Use of postbiotics produced by Lactobacillus spp. as biopreservatives in foods: From probiotics to postbiotics

Yıl 2024, , 1562 - 1575, 01.12.2024

Yasemin Kaya , Mustafa Şengül , Enes Dertli

https://doi.org/10.21597/jist.1487261

Öz

In food applications, undesirable conditions occur when appropriate conditions for primary and secondary cultures are not provided. Postbiotics have recently emerged as an alternatives to this issue. Postbiotics are bioactive substances produced by live microorganisms during fermentation that have positive effects on health. These substances include bioactive peptides, teichoic acid, B vitamins and organic acids. These complex compounds can provide significant benefits in the food and health sectors. This study evaluates current research on Lactobacillus spp. postbiotics that have been confirmed to be safe. Additionally, specific metabolites responsible for antimicrobial activity have been examined. Bioactive compounds responsible for preventing biofilm formation, and their antibacterial and antifungal activities have been assessed. Finally, the potential use of these postbiotics as biologically degradable alternatives to industrial disinfectants has been thoroughly investigated. The review discusses the effects of Lactobacillus spp postbiotics on food quality and safety, depending on their use in dairy products, meat products, fruits, vegetables and other foods, and explores the metabolites responsible for antagonistic activity.

Anahtar Kelimeler

Postbiotic, Food safety, Antimicrobial

Lactobacillus spp. Tarafından Üretilen Postbiyotiklerin Gıdalarda Biyokoruyucu Olarak Kullanımı: Probiyotiklerden Postbiyotiklere Geçiş

Öz

Gıda uygulamalarında kullanılan birincil ve ikincil kültürlere uygun şartlar sağlanmadığında üründe istenmeyen durumlar meydana gelmektedir. Postbiyotikler, son zamanlarda bu duruma alternatif olarak ortaya çıkmıştır. Postbiyotikler, fermantasyon sırasında canlı mikroorganizmalar tarafından üretilen ve sağlık üzerinde olumlu etkiler oluşturan biyoaktif maddelerdir. Bu maddeler arasında biyoaktif peptitler, teikoik asitler, B grubu vitaminleri ve organik asitler bulunur. Kompleks yapılı bu bileşenler, gıda ve sağlık alanlarında önemli faydalar sağlayabilirler. Yapılan çalışma ile, güvenli olduğu doğrulanmış Lactobacillus spp. postbiyotiklerinin mevcut güncel çalışmaları değerlendirilmiştir. Ayrıca antimikrobiyal aktiviteden sorumlu belirli metabolitler incelenmiştir. Gıdalarda biyofilm oluşumunun önlenmesi, antibakteriyel ve antifungal aktiviteleri sağlayan biyoaktif bileşenler değerlendirilmiştir. Son olarak, bu postbiyotiklerin endüstriyel dezenfektan yerine biyolojik olarak parçalanabilen alternatif santizer olarak kullanımı detaylı bir şeklide incelenmiştir. Derlemede, Lactobacillus spp. postbiyotiklerinin süt ürünleri, et ürünleri, meyve, sebze ve diğer bazı gıdalarda kullanımına bağlı olarak gıda kalitesi ve güvenliği üzerindeki etkileri değerlendirilmekte ve antagonistik aktiviteden sorumlu metabolitler tartışılmaktadır.

