Enhancing safety and prebiotic functionality of fresh amaranth sprout salad using low-concentration limonene pretreatment
Main Article Content
Keywords
Red amaranth sprouts; Limonene; Bioactive compounds; Prebiotic; Antibacterial
Abstract
Red amaranth sprouts are rich in nutrients and bioactive compounds that promote health. They are suitable for home growing or commercial production for use in fresh salads. In this study, red amaranth seeds were pretreated with limonene nano-emulsions at concentrations between 5 and 20 µL/mL to improve germination and enhance functionality. At 5 µL/mL, limonene had no negative effect on germination, with sprouts reaching an average length of 3.3 cm and showing a 96 percent germination rate. Higher concentrations reduced germination significantly. Microscopy showed that limonene at 5 µL/mL was absorbed and moved into stems and leaves, forming a thin surface film and encouraging vascular growth. This led to deeper red coloration, with anthocyanin content of 57.1 ∆A /100g and an a* value of 1.29, higher than the control. Sprouts also had increased phenolics at 53.7 milligrams GAE and flavonoids to 16.2 mg QE/100g, along with strong antioxidant activity. Nutritional content supported the growth of beneficial probiotics including Lactobacillus plantarum, L. acidophilus, and Bifidobacterium longum. Antibacterial activity was more effective against Gram-positive bacteria. This simple seed-soaking method provides a novel approach to producing bioactive-rich, high-quality sprouts that resist microbial contamination, support probiotic growth, and can be sustainably applied in industry using limonene from citrus peels.
References
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Dos Santos, D.L.S., Almeida, N.A., de Almeida, J.M., Oliveira, M.E.A.S., de Oliveira Rocha, L. and Silva, N.C.C., 2024. Antimicrobial activity evaluation of combinations of essential oils, thymol and R-limonene against food-borne pathogens and spoilage agents. Food Bioscience 62: 105035.
Feng, S., Tian, Y., Yu, J., Lin, Y., de Oliveira Sartori, A.G. and Shao, P., 2025. Limonene-loaded nanostructured lipid carriers incorporated in hydrogels: Enhanced acid stability and controlled release properties. Food Research International 214(1): 116601. https://doi.org/10.1016/j.foodres.2025.116601
Gaur, G. and Gänzle, M.G., 2023. Conversion of (poly) phenolic compounds in food fermentations by lactic acid bacteria: Novel insights into metabolic pathways and functional metabolites. Current Research in Food Science 6: 100448. https://doi.org/10.1016/j.crfs.2023.100448
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Jahan, F., Bhuiyan, M.N.H., Islam, M.J., Ahmed, S., Hasan, M.S., Al Bashera, M., et al. 2022. Amaranthus tricolor (red amaranth), an indigenous source of nutrients, minerals, amino acids, phytochemicals, and assessment of its antibacterial activity. Journal of Agriculture and Food Research 10: 100419. https://doi.org/10.1016/j.jafr.2022.100419
Kamalpour, R., Koocheki, A. and Ghorani, B., 2025. Encapsulation of D-limonene in Lepidium perfoliatum seed gum/PVA electrospun nanofibers: Physicochemical characterization and modeling the kinetics of release. Current Research in Food Science 10: 100966. https://doi.org/10.1016/j.crfs.2024.100966
Klištincová, N., Pin, L., Puškárová, A., Giannino, D., Bučková, M., Lambreva, M.D., et al. 2024. From farm to fork: Fungal and bacterial contaminants and their diagnostics in the production steps of ready-to-eat salads. Trends in Food Science and Technology 150: 104573. https://doi.org/10.1016/j.tifs.2024.104573
Mahmmodi, P., Khoshkhoo, Z., Basti A.A., Shotorbani, P.M., Khanjari, A., 2021. Effect of Bunium persicum essential oil, NaCl, Bile Salts, and their combinations on the viability of Lactobacillus acidophilus in probiotic yogurt. Quality Assurance and Safety of Crops and Foods 13(1): 37–48. https://doi.org/10.15586/qas.v13i1.858
Maqsood, S., Bordiga, M. and Xu, B., 2025. The role of fiber in gut health and chronic diseases: A comprehensive review. Quality Assurance and Safety of Crops and Foods 17(2): 165–199. https://doi.org/10.15586/qas.v17i2.1534
