Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: