臺灣博碩士論文加值系統

Currently, therapeutic treatments for diabetic patients are giving insulin injections or oral medications, but chronical treatments often have multiple side effects. One of an alternative solution is to modify the regular diet by substituting functional food such as soybean made products containing rich polyphenolic compounds. The streptozotozin (STZ) induced hyperglycemia mice was established to evaluate the protective effects of fermented soybean residue on the solid-state fermentation of soybean residue. In the result of experiment 1. Rhizopus oligosporus or combinations of the selected lactic acid bacteria were utilized for either a short period (24 hour) or a long period (48 hour) solid state fermentation. The long period fermentation resulted in significant increase (p<0.05) in total antioxidants, GABA and isoflavones levels are compared with non-fermented soybean residue. MDCK cells was cultured in high glucose condition to screen the beast protective effects of the various microbial fermented soybean residue. In the experiment 2, the combined Rhizopus oligosporus and Lactobacillus plantarum fermented soybean residue was supplied with diet daily for 4 weeks experimental period. The body weight, blood glucose level, glucose tolerance test, reactive oxygen species (ROS) level of blood leukocytes and serum biochemical were assessed. There was not significantly different the body weight and blood glucose, but there were significantly in glucose tolerance test, kidney weight and biochemical analysis (HbA1c, GOT, GPT, BUN, Alb, T-Chol, T-Protein) (p<0.05). The reactive oxygen species (ROS) level blood leukocytes and the histopathology of the kidney showed protective property of the RLSR-48. This study suggested that the combined Rhizopus oligosporus and Lactobacillus plantarum fermented soybean residue (FSR) had improved bioavailability of isoflavone genistein (Ge); daidzein (De) and GABA in the longer fermentation and exhibiting ameliorative property in STZ-induced hyperglycemia mice and it is possibly attribute to the significant GABA content which may promote pancreatic β-cell regeneration.

Currently, therapeutic treatments for diabetic patients are giving insulin injections or oral medications, but chronical treatments often have multiple side effects. One of an alternative solution is to modify the regular diet by substituting functional food such as soybean made products containing rich polyphenolic compounds. The streptozotozin (STZ) induced hyperglycemia mice was established to evaluate the protective effects of fermented soybean residue on the solid-state fermentation of soybean residue. In the result of experiment 1. Rhizopus oligosporus or combinations of the selected lactic acid bacteria were utilized for either a short period (24 hour) or a long period (48 hour) solid state fermentation. The long period fermentation resulted in significant increase (p<0.05) in total antioxidants, GABA and isoflavones levels are compared with non-fermented soybean residue. MDCK cells was cultured in high glucose condition to screen the beast protective effects of the various microbial fermented soybean residue. In the experiment 2, the combined Rhizopus oligosporus and Lactobacillus plantarum fermented soybean residue was supplied with diet daily for 4 weeks experimental period. The body weight, blood glucose level, glucose tolerance test, reactive oxygen species (ROS) level of blood leukocytes and serum biochemical were assessed. There was not significantly different the body weight and blood glucose, but there were significantly in glucose tolerance test, kidney weight and biochemical analysis (HbA1c, GOT, GPT, BUN, Alb, T-Chol, T-Protein) (p<0.05). The reactive oxygen species (ROS) level blood leukocytes and the histopathology of the kidney showed protective property of the RLSR-48. This study suggested that the combined Rhizopus oligosporus and Lactobacillus plantarum fermented soybean residue (FSR) had improved bioavailability of isoflavone genistein (Ge); daidzein (De) and GABA in the longer fermentation and exhibiting ameliorative property in STZ-induced hyperglycemia mice and it is possibly attribute to the significant GABA content which may promote pancreatic β-cell regeneration.

ABSTRACT i
Acknowledgements ii
CONTENTS iii
SCHEMES AND TABLES vi
FIGURES vii
Chapter 1. Literature review 1
1.Soybean residue 2
2.Solid state fermentation 3
2.1 Rhizopus oligosporus and lactic acid bacteria 4
2.2 Tempeh and Rhizopus oligosporus 4
2.3 Lactic acid bacteria 5
3. The endocrine system in blood glucose utilization 6
4. Hyperglycemia symptoms 9
4.1 Diabetes cause chronic hyperglycemia 9
4.2 Complication of diabetes 11
4.3 Diabetes and kidney disease 13
4.4 Diabetes treatment and therapy 14
5. Soybean product as functional food 14
5.1 Isoflavones 15
5.2 GABA 16
5.3 Antioxidant 17
6. Streptozotocin-induced hyperglycemia mouse models 18
References 19
Experiment 1. Soybean residue improved by solid-state fermentation as functional food
1. Abstract 27
2. Introduction 28
3. Materials and methods 30
3.1 Sample preparation 30
3.2 Combined fungal and bacterial fermentation of soybean residue 30
3.3 Experimental groups 31
3.4 Measurement of pH, temperature and moisture content of fermentation 31
3.5 Extraction for antioxidant assay 31
a. Antioxidant activity assay 31
b. GABA of fermented soybean residue 33
c. HPLC analysis of GABA 33
d. Isoflavone of fermented soybean residue 34
e. HPLC analysis of isoflavones 34
3.6 Cell culture and reagents 35
3.7 Cytotoxicity 35
3.8 Measurement of intracellular ROS 35
3.9 Statistical analysis 36
4. Results and discussions 36
4.1 Changes in the general appearance of fermented soybean residue 36
4.2 Antioxidant scavenging activity 40
4.3 Production of γ-amino butyric acid (GABA) 42
4.4 Determination of isoflavone components 44
4.5 Cytotoxicity of fermented soybean residue products on the High-Glucose conditioned MDCK cells 45
4.6 Evaluation of intracellular ROS on the fermented soybean residue treated High-Glucose conditioned MDCK cells 46
5. Conclusion 47
References 49
Experiment 2. Fermented soybean residue as anti-hyperglycemic food for STZ- induced hyperglycemia mice
1. Abstract 53
2. Introduction 54
3. Materials and methods 56
3.1 Sample preparation 55
3.2 Acclimation period of animal and streptozotocin induction 56
3.3 Experimental groups 57
3.4 Monitoring body weight and blood glucose 58
3.5 Glucose tolerance test 58
3.6 Animal sacrifice 59
3.7 Measurement intracellular ROS 59
3.8 Biochemical measurement 59
3.9 Kidney histopathological examination 59
3.10 Statistical analysis 60
4.Results and discussions 61
4.1 Body weight changes 61
4.2 Fasting blood glucose level 62
4.3 Glucose tolerance test 64
4.4 Reactive Oxygen Species (ROS) in hyperglycemia mice 65
4.5 Kidney histopathology of hyperglycemia mice gavage with fermented soybean residues 67
4.6 Biochemical analysis 70
5. Conclusion 72
References 73
Appendix 77

1. Anonymous. 2009. Diagnosis and classification of diabetes mellitus. American Diabetes Association: Diabetes care, 32:S62–S67. doi:10.2337/dc09-S062
2. World Health Organizatrion. 2016. Global report on diabetes. WHO Library Cataloguing-in-Publication data: ISBN 9789241565257, p:21-23. https://www.who.data/globalreportondiabetes/diabetes.eng [ 30 January 2020].
3. Haase, V. H. 2012. Hypoxia-inducible factor signaling in the development of kidney fibrosis, Journal Fibrogenesis & Tissue Repair, 5(1): S16. doi: 10.1186/1755-1536-5-S1-S16.
4. El-Kordy, E. A. and A. M. Alshahrani. 2015. Effect of genistein, a natural soy isoflavone, on pancreatic β-cells of streptozotocin-induced diabetic rats: Histological and immunohistochemical study. Journal of Microscopy and Ultrastructure, 3(3): 108-119.