Matan, N. and Matan, N., 2025. Effect of a home-grown palm wood germination tray with lime oil on enhancing GABA and inhibiting foodborne pathogens in RTE sunflower sprout salad and its mode of action. Food Control 172: 111159. https://doi.org/10.1016/j.foodcont.2025.111159
Mclauchlin, J., Aird, H., Amar, C.F.L., Jenkins, C., Jørgensen, F., Lai, S., et al. 2022. Microbiological quality of ready-to-eat salad products collected from retail and catering settings in England during 2020 to 2021. Journal of Food Protection 85(12): 1680–1689. https://doi.org/10.4315/JFP-22-116
Mir, S.A., Farooq, S., Shah, M.A., Sofi, S.A., Dar, B.N., Hamdani, A.M., et al. 2021. An overview of sprouts nutritional properties, pathogens and decontamination technologies. LWT 141(2): 110900. https://doi.org/10.1016/j.lwt.2021.110900
Niroula, A., Khatri, S., Khadka, D. and Timilsina, R., 2019. Total phenolic contents and antioxidant activity profile of selected cereal sprouts and grasses. International Journal of Food Properties 22(1): 427–437. https://doi.org/10.1080/10942912.2019.1588297
Novais, C., Molina, A.K., Abreu, R.M., Santo-Buelga, C., Ferreira, I.C., Pereira, C., et al. 2022. Natural food colorants and preservatives: A review, a demand, and a challenge. Journal of Agriculture and Food Chemistry 70(9): 2789–2805. https://doi.org/10.1021/acs.jafc.1c07533
Patade, V.Y., Singh, N., Grover, A. and Bala, M., 2025. Effect of pretreatments on seed germination of musk rose (Rosa moschata Herrm.). Journal of Applied Research on Medicinal and Aromatic Plants 45: 100628. https://doi.org/10.1016/j.jarmap.2025.100628
Phothisuwan, S., Matan, N. and Matan, N., 2021. The influence of a closed system combining orange oil and mode of action on quality preservation of Salacca fruit. Food Control 130(1): 108265. https://doi.org/10.1016/j.foodcont.2021.108265
Sanna, A., Pani, S.M. and Coroneo, V., 2025. Risk management in fruits and vegetables production and supply: Constructing a control chart for Enterobacteriaceae. Quality Assurance and Safety of Crops and Foods 17(3): 308–318. https://doi.org/10.15586/qas.v17i3.1563
Sarker, U., Lin, Y.P., Oba, S., Yoshioka, Y., Hoshikawa, K., 2022. Prospects and potentials of underutilized leafy amaranths as vegetable use for health-promotion. Plant Physiology and Biochemistry 182: 104–123. https://doi.org/10.1016/j.plaphy.2022.04.011
Sarker, U. and Oba, S., 2019. Protein, dietary fiber, minerals, antioxidant pigments and phytochemicals, and antioxidant activity in selected red morph amaranthus leafy vegetable. PLoS One 14(12): 0222517. https://doi.org/10.1371/journal.pone.0222517
Schliemann, W., Cai, Y., Degenkolb, T., Schmidt, J., and Corke, H., 2001. Betalains of Celosia argentea. Phytochemistry 58(1): 159–165. https://doi.org/10.1016/S0031-9422(01)00141-8
Sperandeo, P., Martorana, A.M. and Polissi, A., 2019. Lipopolysaccharide biosynthesis and transport to the outer membrane of gram-negative bacteria. In: Kuhn, A., editor. Bacterial cell walls and membranes, subcellular biochemistry. Cham: Springer. pp. 9–37.
Van der Wolf, J.M., Birnbaum, Y., Van der Zouwen, P.S. and Groot, S.P.C., 2008. Disinfection of vegetable seed by treatment with essential oils, organic acids and plant extracts. Seed Science and Technology 36(1): 76–88.
Xu, Y., Gao, C., Hou, K., Zhang, Y., Chen, Y., Feng, X., et al. 2026. The preparation and antimicrobial activity of lauric arginate/hydroxypropyl-β-cyclodextrin stabilized essential oil nanoemulsion. Journal of Future Foods 6(1): 82–89. https://doi.org/10.1016/j.jfutfo.2024.04.004
Zahi, M.R., Liang, H. and Yuan, Q., 2015. Improving the antimicrobial activity of d-limonene using a novel organogel-based nanoemulsion. Food Control 50: 554–559. https://doi.org/10.1016/j.foodcont.2014.10.001
Zancan, A.M., da Silva, J.L. and Stradiotto, N.R., 2025. Limonene monitoring in citrus industry wastewater using molecularly imprinted voltammetric sensor. Talanta 292: 127831.