Anahtar Kelimeler

Postbiyotik, Gıda Güvenliği, Antimikrobiyal

Kaynakça

• Abouloifa, H., Gaamouche, S., Rokni, Y., Hasnaoui, I., Bellaouchi, R., Ghabbour, N., Salah, R. B. (2021). Antifungal activity of probiotic Lactobacillus strains isolated from natural fermented green olives and their application as food bio-preservative. Biological Control, 152, 104450.
• Adesina, I., Ojokoh, A., & Arotupin, D. (2017). Inhibitory properties of lactic acid bacteria against moulds associated with spoilage of bakery products. Journal of Advances in Microbiology, 4(3), 1-8.
• Ahmad Rather, I., Seo, B., Rejish Kumar, V., Choi, U. H., Choi, K. H., Lim, J., & Park, Y. H. (2013). Isolation and characterization of a proteinaceous antifungal compound from Lactobacillus plantarum YML007 and its application as a food preservative. Letters in Applied Microbiology, 57(1), 69-76.
• Álvarez, A., Manjarres, J. J., Ramírez, C., & Bolívar, G. (2021). Use of an exopolysaccharide-based edible coating and lactic acid bacteria with antifungal activity to preserve the postharvest quality of cherry tomato. Lwt, 151, 112225
• Axel, C., Zannini, E., & Arendt, E. K. (2017). Mold spoilage of bread and its biopreservation: A review of current strategies for bread shelf life extension. Critical reviews in food science and nutrition, 57(16), 3528-3542
• Barros, C. P., Guimaraes, J. T., Esmerino, E. A., Duarte, M. C. K., Silva, M. C., Silva, R., Cruz, A. G. (2020). Paraprobiotics and postbiotics: Concepts and potential applications in dairy products. Current opinion in food science, 32, 1-8.
• Bernet-Camard, M.-F., Liévin, V., Brassart, D., Neeser, J.-R., Servin, A. L., & Hudault, S. (1997). The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance (s) active in vitro and in vivo. Applied and environmental microbiology, 63(7), 2747-2753.
• Bomfim, V. B., Neto, J. H. P. L., Leite, K. S., de Andrade Vieira, É., Iacomini, M., Silva, C. M., Cardarelli, H. R. (2020). Partial characterization and antioxidant activity of exopolysaccharides produced by Lactobacillus plantarum CNPC003. Lwt, 127, 109349.
• Brown, S., Santa Maria Jr, J. P., & Walker, S. (2013). Wall teichoic acids of gram-positive bacteria. Annual review of microbiology, 67, 313-336.
• Chaluvadi, S., Hotchkiss, A. T., & Yam, K. L. (2015). Gut microbiota: Impact of probiotics, prebiotics, synbiotics, pharmabiotics, and postbiotics on human health. In Probiotics, prebiotics, and synbiotics: Bioactive foods in health promotion (pp. 515-523): Elsevier Inc.
• Cicenia, A., Scirocco, A., Carabotti, M., Pallotta, L., Marignani, M., & Severi, C. (2014). Postbiotic activities of lactobacilli-derived factors. Journal of clinical gastroenterology, 48, S18-S22.
• Coppola, S., Avagliano, C., Calignano, A., & Berni Canani, R. (2021). The protective role of butyrate against obesity and obesity-related diseases. Molecules, 26(3), 682.
• Corsetti, A., Gobbetti, M., Rossi, J., & Damiani, P. (1998). Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Applied microbiology and biotechnology, 50, 253-256.
• Cotter, P. D., Hill, C., & Ross, R. P. (2005). Bacteriocins: developing innate immunity for food. Nature Reviews Microbiology, 3(10), 777-788.
• Czatzkowska, M., Harnisz, M., Korzeniewska, E., & Koniuszewska, I. (2020). Inhibitors of the methane fermentation process with particular emphasis on the microbiological aspect: A review. Energy Science & Engineering, 8(5), 1880-1897.
• Dash, G., Raman, R. P., Prasad, K. P., Makesh, M., Pradeep, M., & Sen, S. (2015). Evaluation of paraprobiotic applicability of Lactobacillus plantarum in improving the immune response and disease protection in giant freshwater prawn, Macrobrachium rosenbergii (de Man, 1879). Fish & shellfish immunology, 43(1), 167-174.
• da Silva Sabo, S., Pérez-Rodríguez, N., Domínguez, J. M., & de Souza Oliveira, R. P. (2017). Inhibitory substances production by Lactobacillus plantarum ST16Pa cultured in hydrolyzed cheese whey supplemented with soybean flour and their antimicrobial efficiency as biopreservatives on fresh chicken meat. Food Research International, 99, 762-769.
• Dalié, D., Deschamps, A., & Richard-Forget, F. (2010). Lactic acid bacteria–Potential for control of mould growth and mycotoxins: A review. Food control, 21(4), 370-380.
• Delavenne, E., Mounier, J., Déniel, F., Barbier, G., & Le Blay, G. (2012). Biodiversity of antifungal lactic acid bacteria isolated from raw milk samples from cow, ewe and goat over one-year period. International journal of food microbiology, 155(3), 185-190.
• De Marco, S., Sichetti, M., Muradyan, D., Piccioni, M., Traina, G., Pagiotti, R., & Pietrella, D. (2018). Probiotic cell‐free supernatants exhibited anti‐inflammatory and antioxidant activity on human gut epithelial cells and macrophages stimulated with LPS. Evidence‐Based Complementary and Alternative Medicine, 2018(1), 1756308.
• Deseta, M. L., Sponton, O. E., Erben, M., Osella, C. A., Frisón, L. N., Fenoglio, C., . . . Perez, A. A. (2021). Nanocomplexes based on egg white protein nanoparticles and bioactive compounds as antifungal edible coatings to extend bread shelf life. Food Research International, 148, 110597.
• Drewnowski, A. (2012). The economics of food choice behavior: why poverty and obesity are linked. In Obesity treatment and prevention: new directions (Vol. 73, pp. 95-112): Karger Publishers.
• Flórez, A. B., Delgado, S., & Mayo, B. (2005). Antimicrobial susceptibility of lactic acid bacteria isolated from a cheese environment. Canadian journal of microbiology, 51(1), 51-58.
• George-Okafor, U., Nwachukwu, U., & Mike-Anosike, E. (2018). Screening and identification of predominant Lactobacillus spp from fermented milled-mixed corn–soybean wastes. Int. J. Sci. & Eng. Res, 9(2), 2050-2060.
• Gill, P., Van Zelm, M., Muir, J., & Gibson, P. (2018). Short chain fatty acids as potential therapeutic agents in human gastrointestinal and inflammatory disorders. Alimentary pharmacology & therapeutics, 48(1), 15-34.
• Gómez-Sala, B., Herranz, C., Díaz-Freitas, B., Hernández, P. E., Sala, A., & Cintas, L. M. (2016). Strategies to increase the hygienic and economic value of fresh fish: Biopreservation using lactic acid bacteria of marine origin. International journal of food microbiology, 223, 41-49.
• Gueniche, A., Perin, O., Bouslimani, A., Landemaine, L., Misra, N., Cupferman, S., Khodr, A. (2022). Advances in microbiome-derived solutions and methodologies are founding a new era in skin health and care. Pathogens, 11(2), 121.
• Guilhaumou, R., Benaboud, S., Bennis, Y., Dahyot-Fizelier, C., Dailly, E., Gandia, P.,Roger, C. (2019). Optimization of the treatment with beta-lactam antibiotics in critically ill patients—guidelines from the French Society of Pharmacology and Therapeutics (Société Française de Pharmacologie et Thérapeutique—SFPT) and the French Society of Anaesthesia and Intensive Care Medicine (Société Française d’Anesthésie et Réanimation—SFAR). Critical Care, 23(1), 1-20.
• Hamad, G., Botros, W., & Hafez, E. (2017). Combination of probiotic filtrates as antibacterial agent against selected some pathogenic bacteria in milk and cheese. Int J Dairy Sci, 12(6), 368-376.
• Hamad, G. M., Abdelmotilib, N. M., Darwish, A. M., & Zeitoun, A. M. (2020). Commercial probiotic cell-free supernatants for inhibition of Clostridium perfringens poultry meat infection in Egypt. Anaerobe, 62, 102181.
• Hasan, F. B., Reza, M., Al Masud, H., Uddin, M. K., & Uddin, M. S. (2019). Preliminary characterization and inhibitory activity of bacteriocin like substances from Lactobacillus casei against multi-drug resistant bacteria. Bangladesh J Microbiol, 36(1), 1-6.
• Hepatology, T. L. G. (2019). Food safety really is everyone's business. In (Vol. 4, pp. 571).
• Homayouni Rad, A., Aghebati Maleki, L., Samadi Kafil, H., & Abbasi, A. (2021). Postbiotics: A novel strategy in food allergy treatment. Critical reviews in food science and nutrition, 61(3), 492-499.
• Iordache, F., Gheorghe, I., Lazar, V., Curutiu, C., Ditu, L. M., Grumezescu, A. M., & Holban, A. M. (2017). Nanostructurated materials for prolonged and safe food preservation. In Food preservation (pp. 305-335): Elsevier.
• İncili, G. K., Karatepe, P., Akgöl, M., Kaya, B., Kanmaz, H., & Hayaloğlu, A. A. (2021). Characterization of Pediococcus acidilactici postbiotic and impact of postbiotic-fortified chitosan coating on the microbial and chemical quality of chicken breast fillets. International journal of biological macromolecules, 184, 429-437.
• İncili, G. K., Karatepe, P., Akgöl, M., Tekin, A., Kanmaz, H., Kaya, B., Hayaloğlu, A. A. (2022). Impact of chitosan embedded with postbiotics from Pediococcus acidilactici against emerging foodborne pathogens in vacuum-packaged frankfurters during refrigerated storage. Meat Science, 188, 108786.
• Izuddin, W. I., Humam, A. M., Loh, T. C., Foo, H. L., & Samsudin, A. A. (2020). Dietary postbiotic Lactobacillus plantarum improves serum and ruminal antioxidant activity and upregulates hepatic antioxidant enzymes and ruminal barrier function in post-weaning lambs. Antioxidants, 9(3), 250.
• Jonkuvienė, D., Vaičiulytė-Funk, L., Šalomskienė, J., Alenčikienė, G., & Mieželienė, A. (2016). Potential of Lactobacillus reuteri from spontaneous sourdough as a starter additive for improving quality parameters of bread. Food Technology and Biotechnology, 54(3), 342.
• Khaneghah, A. M., Hashemi, S. M. B., & Limbo, S. (2018). Antimicrobial agents and packaging systems in antimicrobial active food packaging: An overview of approaches and interactions. Food and Bioproducts Processing, 111, 1-19.
• Khaneghah, A. M., Abhari, K., Eş, I., Soares, M. B., Oliveira, R. B., Hosseini, H.,Cruz, A. G. (2020). Interactions between probiotics and pathogenic microorganisms in hosts and foods: A review. Trends in Food Science & Technology, 95, 205-218.
• Khodaii, Z., Ghaderian, S. M. H., & Natanzi, M. M. (2017). Probiotic bacteria and their supernatants protect enterocyte cell lines from enteroinvasive Escherichia coli (EIEC) invasion. International journal of molecular and cellular medicine, 6(3), 183.
• Lee, K. J., Park, H. W., Choi, E. J., & Chun, H. H. (2016). Effects of CFSs produced by lactic acid bacteria in combination with grape seed extract on the microbial quality of ready-to-eat baby leaf vegetables. Cogent Food & Agriculture, 2(1), 1268742.
• Lücke, F.-K. (2023). Overview of Biopreservation.
• Mani-López, E., García, H., & López-Malo, A. (2012). Organic acids as antimicrobials to control Salmonella in meat and poultry products. Food Research International, 45(2), 713-721
• Moll, G. N., Konings, W. N., & Driessen, A. J. (1999). Bacteriocins: mechanism of membrane insertion and pore formation. Antonie Van Leeuwenhoek, 76, 185-198.
• Mokoena, M. P. (2017). Lactic acid bacteria and their bacteriocins: classification, biosynthesis and applications against uropathogens: a mini-review. Molecules, 22(8), 1255.
• Moradi, M., Kousheh, S. A., Almasi, H., Alizadeh, A., Guimarães, J. T., Yılmaz, N., & Lotfi, A. (2020). Postbiotics produced by lactic acid bacteria: The next frontier in food safety. Comprehensive Reviews in Food Science and Food Safety, 19(6), 3390-3415.
• Moradi, M., Mardani, K., & Tajik, H. (2019). Characterization and application of postbiotics of Lactobacillus spp. on Listeria monocytogenes in vitro and in food models. Lwt, 111, 457-464.
• Moradi, M., Tajik, H., Mardani, K., & Ezati, P. (2019). Efficacy of lyophilized cell-free supernatant of Lactobacillus salivarius (Ls-BU2) on Escherichia coli and shelf life of ground beef. Paper presented at the Veterinary Research Forum.
