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Improvement of Aglycone Content in Soy Isoflavones Extract by Free and Immobilized Β-Glucosidase and their Effects in Lipid Accumulation

Abstract

Soybean is one of the most important commodities in the world, being applied in feed crops and food, pharmaceutical industries in different ways. Soy is rich in isoflavones that in aglycone forms have exhibited significant anti-obesity and anti-lipogenic effects. Obesity is a global problem as several diseases have been related to this worldwide epidemic. The aim of this work was to verify the effect of free and immobilized β-glucosidase, testing Lentikats, and sol–gel as carriers. Moreover, we wanted to examine if the different types of hydrolysis would generate extracts with distinct biological activity concerning lipid accumulation, PPAR-α regulation, and TNF-α, IL-6, and IL-10 concentrations using in vitro assays. Our results show that all formulations of β-glucosidase could hydrolyze soy isoflavones. Thus, after 24 h of incubation, daidzein content increased 2.6-, 10.8-, and 12.2-fold; and genistein content increased 11.7, 11.4, and 11.4 times with the use of free enzyme, Lentikats®, and sol–gel immobilized enzyme, respectively. Moreover, both methodologies for enzyme immobilization led to promising forms of biocatalysts for application in the production of soy extracts rich in isoflavones aglycones, which are expected to bring about health benefits. A mild lipogenic effect was observed for some concentrations of extracts, as well as a slight inhibition in PPAR-α expression, although no significant differences were noticeable in the cytokines TNF-α, IL-10, and IL-6 as compared with the control.

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References

  1. 1.

    Nielsen, I. L. F., & Williamson, G. (2007). Review of the factors affecting bioavailability of soy isoflavones in humans. Nutrition and Cancer, 57(1), 1–10. https://doi.org/10.1080/01635580701267677.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Cederroth, C. R., Zimmermann, C., & Nef, S. (2012). Soy, phytoestrogens and their impact on reproductive health. Molecular and Cellular Endocrinology, 355(2), 192–200. https://doi.org/10.1016/j.mce.2011.05.049.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Benassayag, C., Ferre, F., & Perrot-Applanat, M. (2002). Phytoestrogens as modulators of steroid action in target cells. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 777(1–2), 233–248. https://doi.org/10.1016/S1570-0232(02)00340-9.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Matsuura, M., Obata, A., & Fukushima, D. (1989). Objectionable flavor of soy Milk developed during the soaking of soybeans and its control. Journal of Food Science, 54(3), 602–605. https://doi.org/10.1111/j.1365-2621.1989.tb04662.x.

    CAS  Article  Google Scholar 

  5. 5.

    Lijun, W., Saito, M., Tatsumi, E., & Lite, L. I. (2003). Antioxidative and Angiotensin I-Converting Enzyme Inhibitory Activities of Sufu (Fermented Tofu) Extracts. Japan Agricultural Research Quartely, 37(2), 129–132. http://www.jircas.affrc.go.jp.

  6. 6.

    Lin, I. C., Yamashita, S., Murata, M., Kumazoe, M., & Tachibana, H. (2016). Equol suppresses inflammatory response and bone erosion due to rheumatoid arthritis in mice. Journal of Nutritional Biochemistry, 32, 101–106. https://doi.org/10.1016/j.jnutbio.2016.02.012.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Rostagno, M. A., Villares, A., Guillamón, E., García-Lafuente, A., & Martínez, J. A. (2009). Sample preparation for the analysis of isoflavones from soybeans and soy foods. Journal of Chromatography A, 1216(1), 2–29. https://doi.org/10.1016/j.chroma.2008.11.035.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    El-Shazly, A., Noor El-Deen, A., Ibrahim, N., Abdelwahed, N., El-Beih, A., Shetaia, Y., & Farid, M. (2017). Assessment of Genistein and Daidzein production by some local fungal and bacterial isolates. Egyptian Journal of Microbiology, 0(0), 49–61. https://doi.org/10.21608/ejm.2017.1134.1023.

    Article  Google Scholar 

  9. 9.