Zhang, G., Jiang, X., Tan, Q. and Li, L., 2024. Preparation and characterization of Pummelo essential oil microcapsules and their application to preservation of Agricus bisporus (white mushrooms). Quality Assurance and Safety of Crops and Foods 16(4): 28–39. https://doi.org/10.15586/qas.v16i4.1505
Zhang, H., Yamamoto, E., Murphy, J. and Locas, A., 2020. Microbiological safety of ready-to-eat fresh-cut fruits and vegetables sold on the Canadian retail market. International Journal of Food Microbiology 335: 108855. https://doi.org/10.1016/j.ijfoodmicro.2020.108855
Zhang, Z., Vriesekoop, F., Yuan, Q. and Liang, H., 2014. Effects of nisin on the antimicrobial activity of D-limonene and its nanoemulsion. Food Chemistry 150: 307–312. https://doi.org/10.1016/j.foodchem.2013.10.160
Zhou, P., Chen, C., Patil, S. and Dong, S., 2024. Unveiling the therapeutic symphony of probiotics, prebiotics, and postbiotics in gut-immune harmony. Frontiers in Nutrition 11: 1355542. https://doi.org/10.3389/fnut.2024.1355542
Zhu, H., Chen, S., Xue, J., Wang, X., Xiao, Z. and Luo, Y., 2025. Application of nanotechnology in sprouts and microgreens: Current developments and future perspectives. Journal of Agriculture and Food Research 19: 101680. https://doi.org/10.1016/j.jafr.2025.101680
Zielińska-Dawidziak, M., 2021. Influence of stress conditions on the quality of obtained sprouts—Modification of their chemical composition. Quality Assurance and Safety of Crops and Foods 13(2): 1–12. https://doi.org/10.15586/qas.v13i2.836
AOAC, 2019. Official methods of analysis. 21st ed. Rockville, MD: Association of Official Analytical Chemists.
AOAC, 2023. Official methods of analysis. 22nd ed. Rockville, MD: Association of Official Analytical Chemists.
Assogbadjo, A.E., Glèlè Kakaï, R., Edon, S., Kyndt, T. and Sinsin, B., 2011. Natural variation in fruit characteristics, seed germination and seedling growth of Adansonia digitata L. in Benin. New Forests 41(1): 113–125. https://doi.org/10.1007/s11056-010-9214-z
Chadathong, N., Chancha, P., Siripornadulsil, S. and Siripornadulsil, W., 2025. Prebiotics extracted from fruits and vegetables can be applied to lactic acid bacteria to improve their antioxidant activity. Food Bioscience 68: 106577. https://doi.org/10.1016/j.fbio.2025.106577
Chaidech, P., Suhem, K., Kongchoosi, N. and Matan, N., 2024. Prebiotic potential of rambutan peel with cardamom oil and its application to extend the shelf-life of plant-base rice milk. Bioactive Carbohydrates and Dietary Fibre 32(2): 100438. https://doi.org/10.1016/j.bcdf.2024.100438
Dos Santos, D.L.S., Almeida, N.A., de Almeida, J.M., Oliveira, M.E.A.S., de Oliveira Rocha, L. and Silva, N.C.C., 2024. Antimicrobial activity evaluation of combinations of essential oils, thymol and R-limonene against food-borne pathogens and spoilage agents. Food Bioscience 62: 105035.
Feng, S., Tian, Y., Yu, J., Lin, Y., de Oliveira Sartori, A.G. and Shao, P., 2025. Limonene-loaded nanostructured lipid carriers incorporated in hydrogels: Enhanced acid stability and controlled release properties. Food Research International 214(1): 116601. https://doi.org/10.1016/j.foodres.2025.116601
Gaur, G. and Gänzle, M.G., 2023. Conversion of (poly) phenolic compounds in food fermentations by lactic acid bacteria: Novel insights into metabolic pathways and functional metabolites. Current Research in Food Science 6: 100448. https://doi.org/10.1016/j.crfs.2023.100448
Idris, L.M., Nulit, R., Zaman, F.Q. and Arifin, F.K., 2020. Hydrotime analysis of Amaranthus spp. seed germination under salinity condition. Journal of Applied Research on Medicinal and Aromatic Plants 17(2): 100249. https://doi.org/10.1016/j.jarmap.2020.100249
Jahan, F., Bhuiyan, M.N.H., Islam, M.J., Ahmed, S., Hasan, M.S., Al Bashera, M., et al. 2022. Amaranthus tricolor (red amaranth), an indigenous source of nutrients, minerals, amino acids, phytochemicals, and assessment of its antibacterial activity. Journal of Agriculture and Food Research 10: 100419. https://doi.org/10.1016/j.jafr.2022.100419
Kamalpour, R., Koocheki, A. and Ghorani, B., 2025. Encapsulation of D-limonene in Lepidium perfoliatum seed gum/PVA electrospun nanofibers: Physicochemical characterization and modeling the kinetics of release. Current Research in Food Science 10: 100966. https://doi.org/10.1016/j.crfs.2024.100966
Klištincová, N., Pin, L., Puškárová, A., Giannino, D., Bučková, M., Lambreva, M.D., et al. 2024. From farm to fork: Fungal and bacterial contaminants and their diagnostics in the production steps of ready-to-eat salads. Trends in Food Science and Technology 150: 104573. https://doi.org/10.1016/j.tifs.2024.104573
Mahmmodi, P., Khoshkhoo, Z., Basti A.A., Shotorbani, P.M., Khanjari, A., 2021. Effect of Bunium persicum essential oil, NaCl, Bile Salts, and their combinations on the viability of Lactobacillus acidophilus in probiotic yogurt. Quality Assurance and Safety of Crops and Foods 13(1): 37–48. https://doi.org/10.15586/qas.v13i1.858
Maqsood, S., Bordiga, M. and Xu, B., 2025. The role of fiber in gut health and chronic diseases: A comprehensive review. Quality Assurance and Safety of Crops and Foods 17(2): 165–199. https://doi.org/10.15586/qas.v17i2.1534
Matan, N. and Matan, N., 2025. Effect of a home-grown palm wood germination tray with lime oil on enhancing GABA and inhibiting foodborne pathogens in RTE sunflower sprout salad and its mode of action. Food Control 172: 111159. https://doi.org/10.1016/j.foodcont.2025.111159
Mclauchlin, J., Aird, H., Amar, C.F.L., Jenkins, C., Jørgensen, F., Lai, S., et al. 2022. Microbiological quality of ready-to-eat salad products collected from retail and catering settings in England during 2020 to 2021. Journal of Food Protection 85(12): 1680–1689. https://doi.org/10.4315/JFP-22-116
Mir, S.A., Farooq, S., Shah, M.A., Sofi, S.A., Dar, B.N., Hamdani, A.M., et al. 2021. An overview of sprouts nutritional properties, pathogens and decontamination technologies. LWT 141(2): 110900. https://doi.org/10.1016/j.lwt.2021.110900
Niroula, A., Khatri, S., Khadka, D. and Timilsina, R., 2019. Total phenolic contents and antioxidant activity profile of selected cereal sprouts and grasses. International Journal of Food Properties 22(1): 427–437. https://doi.org/10.1080/10942912.2019.1588297
Novais, C., Molina, A.K., Abreu, R.M., Santo-Buelga, C., Ferreira, I.C., Pereira, C., et al. 2022. Natural food colorants and preservatives: A review, a demand, and a challenge. Journal of Agriculture and Food Chemistry 70(9): 2789–2805. https://doi.org/10.1021/acs.jafc.1c07533
Patade, V.Y., Singh, N., Grover, A. and Bala, M., 2025. Effect of pretreatments on seed germination of musk rose (Rosa moschata Herrm.). Journal of Applied Research on Medicinal and Aromatic Plants 45: 100628. https://doi.org/10.1016/j.jarmap.2025.100628
Phothisuwan, S., Matan, N. and Matan, N., 2021. The influence of a closed system combining orange oil and mode of action on quality preservation of Salacca fruit. Food Control 130(1): 108265. https://doi.org/10.1016/j.foodcont.2021.108265
Sanna, A., Pani, S.M. and Coroneo, V., 2025. Risk management in fruits and vegetables production and supply: Constructing a control chart for Enterobacteriaceae. Quality Assurance and Safety of Crops and Foods 17(3): 308–318. https://doi.org/10.15586/qas.v17i3.1563
Sarker, U., Lin, Y.P., Oba, S., Yoshioka, Y., Hoshikawa, K., 2022. Prospects and potentials of underutilized leafy amaranths as vegetable use for health-promotion. Plant Physiology and Biochemistry 182: 104–123. https://doi.org/10.1016/j.plaphy.2022.04.011
Sarker, U. and Oba, S., 2019. Protein, dietary fiber, minerals, antioxidant pigments and phytochemicals, and antioxidant activity in selected red morph amaranthus leafy vegetable. PLoS One 14(12): 0222517. https://doi.org/10.1371/journal.pone.0222517
Schliemann, W., Cai, Y., Degenkolb, T., Schmidt, J., and Corke, H., 2001. Betalains of Celosia argentea. Phytochemistry 58(1): 159–165. https://doi.org/10.1016/S0031-9422(01)00141-8
Sperandeo, P., Martorana, A.M. and Polissi, A., 2019. Lipopolysaccharide biosynthesis and transport to the outer membrane of gram-negative bacteria. In: Kuhn, A., editor. Bacterial cell walls and membranes, subcellular biochemistry. Cham: Springer. pp. 9–37.
Van der Wolf, J.M., Birnbaum, Y., Van der Zouwen, P.S. and Groot, S.P.C., 2008. Disinfection of vegetable seed by treatment with essential oils, organic acids and plant extracts. Seed Science and Technology 36(1): 76–88.
Xu, Y., Gao, C., Hou, K., Zhang, Y., Chen, Y., Feng, X., et al. 2026. The preparation and antimicrobial activity of lauric arginate/hydroxypropyl-β-cyclodextrin stabilized essential oil nanoemulsion. Journal of Future Foods 6(1): 82–89. https://doi.org/10.1016/j.jfutfo.2024.04.004
Zahi, M.R., Liang, H. and Yuan, Q., 2015. Improving the antimicrobial activity of d-limonene using a novel organogel-based nanoemulsion. Food Control 50: 554–559. https://doi.org/10.1016/j.foodcont.2014.10.001
Zancan, A.M., da Silva, J.L. and Stradiotto, N.R., 2025. Limonene monitoring in citrus industry wastewater using molecularly imprinted voltammetric sensor. Talanta 292: 127831.
Zhang, G., Jiang, X., Tan, Q. and Li, L., 2024. Preparation and characterization of Pummelo essential oil microcapsules and their application to preservation of Agricus bisporus (white mushrooms). Quality Assurance and Safety of Crops and Foods 16(4): 28–39. https://doi.org/10.15586/qas.v16i4.1505
Zhang, H., Yamamoto, E., Murphy, J. and Locas, A., 2020. Microbiological safety of ready-to-eat fresh-cut fruits and vegetables sold on the Canadian retail market. International Journal of Food Microbiology 335: 108855. https://doi.org/10.1016/j.ijfoodmicro.2020.108855
Zhang, Z., Vriesekoop, F., Yuan, Q. and Liang, H., 2014. Effects of nisin on the antimicrobial activity of D-limonene and its nanoemulsion. Food Chemistry 150: 307–312. https://doi.org/10.1016/j.foodchem.2013.10.160
Zhou, P., Chen, C., Patil, S. and Dong, S., 2024. Unveiling the therapeutic symphony of probiotics, prebiotics, and postbiotics in gut-immune harmony. Frontiers in Nutrition 11: 1355542. https://doi.org/10.3389/fnut.2024.1355542
Zhu, H., Chen, S., Xue, J., Wang, X., Xiao, Z. and Luo, Y., 2025. Application of nanotechnology in sprouts and microgreens: Current developments and future perspectives. Journal of Agriculture and Food Research 19: 101680. https://doi.org/10.1016/j.jafr.2025.101680
Zielińska-Dawidziak, M., 2021. Influence of stress conditions on the quality of obtained sprouts—Modification of their chemical composition. Quality Assurance and Safety of Crops and Foods 13(2): 1–12. https://doi.org/10.15586/qas.v13i2.836
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Dec 9, 2025
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