• Muhialdin, B. J., Algboory, H. L., Kadum, H., Mohammed, N. K., Saari, N., Hassan, Z., & Hussin, A. S. M. (2020). Antifungal activity determination for the peptides generated by Lactobacillus plantarum TE10 against Aspergillus flavus in maize seeds. Food control, 109, 106898.
• Nielsen, P. V. (2004). Packaging, quality control, and sanitation of bakery products. In Handbook of food and beverage fermentation technology (pp. 922-939): CRC Press.
• Ołdak, A., Zielińska, D., Łepecka, A., Długosz, E., & Kołożyn-Krajewska, D. (2020). Lactobacillus plantarum strains isolated from polish regional cheeses exhibit anti-staphylococcal activity and selected probiotic properties. Probiotics and antimicrobial proteins, 12, 1025-1038.
• Pan, L., Hu, X., & Wang, X. (2011). Assessment of antibiotic resistance of lactic acid bacteria in Chinese fermented foods. Food control, 22(8), 1316-1321.
• Paulino do Nascimento, L. C., Lacerda, D. C., Ferreira, D. J. S., de Souza, E. L., & de Brito Alves, J. L. (2022). Limosilactobacillus fermentum, current evidence on the antioxidant properties and opportunities to be exploited as a probiotic microorganism. Probiotics and antimicrobial proteins, 14(5), 960-979.
• Pimentel, T. C., Cruz, A. G., Pereira, E., da Costa, W. K. A., da Silva Rocha, R., de Souza Pedrosa, G. T., . . . Sant’Ana, A. S. (2023). Postbiotics: An overview of concepts, inactivation technologies, health effects, and driver trends. Trends in Food Science & Technology, 138, 199-214
• Prema, P., Smila, D., Palavesam, A., & Immanuel, G. (2010). Production and characterization of an antifungal compound (3-phenyllactic acid) produced by Lactobacillus plantarum strain. Food and Bioprocess Technology, 3, 379-386.
• Rad, A. H., Abbasi, A., Kafil, H. S., & Ganbarov, K. (2020). Potential pharmaceutical and food applications of postbiotics: a review. Current pharmaceutical biotechnology, 21(15), 1576-1587
• Rad, A. H., Aghebati-Maleki, L., Kafil, H. S., Gilani, N., Abbasi, A., & Khani, N. (2021a). Postbiotics, as dynamic biomolecules, and their promising role in promoting food safety. Biointerface Res Appl Chem, 11(6), 14529-14544.
• Rad, A. H., Maleki, L. A., Kafil, H. S., Zavoshti, H. F., & Abbasi, A. (2021b). Postbiotics as promising tools for cancer adjuvant therapy. Advanced Pharmaceutical Bulletin, 11(1), 1.
• Rafique, N., Jan, S. Y., Dar, A. H., Dash, K. K., Sarkar, A., Shams, R., Hussain, S. Z. (2023). Promising bioactivities of postbiotics: A comprehensive review. Journal of Agriculture and Food Research, 100708.
• Ranadheera, C., Evans, C., Baines, S. K., Balthazar, C. F., Cruz, A. G., Esmerino, E. A., Naumovski, N. (2019). Probiotics in goat milk products: Delivery capacity and ability to improve sensory attributes. Comprehensive Reviews in Food Science and Food Safety, 18(4), 867-882.
• Rendueles, O., Kaplan, J. B., & Ghigo, J. M. (2013). Antibiofilm polysaccharides. Environmental microbiology, 15(2), 334-346.
• Rolim, F. R. L., dos Santos, K. M. O., de Barcelos, S. C., do Egito, A. S., Ribeiro, T. S., da Conceição, M. L., do Egypto, R. d. C. R. (2015). Survival of Lactobacillus rhamnosus EM1107 in simulated gastrointestinal conditions and its inhibitory effect against pathogenic bacteria in semi-hard goat cheese. LWT-Food science and Technology, 63(2), 807-813.
• Sarkar, S. (2018). Whether viable and dead probiotic are equally efficacious? Nutrition & Food Science, 48(2), 285-300.
• Sarikhani, M., Kermanshahi, R. K., Ghadam, P., & Gharavi, S. (2018). The role of probiotic Lactobacillus acidophilus ATCC 4356 bacteriocin on effect of HBsu on planktonic cells and biofilm formation of Bacillus subtilis. International journal of biological macromolecules, 115, 762-766.
• Sharma, V., Harjai, K., & Shukla, G. (2018). Effect of bacteriocin and exopolysaccharides isolated from probiotic on P. aeruginosa PAO1 biofilm. Folia microbiologica, 63, 181-190.