    Piskula, M. K., Yamakoshi, J., & Iwai, Y. (1999). Daidzein and genistein but not their glucosides are absorbed from the rat stomach. FEBS Letters, 447(2–3), 287–291. https://doi.org/10.1016/S0014-5793(99)00307-5.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Chang, F., Xue, S., Xie, X., Fang, W., Fang, Z., & Xiao, Y. (2018). Carbohydrate-binding module assisted purification and immobilization of β-glucosidase onto cellulose and application in hydrolysis of soybean isoflavone glycosides. Journal of Bioscience and Bioengineering, 125(2), 185–191. https://doi.org/10.1016/j.jbiosc.2017.09.001.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Izumi, T., Piskula, M. K., Osawa, S., Obata, A., Tobe, K., Saito, M., Kataoka, S., Kubota, Y., & Kikuchi, M. (2000). Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. The Journal of Nutrition, 130(7), 1695–1699. https://doi.org/10.1093/jn/130.7.1695.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Dang, Z. C., & Löwik, C. W. G. M. (2004). The balance between concurrent activation of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis. Journal of Bone and Mineral Research, 19(5), 853–861. https://doi.org/10.1359/JBMR.040120.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Lesinski, G. B., Reville, P. K., Mace, T. A., Young, G. S., Ahn-Jarvis, J., Thomas-Ahner, J., Vodovotz, Y., Ameen, Z., Grainger, E., Riedl, K., Schwartz, S., & Clinton, S. K. (2015). Consumption of soy isoflavone enriched bread in men with prostate cancer is associated with reduced proinflammatory cytokines and immunosuppressive cells. Cancer Prevention Research, 8(11), 1036–1044. https://doi.org/10.1158/1940-6207.CAPR-14-0464.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Ahn-Jarvis, J. H., Teegarden, M. D., Schwartz, S. J., Lee, K., & Vodovotz, Y. (2017). Modulating conversion of isoflavone glycosides to aglycones using crude beta-glycosidase extracts from almonds and processed soy. Food Chemistry, 237, 685–692. https://doi.org/10.1016/j.foodchem.2017.05.122.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Sakamoto, Y., Naka, A., Ohara, N., Kondo, K., & Iida, K. (2014). Daidzein regulates proinflammatory adipokines thereby improving obesity-related inflammation through PPARγ. Molecular Nutrition and Food Research, 58(4), 718–726. https://doi.org/10.1002/mnfr.201300482.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Seo, S. G., Yang, H., Shin, S. H., Min, S., Kim, Y. A., Yu, J. G., Lee, D. E., Chung, M. Y., Heo, Y. S., Kwon, J. Y., Yue, S., Kim, K. H., Cheng, J. X., Lee, K. W., & Lee, H. J. (2013). A metabolite of daidzein, 6,7,4′-trihydroxyisoflavone, suppresses adipogenesis in 3T3-L1 preadipocytes via ATP-competitive inhibition of PI3K. Molecular Nutrition and Food Research, 57(8), 1446–1455. https://doi.org/10.1002/mnfr.201200593.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Coughlan, M. P. (1991). Mechanisms of cellulose degradation by fungi and bacteria. Animal Feed Science and Technology, 32(1–3), 77–100. https://doi.org/10.1016/0377-8401(91)90012-H.

    CAS  Article  Google Scholar 

  18. 18.

    Chang, M. Y., & Juang, R. S. (2007). Use of chitosan-clay composite as immobilization support for improved activity and stability of β-glucosidase. Biochemical Engineering Journal, 35(1), 93–98. https://doi.org/10.1016/j.bej.2007.01.003.

    CAS  Article  Google Scholar 

  19. 19.

    Singh, G., Verma, A. K., & Kumar, V. (2016). Catalytic properties, functional attributes and industrial applications of β-glucosidases. 3 Biotech, 6(1), 1–14. https://doi.org/10.1007/s13205-015-0328-z.

    Article  PubMed  Google Scholar 

  20. 20.

    Sheldon, R. A., & van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: Why, what and how. Chemical Society Reviews, 42(15), 6223–6235. https://doi.org/10.1039/c3cs60075k.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Bommarius, A. S., & Paye, M. F. (2013). Stabilizing biocatalysts. Chemical Society Reviews, 42(15), 6534–6565. https://doi.org/10.1039/c3cs60137d.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Chen, K. I., Erh, M. H., Su, N. W., Liu, W. H., Chou, C. C., & Cheng, K. C. (2012). Soyfoods and soybean products: From traditional use to modern applications. Applied Microbiology and Biotechnology, 96(1), 9–22. https://doi.org/10.1007/s00253-012-4330-7.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Grade, L. C., Moreira, A. A., Varea, G. d. S., Mandarino, J. M. G., da Silva, J. B., Ida, E. I., & Ribeiro, M. L. L. (2014). Soybean β-glucosidase immobilisated on chitosan beads and its application in soy drink increase the aglycones. Brazilian Archives of Biology and Technology, 57(5), 766–773. https://doi.org/10.1590/S1516-8913201402331.

    CAS  Article  Google Scholar 

  24. 24.

    Durieux, A., Nicolay, X., & Simon, J. P. (2000). Continuous malolactic fermentation by Oenococcus oeni entrapped in Lentikats. Biotechnology Letters, 22(21), 1679–1684. https://doi.org/10.1023/A:1005667611732.

    CAS  Article  Google Scholar 

  25. 25.

    Krasňan, V., Stloukal, R., Rosenberg, M., & Rebroš, M. (2016). Immobilization of cells and enzymes to LentiKats®. Applied Microbiology and Biotechnology, 100(6), 2535–2553. https://doi.org/10.1007/s00253-016-7283-4.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Hench, L. L., & West, J. K. (1990). The sol-gel process. Chemical Reviews, 90(1), 33–72. https://doi.org/10.1021/cr00099a003.

    CAS  Article  Google Scholar 

  27. 27.

    Avnir, D., Lev, O., & Livage, J. (2006). Recent bio-applications of sol-gel materials. Journal of Materials Chemistry, 16(11), 1013–1030. https://doi.org/10.1039/b512706h.

    CAS  Article  Google Scholar 

  28. 28.

    Wang, X., Ahmed, N., Alvarez, G., Tuttolomondo, M., Helary, C., Desimone, M., & Coradin, T. (2015). Sol-gel encapsulation of biomolecules and cells for medicinal applications. Current Topics in Medicinal Chemistry, 15(3), 223–244. https://doi.org/10.2174/1568026614666141229112734.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Wang, S., Wang, Y., Pan, M. H., & Ho, C. T. (2017). Anti-obesity molecular mechanism of soy isoflavones: Weaving the way to new therapeutic routes. Food and Function, 8(11), 3831–3846. https://doi.org/10.1039/c7fo01094j.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Qatanani, M., & Lazar, M. A. (2007). Mechanisms of obesity-associated insulin resistance: Many choices on the menu. Genes and Development, 21(12), 1443–1455. https://doi.org/10.1101/gad.1550907.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Gregoire, F. M., Smas, C. M., & Sul, H. S. (1998). Understanding adipocyte differentiation. Physiological Reviews, 78(3), 783–809. https://doi.org/10.1152/physrev.1998.78.3.783.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    De Alencar Figueira, J., Dias, F. F. G., Sato, H. H., & Fernandes, P. (2011). Screening of supports for the immobilization of β-glucosidase. Enzyme Research, 2011(1), 1–8. https://doi.org/10.4061/2011/642460.

    CAS  Article  Google Scholar 

  33. 33.

    Matsuura, M., Sasaki, J., & Murao, S. (1995). Studies on β-Glucosidases from soybeans that hydrolyze daidzin and genistin: isolation and characterization of an Isozyme. Bioscience, Biotechnology, and Biochemistry, 59(9), 1623–1627. https://doi.org/10.1271/bbb.59.1623.

    CAS  Article  Google Scholar 

  34. 34.

    Aguiar, C L de. (2004). Transformações fisica e bioquimica de isoflavonas conjugadas de soja (Glycine max L.) e o efeito na atividade biologica in vitro. Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos, Campinas.

  35. 35.

    Park, Y. K., Aguiar, C. L., Alencar, S. M., Mascarenhas, H. A. A., & Scamparini, A. R. P. (2002). Conversão de malonil-beta-glicosil isoflavonas em isoflavonas glicosadas presentes em alguns cultivares de soja brasileira. Ciência e Tecnologia de Alimentos, 22(2), 130–135. https://doi.org/10.1590/s0101-20612002000200005.

    CAS  Article  Google Scholar 

  36. 36.

    Coward, L., Barnes, N. C., Setchell, K. D. R., & Barnes, S. (1993). Genistein, daidzein, and their β-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets. Journal of Agricultural and Food Chemistry, 41(11), 1961–1967. https://doi.org/10.1021/jf00035a027.

    CAS  Article  Google Scholar 

  37. 37.

    Axelson, M., Sjövall, J., Gustafsson, B. E., & Setchell, K. D. R. (1984). Soya–a dietary source of the non-steroidal oestrogen equol in man and animals. Journal of Endocrinology, 102(1), 49–56. https://doi.org/10.1677/joe.0.1020049.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Day, A. J., Dupont, M. S., Ridley, S., Rhodes, M., Rhodes, M. J. c., Morgan, M. R. a., & Williamson, G. (1998). Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver β-glucosidase activity. FEBS Letters, 436(1), 71–75. https://doi.org/10.1016/S0014-5793(98)01101-6.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Kawakami, Y., Tsurugasaki, W., Nakamura, S., & Osada, K. (2005). Comparison of regulative functions between dietary soy isoflavones aglycone and glucoside on lipid metabolism in rats fed cholesterol. Journal of Nutritional Biochemistry, 16(4), 205–212. https://doi.org/10.1016/j.jnutbio.2004.11.005.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Vong, W. C., Au Yang, K. L. C., & Liu, S. Q. (2016). Okara (soybean residue) biotransformation by yeast Yarrowia lipolytica. International Journal of Food Microbiology, 235, 1–9. https://doi.org/10.1016/j.ijfoodmicro.2016.06.039.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Jackson, C. J. C., Dini, J. P., Lavandier, C., Rupasinghe, H. P. V., Faulkner, H., Poysa, V., Buzzell, D., & DeGrandis, S. (2002). Effects of processing on the content and composition of isoflavones during manufacturing of soy beverage and tofu. Process Biochemistry, 37(10), 1117–1123. https://doi.org/10.1016/S0032-9592(01)00323-5.