5. Forbes, J. M. and M. E. Cooper. 2013a. Mechanisms of diabetic complications. Physiol Rev: 93(1):137-88.
6. Fernandes, S. M., Cordeiro, P. M., Watanabe, M., Fonseca, C.D. and M. F. Vattimo. 2016. The role of oxidative stress in strepto¬zotocin induced diabetic nephropathy in rats. Journal Arch Endocrinol Metab 60: 443 449.
7. Jia, Q., Yang, R., Liu, X. F., Ma, S. F. and L. Wang. 2019. Genistein attenuates renal fibrosis in streptozotocininduced diabetic rats. Mol Med Rep 19(1): 423-431.
8. Li, X., Wang, N., Yin, B., Fang, D., Jiang, T., Fang, S., and W. Chen. 2016. Effects of Lactobacillus plantarum CCFM0236 on hyperglycaemia and insulin resistance in high-fat and streptozotocin-induced type 2 diabetic mice. Journal Applied Microbiology, 121(6), 1727-1736. doi:10.1111/jam.13276.
9. Zhang, R. F., Zhang, F. X., Zhang, M. W., Wei, Z. C., Yang, C.Y., and Y. Zhang Y. 2011. Phenolic composition and antioxidant activity in seed coats of 60 Chinese black soybean (Glycine max L. Merr.) varieties. J Agric Food Chem, 59: 544-593.
10. Kitawaki, R., Nishimura, Y., Takagi, N., Iwasaki, M., Tsuzuki, K. and M. Fukuda. 2009. Effects of Lactobacillus fermented soymilk and soy yogurt on hepatic lipid accumulation in rats fed a cholesterol-free diet. Journal Biosci Biotechnol Biochem, 73(7):1484-1488. doi:10.1271/bbb.80753
11. Huang, H., Krishnan, H. B., Pham, Q., Yu, L. L. and T.T. Wang. 2016. Soy and gut microbiota: interaction and implication for human health. Journal of agricultural and food chemistry, 64(46), 8695-8709.
12. Queiroz Santos, V. A., Nascimento, C. G., Schmidt, C. A. P., Mantovani, D., Dekker, R. F. H., and M. A. A. da Cunha. 2018. Solid-state fermentation of soybean okara: Isoflavones biotransformation, antioxidant activity and enhancement of nutritional quality. LWT, 92:509-515. doi:https://doi.org/10.1016/j.lwt.2018.02.067.
13. Soltani, N., Qiu, H., Aleksic, M. Glinka, Y., Zhao, F., Liu, R., Li, Y., Zhang, N. and Q. Wang. 2011. GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. Proceedings of the National Academy of Sciences of the United States of America, 108(28):11692–11697. doi: 10.1073/pnas.1102715108.
14. Pandey, A. 2003. Solid-state fermentation. Biochemical Engineering Journal, 13(2-3):81-84.
15. Musaalbakri A. B. and C. Webb. 2017. Modern microbial solid state fermentation technology for future biorefineries for the production of added-value products. Biofuel Research Journal, 4 (4), 730-740. doi:10.18331/brj2017.4.4.5
16. Aoki, H., Uda, I., Tagami, K., Furuya, Y., Endo, Y. and K. Fujimoto. 2003. The production of a new tempeh-like fermented soybean containing a high level of γ-aminobutyric acid by anaerobic incubation with Rhizopus. Bioscience, biotechnology, and biochemistry, 67(5), 1018-1023.
17. Vong, W. C., Hua, X. Y. and S.-Q. Liu. 2018. Solid-state fermentation with Rhizopus oligosporus and Yarrowia lipolytica improved nutritional and flavour properties of okara. LWT, 90:316-32.
18. Tian, J., Dang, H., Chen, Z., Guan, A., Jin, Y., Atkinson, M. A., and D. L. Kaufman. 2013. γ-Aminobutyric acid regulates both the survival and replication of human β-cells. Journal Diabetes, 62(11): 3760-3765. doi:10.2337/db13-0931
19. Gilbert, E. R. and D. Liu. 2013. Anti-diabetic functions of soy isoflavone genistein: mechanisms underlying its effects on pancreatic β-cell function. Journal Food & Function, 4(2), 200-212. doi:10.1039/C2FO30199G.
20. Furman, B. L. 2015. Streptozotocin‐induced diabetic models in mice and rats. Current protocols in pharmacology, 70(1):5.47. 41-45.47. 20.
21. Pepato, M.T., Migliorini, R. H., Goldberg, A.L. and L.C. Kettelhut. 1996. Role of different proteolytic pathways in degradation of muscle protein from streptozotocin-diabetic rats. America Journal Physiology, 271(2): 340-347.
22. Choi, M. S., Jung, U. J., Yeo, J., Kim, M. J. and M. K. Lee. 2008. Genistein and daidzein prevent diabetes onset by elevating insulin level and altering hepatic gluconeogenic and lipogenic enzyme activities in non-obese diabetic (NOD) mice. Diabetes Metab Res Rev, 24(1), 74-81. doi:10.1002/dmrr.780.
23. Wu, K. K. and Y. Huan. 2007. Diabetic atherosclerosis mouse models. Journal Atherosclerosis 191:241-249. doi: 10.1016/j.atherosclerosis.2006.08.030
24. McCue, P., Kwon, Y.I. and K. Shetty. 2005. Anti-diabetic and anti-hypertensive potential of sprouted and solid-state bioprocessed soybean. Asia Pac. J. Clin. Nutr, 14:145-152.
25. Korkmaz, O. A., Sumlu, E., Koca, H. B., Pektas, M. B., Kocabas, A., Sadi, G. and F. Akar. 2019. Effects of Lactobacillus Plantarum and Lactobacillus Helveticus on Renal Insulin Signaling, Inflammatory Markers, and Glucose Transporters in High-Fructose-Fed Rats. Journal Medicina (Kaunas), 55(5). doi: 10.3390/medicina55050207.
26. Das, D., Sarkar, S., Bordoloi, J., Wann, S. B., Kalita, J., and P. Manna. 2018. Daidzein, its effects on impaired glucose and lipid metabolism and vascular inflammation associated with type 2 diabetes. Biofactors, 44(5), 407-417. doi:10.1002/biof.1439
27. Cheong, S. H., Furuhashi, K., Ito, K., Nagaoka, M., and T.Yonezawa. 2014. Antihyperglycemic effect of equol, a daidzein derivative, in cultured L6 myocytes and ob/ob mice. Mol. Nutr. Food Res. 58, 267–277.
28. Balakumar, M., Prabhu, D., Sathishkumar, C., Prabu, P., Rokana, N., Kumar, R., and M. Balasubramanyam. 2018. Improvement in glucose tolerance and insulin sensitivity by probiotic strains of Indian gut origin in high-fat diet-fed C57BL/6J mice. Eur J Nutr, 57(1): 279-295. doi:10.1007/s00394-016-1317-7.
29. Tian, J., Dang, H. N., Yong, J. Chui, W. S., Dizon, M. P, Yaw, C.K. and D. L. Kaufman. 2011b. Oral treatment with γ-aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in high fat diet-fed mice. PLoS ONE, 6(9):e25338. doi: 10.1371/journal.pone.0025338.
30. Liu, Y., Weng, W., Wang, S., Long, R., Li, H., Li, H., and M. Wu. 2018. Effect of gamma-Aminobutyric Acid-Chitosan Nanoparticles on Glucose Homeostasis in Mice. ACS Omega, 3(3), 2492-2497. doi:10.1021/acsomega.7b01988.
31. Bisht, S., Sisodia, S., Mahendra, P. and N. Sharma. 2011. Oral glucose tolerance test: An essential tool to make the diagnosis of diabetes. International Journal of Pharmaceutical Sciences Review and Research, 6: 48-55.
32. Ali A. A., Velasquez, M.T., Hansen, C.T., Mohamed, A.I. and S. J. Bhathena. 2005. Modulation of carbohydrate metabolism and peptide hormones by soybean isoflavones and probiotics in obesity and diabetes. Journal Nutrition Biochemical, 16:6993–6999. doi: 10.1016/j.jnutbio.2005.03.011.
33. Fu, Z., Gilbert, E.Z. and D. Liu. 2013. Regulation of insulin synthesis and secretion and pancreatic beta-cell dysfunction in diabetes. Journal Curr Diabetes, 9(1): 25-53.
34. Dhakal, R., Bajpai, V. K., and K. H. Baek. 2012. Production of gaba (γ - Aminobutyric acid) by microorganisms: a review. Brazilian journal of microbiology, 43(4), 1230–1241. doi:10.1590/S1517-83822012000400001
35. Martensson, U.E, Salehi, S.A., Windahl, S., Gomez, M. F., Daszkiewicz-Nilsson, J., Wendt, A., Andersson, N., Hellstrand, P., Grände, P.O., Owman, C. and L.M. Leeb-Lundberg. 2009. Deletion of the G-protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice. Journal Endocrinology 150:687–698. doi: 10.1210/en.2008-0623.
36. Thomas, P. and J. Dong. 2006. Binding and activation of the seven-transmembrane estrogen receptor GPR30 by environmental estrogens: a potential novel mechanism of endocrine disruption. J Steroid Biochem Mol Biol 102:175–179. doi: 10.1016/j.jsbmb.2006.09.017.
37. Fu, Z., Zhang, W., Zhen, W., Lum, H., Nadler, J., Bassaganya-Riera, J. and D. Liu. 2010. Genistein induces pancreatic beta-cell proliferation through activation of multiple signaling pathways and prevents insulin-deficient diabetes in mice. Journal Endocrinology, 151(7): 3026-3037. doi:10.1210/en.2009-1294
38. Palak, P. and I, Dipa. 2013. GABA as potential target in the treatment of Type-1 Diabetes Mellitus. American Journal of Pharm Research, 3(3).
39. Matough, F. A., Budin, S. B., Hamid, Z. A., Alwahaibi, N. and J. Mohamed. 2012. The role of oxidative stress and antioxidants in diabetic complications. Sultan Qaboos University medical journal, 12(1): 5–18. doi:10.12816/0003082.
40. Pham-Huy, L. A., He, H. and C. Pham-Huy. 2008. Free radicals, antioxidants in disease and health. International journal of biomedical science: IJBS, 4(2): 89-96.
41. Asmat, U., Abad, K., and Ismail, K. 2016. Diabetes mellitus and oxidative stress a concise review. Saudi Pharmaceutical Journal, 24(5), 547-553.
42. Sun, M-Y., Ye, Y., Xiao, L., Rahman, K., Xia, W. and H. Zhang. 2016. Daidzein: A review of pharmacological effects. African Journal of Traditional, Complementary and Alternative Medicines 13: 117. Doi: 10.4314/ajtcam.v13i3.15.
43. Fakhruddin, S., Alanazi, W. and K. E. Jackson. 2017. Diabetes-induced reactive oxygen species: mechanism of their generation and role in renal injury. Journal Diabetes Res, 8379327. doi:10.1155/2017/8379327.
44. Mestry, S. N., Dhodi J. B., Kumbhar S. B. and A. R. Juvekar. 2017. Attenuation of diabetic nephropathy in streptozotocin-induced diabetic rats by Punica granatum Linn. Leaves extract. J Tradit Complement Med 7(3): 273-280.
45. Lee, S-J., Lee, J. H., Lee, H. H., Kim, S. H., Chun, T. and J.Y. Imm. 2011. Effect of mung bean ethanol extract on pro-inflammtory cytokines in LPS stimulated macrophages. Journal Food Science Biotechnology, 20 (2): 519–524. doi: 10.1007/s10068-011-0072-z.
46. Forbes, J. M., Coughlan, M.T. and M.E. Cooper. 2008. Oxidative stress as a major culprit in kidney disease in diabetes. Journal Diabetes, 57(6):1446-54.