• Soomro, A., Masud, T., & Anwaar, K. (2002). Role of lactic acid bacteria (LAB) in food preservation and human health-a review. Pakistan Journal of Nutrition, 1(1), 20-24.
• Scarpellini, E., Rinninella, E., Basilico, M., Colomier, E., Rasetti, C., Larussa, T., Abenavoli, L. (2021). From pre-and probiotics to post-biotics: a narrative review. International journal of environmental research and public health, 19(1), 37.
• Schaefer, L., Auchtung, T. A., Hermans, K. E., Whitehead, D., Borhan, B., & Britton, R. A. (2010). The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups. Microbiology, 156(6), 1589-1599.
• Sukmarını, L., Mustopa, A. Z., Normawatı, M., & Muzdalifah, I. (2014). Identification of antibiotic-resistance genes from lactic acid bacteria in Indonesian fermented foods. HAYATI Journal of Biosciences, 21(3), 144-150
• Sun, J., Rutherford, S. T., Silhavy, T. J., & Huang, K. C. (2022). Physical properties of the bacterial outer membrane. Nature Reviews Microbiology, 20(4), 236-248.
• Šušković, J., Kos, B., Beganović, J., Leboš Pavunc, A., Habjanič, K., & Matošić, S. (2010). Antimicrobial activity–the most important property of probiotic and starter lactic acid bacteria. Food Technology and Biotechnology, 48(3), 296-307.
• Tenea, G., & Barrigas, A. (2018). The efficacy of bacteriocin-containing cell-free supernatant from Lactobacillus plantarum Cys5-4 to control pathogenic bacteria growth in artisanal beverages. International Food Research Journal, 25(5), 2031-2037.
• Tomkovich, S., & Jobin, C. (2016). Microbiota and host immune responses: a love–hate relationship. Immunology, 147(1), 1-10.
• Toushik, S. H., Kim, K., Park, S.-H., Park, J.-H., Ashrafudoulla, M., Ulrich, M. S. I., . . . Kang, I. (2023). Prophylactic efficacy of Lactobacillus curvatus B67-derived postbiotic and quercetin, separately and combined, against Listeria monocytogenes and Salmonella enterica ser. Typhimurium on processed meat sausage. Meat Science, 197, 109065
• Valan Arasu, M., Jung, M. W., Ilavenil, S., Jane, M., Kim, D. H., Lee, K. D., Lim, Y. C. (2013). Isolation and characterization of antifungal compound from Lactobacillus plantarum KCC‐10 from forage silage with potential beneficial properties. Journal of applied microbiology, 115(5), 1172-1185.
• Verraes, C., Van Boxstael, S., Van Meervenne, E., Van Coillie, E., Butaye, P., Catry, B., Dierick, K. (2013). Antimicrobial resistance in the food chain: a review. International journal of environmental research and public health, 10(7), 2643-2669.
• Wang, K., Niu, M., Song, D., Song, X., Zhao, J., Wu, Y.,Niu, G. (2020). Preparation, partial characterization and biological activity of exopolysaccharides produced from Lactobacillus fermentum S1. Journal of bioscience and bioengineering, 129(2), 206-214.
• Yang, V. W., & Clausen, C. A. (2005). Determining the suitability of Lactobacilli antifungal metabolites for inhibiting mould growth. World Journal of Microbiology and Biotechnology, 21, 977-981.
• Yoon, B. K., Jackman, J. A., Valle-González, E. R., & Cho, N.-J. (2018). Antibacterial free fatty acids and monoglycerides: biological activities, experimental testing, and therapeutic applications. International journal of molecular sciences, 19(4), 1114.
• Yordshahi, A. S., Moradi, M., Tajik, H., & Molaei, R. (2020). Design and preparation of antimicrobial meat wrapping nanopaper with bacterial cellulose and postbiotics of lactic acid bacteria. International journal of food microbiology, 321, 108561.
• Zavišić, G., Ristić, S., Petričević, S., Janković, D., & Petković, B. (2024). Microbial Contamination of Food: Probiotics and Postbiotics as Potential Biopreservatives. Foods, 13(16), 2487.
• Żółkiewicz, J., Marzec, A., Ruszczyński, M., & Feleszko, W. (2020). Postbiotics—a step beyond pre-and probiotics. Nutrients, 12(8), 2189.