    CAS  Article  Google Scholar 

  42. 42.

    Barnes, S., Kirk, M., & Coward, L. (1994). Isoflavones and their conjugates in soy foods: Extraction conditions and analysis by HPLC-mass spectrometry. Journal of Agricultural and Food Chemistry, 42(11), 2466–2474. https://doi.org/10.1021/jf00047a019.

    CAS  Article  Google Scholar 

  43. 43.

    Murphy, P. A., Song, T., Buseman, G., Barua, K., Beecher, G. R., Trainer, D., & Holden, J. (1999). Isoflavones in retail and institutional soy foods. Journal of Agricultural and Food Chemistry, 47(7), 2697–2704. https://doi.org/10.1021/jf981144o.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Cho, K. M., Lee, J. H., Yun, H. D., Ahn, B. Y., Kim, H., & Seo, W. T. (2011). Changes of phytochemical constituents (isoflavones, flavanols, and phenolic acids) during cheonggukjang soybeans fermentation using potential probiotics Bacillus subtilis CS90. Journal of Food Composition and Analysis, 24(3), 402–410. https://doi.org/10.1016/j.jfca.2010.12.015.

    CAS  Article  Google Scholar 

  45. 45.

    Wu, X., Yue, H., Zhang, Y., Gao, X., Li, X., Wang, L., Cao, Y., Hou, M., An, H., Zhang, L., Li, S., Ma, J., Lin, H., Fu, Y., Gu, H., Lou, W., Wei, W., Zare, R. N., & Ge, J. (2019). Packaging and delivering enzymes by amorphous metal-organic frameworks. Nature Communications, 10(1), 1–8. https://doi.org/10.1038/s41467-019-13153-x.

    CAS  Article  Google Scholar 

  46. 46.

    Hu, C., Bai, Y., Hou, M., Wang, Y., Wang, L., Cao, X., Chan, C. W., Sun, H., Li, W., Ge, J., & Ren, K. (2020). Defect-induced activity enhancement of enzyme-encapsulated metal-organic frameworks revealed in microfluidic gradient mixing synthesis. Science Advances, 6(5), 1–9. https://doi.org/10.1126/sciadv.aax5785.

    Article  Google Scholar 

  47. 47.

    Huang, C. C., Huang, W. C., Hou, C. W., Chi, Y. W., & Huang, H. Y. (2014). Effect of black soybean koji extract on glucose utilization and adipocyte differentiation in 3T3-L1 cells. International Journal of Molecular Sciences, 15(5), 8280–8292. https://doi.org/10.3390/ijms15058280.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Satoh, S., Nakano, R., Ishii, C., Sugiyama, T., Eto, K., Tsubamoto, Y., Okuno, A., Murakami, K., Sekihara, H., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K., Kitamura, T., Fujita, T., Ezaki, O., Aizawa, S., Nagai, R., Tobe, K., Kimura, S., & Kadowaki, T. (1999). PPARγ mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Molecular Cell, 4(4), 597–609. https://doi.org/10.1016/S1097-2765(00)80210-5.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Sakamoto, Y., Kanatsu, J., Toh, M., Naka, A., Kondo, K., & Iida, K. (2016). The dietary isoflavone daidzein reduces expression of pro-inflammatory genes through PPARα/γ and JNK pathways in adipocyte and macrophage co-cultures. PLoS One, 11(2), 1–14. https://doi.org/10.1371/journal.pone.0149676.

    CAS  Article  Google Scholar 

  50. 50.

    Nakajima, V. M., Moala, T., Caria, C. R. E. P., Moura, C. S., Amaya-Farfan, J., Gambero, A., et al. (2017). Biotransformed citrus extract as a source of anti-inflammatory polyphenols: Effects in macrophages and adipocytes. Food Research International, 97, 37–44. https://doi.org/10.1016/j.foodres.2017.03.034.

    CAS  Article  PubMed  Google Scholar 

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Funding

This work was supported by a FAPESP scholarship to Joelise de Alencar Figueira Angelotti (process number 2013/13212-6).

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Angelotti, J.A.F., Dias, F.F.G., Sato, H.H. et al. Improvement of Aglycone Content in Soy Isoflavones Extract by Free and Immobilized Β-Glucosidase and their Effects in Lipid Accumulation. Appl Biochem Biotechnol 192, 734–750 (2020). https://doi.org/10.1007/s12010-020-03351-5

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Keywords

  • Soy isoflavones
  • β-Glucosidase
  • Aglycones
  • Obesity
  • Cell culture