47. Jafari, T. 2016. Nutritional assessment in patients on hemodialysis. Journal of Preventive Epidemiology, 1 (1).
48. Rafieian-kopaei, M., Beigrezaei, S., Nasri, H., and M. Kafeshani. 2017. Soy Protein and Chronic Kidney Disease: An Updated Review. International Journal of Preventive Medicine 8:105. doi: 10.4103/ijpvm.IJPVM_244_17
49. Palsamy, P. and S.Subramanian. 2008. Resveratrol, a natural phytoalexin, normalizes hyperglycemia in streptozotocin-nicotinamide induced experimental diabetic rats. Journal Biomedicine Pharmacotherapy, 62 (9): 598-605. doi: doi.org/10.1016/j.biopha.2008.06.037
50. Choi, M.S., Jung, U. J., Yeo, J., Kim, M. J., and M. K. Lee. 2008. Genistein and daidzein prevent diabetes onset by elevating insulin level and altering hepatic gluconeogenic and lipogenic enzyme activities in non-obese diabetic (NOD) mice. Diabetes Metab. Res, 24:74–81.
51. Ingram, L.R. 2003. Liver function: In Clinical chemistry: concepts and applications. In Anderson, S. C. and Cockayne, S. editor. New York: McGraw-Hill, 285-321.
52. Ramachandran, S., Rajasekaran, A. and K. T. Kumar. 2011. Antidiabetic, antihyperlipidemic and antioxidant potential of methanol extract of Tectona grandis flowers in streptozotocin induced diabetic rats. Asian Pacific Journal Tropical Medicine, 4(8): 624-631.
53. Prabhu, K. S., Lobo, R.. and A. Shirwaikar. 2007. Antidiabetic properties of the alcoholic extract of Sphaeranthus indicus in streptozotocinnicotinamide induced diabetic rats. Journal Pharm Pharmacology; 60: 909-916.
54. Al-Shamaony, L., Al-khazrajoi, S., and H. Twaij. 1994. Hypoglycaemic effect of artemisia herba alba. II. effect of a valuable extract on some blood parameters in diabetic animals. Journal Ethnopharmacol, 43:167–171.
55. Pushparaj, P., Tan, C. and B. Tan. 2000. Effects of Averrhoe bilimli leaf extract on blood glucose and lipids in streptozotocin diabetic rats. Journal Ethnopharmacol, 72:69–76.
56. Ashraf, H., Heidari, R., and N. V. I. Minoo. 2013. Effects of aqueous extract of berberis integerrima root on some physiological parameters in streptozotocin-induced diabetic rats. Iranian journal of pharmaceutical research: IJPR 12(2): 425-434.
57. Glastras, S. J., Chen, H., Teh, R., McGrath, R. T., Chen, J., Pollock, C. A. and S. Saad. 2016. Mouse models of diabetes, obesity and related kidney disease. PloS one, 11(8): e0162131.
58. Liu, Z., Chen, Y., Ho, S. C., Ho, Y. P., and J. Woo. 2010. Effects of soy protein and isoflavones on glycemic control and insulin sensitivity: a 6-mo double-blind, randomized, placebo-controlled trial in postmenopausal Chinese women with prediabetes or untreated early diabetes. The American Journal of Clinical Nutrition, 91(5): 1394–1401. doi:10.3945/ajcn.2009.28813
59. Behloul, N. and G. Wu. 2013. Genistein: a promising therapeutic agent for obesity and diabetes treatment. European Journal of Pharmacology, 698 (1-3): 31-38.
doi: 10.1016/j.ejphar.2012.11.013
60. Cederberg, H., Saukkonen, T., Laakso, M., Jokelainen, J., Harkonen, P., Timonen, M., and U. Rajala. 2010. Postchallenge glucose, A1C, and fasting glucose as predictors of type 2 diabetes and cardiovascular disease: a 10-year prospective cohort study. Journal Diabetes care, 33(9): 2077-2083. doi:10.2337/dc10-0262.
61. Florkowski, C. 2013. HbA1c as a diagnostic test for diabetes mellitus–reviewing the evidence. The Clinical Biochemist Reviews, 34(2): 75.
.