臺灣博碩士論文加值系統

β-葡聚醣是一種由葡萄糖以β-1,3醣苷鍵鍵結構成主鏈，分支為β-1,4或β-1,6鍵結形成的功能性多醣，廣泛存在於植物(燕麥)、藻類、細菌及真菌之中，因具有保水、增稠及乳化等功能，可應用於食品及化妝品業；此多醣亦有免疫調節的功能，市售已有多項萃取自啤酒酵母之保健食品，宣稱可以調節生理機能，不過市售酵母β-葡聚醣產品的原料，常取自啤酒釀造後之廢酵母萃取而成，雖具有成本較低之優勢，然由於其酵母品種以及發酵環境等不一定處在具有最高β-葡聚醣含量之狀態，整體而言仍有改善空間。本研究結果顯示，不同屬酵母每單位菌體重之β-葡聚醣含量不同，其中一株具有厚細胞壁之紅色酵母(Xanthophyllomyces dendrorhous)，每單位乾菌含量高達18.04% (w/w, 乾基)，而相同培養條件下的其中一株釀酒酵母(Saccharomyces cerevisiae)則為10.97%。其次評估並比較以液態搖瓶培養時，改變培養基組成（碳、氮源之種類及濃度）、培養條件（氧氣、溫度）及環境緊迫因子（極端pH值、滲透壓），對篩選菌株之菌體產率及β-葡聚醣含量的影響。實驗結果顯示S. cerevisiae BCRC 21450最佳條件為24℃、72小時，並以果糖為主要之YMB (Yeast Malt Broth)碳源及額外加入1% (w/v)氯化鈉，透過搖瓶培養試驗菌株，可得β-葡聚醣約0.37 g/L。最後，探討從酵母細胞中破壁後並萃取β-葡聚醣之方法，並透過傅立葉轉換紅外光譜(FT-IR)鑑定以製備一定純度之β-葡聚醣粉末。

Glucan from the yeast cell wall is a functional polysaccharide which has properties of water retention, thickening and emulsification, etc. It can be a functional ingredient for functional food preparation. This polysaccharide also has immunomodulatory properties. Most commercial yeast β-glucan products are prepared from the spent brewer’s yeasts, with low in cost but suffering from the species of yeast might not be the best or in the physiological status having high β-glucan content. Data showed that the β-glucan content of a yeast cell varies with species, for example, the β-glucan content of a red yeast, X. dendrorhous, is as high as 18.04% (dry basis), while that of S. cerevisiae is only 10.97%, both were cultured under the same medium and condition. In this study, various types of yeasts including brewer’s yeast, red yeast, and black yeast will be tested and selected. The study covered the following tasks, these include 1) screening of a potent yeast from the above-mentioned yeasts; 2) investigating effect of medium composition especially type and concentration of carbon source and nitrogen source; 3) studying effect of culture conditions such as temperature and presence of oxygen; and 4) examining effect of stress conditions, such as extreme pH and osmotic pressure, on the cell yield, β-glucan content per unit cell weight and β-glucan production, respectively.The best conditions obtained were 24℃ for 72 hours, fructose as the main carbon source of YMB (Yeast Malt Broth) and containing 1% (w/v) sodium chloride, through the shake-flask fermentation of S. cerevisiae BCRC 21450. The β-glucan were about 0.37 g/L. Finally, extraction methods of β-glucan from yeast cells were also investigated, and it were identified by FT-IR to prepare β-glucan powder with certain purity.

摘要 i
Abstract ii
目次 iii
圖目錄 vi
表目錄 ix
第一章 前言 1
第二章 文獻回顧 3
一、酵母之介紹 3
(一)酵母之型態及其應用 3
(二)酵母之生殖 4
(三)酵母之代謝 4
(四)酵母細胞及其細胞壁之組成 5
二、β-葡聚醣之介紹 5
(一)β-葡聚醣之來源 5
(二)β-葡聚醣之結構 9
(三)β-葡聚醣之應用 9
三、酵母β-葡聚醣之生產 13
四、影響酵母β-葡聚醣生產之因子 13
(一)酵母菌種與來源 13
(二)培養基組成之影響 15
(三)培養條件之影響 19
(四)萃取方法之影響 20
(五)乾燥方法之影響 24
五、發酵技術之介紹 25
(一)批次發酵(batch fermentation) 25
(二)饋料批次發酵(fed-batch fermentation) 25
(三)連續式發酵(continuous fermentation) 26
(四)量產酵母β-葡聚醣之發酵策略 26
六、破菌方法之介紹 26
(一)自溶作用(autolysis) 29
(二)珠磨均質法(homogenization in a bead mill) 29
(三)超音波法(ultrasound) 32
第三章 材料與方法 33
一、實驗材料 33
(一)實驗菌株 33
(二)培養基 33
(三)化學藥劑 33
二、儀器設備 34
三、套裝軟體 35
四、實驗架構 36
五、實驗方法 37
(一)菌株活化及保存 37
(二)菌粉製備 37
(三)菌株篩選 37
(四)酵母菌生長曲線之測定 37
(五)搖瓶培養之最適條件探討 38
(六)搖瓶培養之最適條件應用於饋料培養模式 39
(七)酵母菌體之破壁方法 39
(八)β-葡聚醣粉末之萃取與製備 40
(九)分析方法 41
第四章 結果與討論 45
一、最適生產β-葡聚醣菌種/菌株之篩選 45
二、最適菌株之培養時間與培養方式 45
三、搖瓶培養之最適生產β-葡聚醣條件 50
(一)較適碳源種類之探討 50
(二)較適碳源濃度之探討 55
(三)滲透壓對β-葡聚醣含量之影響 61
(四)pH值對β-葡聚醣含量之影響 65
(五)最適培養基組成及培養條件之分析 65
四、改變培養模式之探討 70
(一)批次及饋料批次培養之差異 70
五、破菌方式對酵母細胞及其β-葡聚醣含量之影響 76
(一)自溶作用 76
(二)超音波法 79
(三)珠磨均質法 79
(四)破菌效力與影響β-葡聚醣含量之分析 79
(五)以傅立葉轉換紅外線光譜圖比較破菌效力 87
六、製備酵母β-葡聚醣粉末 90
(一)萃取流程之適當性分析 90
(二)β-葡聚醣粉末之純度鑑定 90
(三)不同屬酵母生產之β-葡聚醣 94
第五章 結論 99
一、最適生產β-葡聚醣之篩選菌株 99
二、搖瓶培養之最適生產β-葡聚醣條件 99
三、改變培養模式之探討 99
四、細胞之破壁及製備酵母β-葡聚醣粉末 99
第六章 未來展望 101
一、X. dendrorhous於搖瓶培養條件之改善 101
二、培養基質之替代方案 101
三、生產酵母菌β-葡聚醣之副產物 101
第七章 參考文獻 103
附錄一、蝦紅素及β-胡蘿蔔素等類胡蘿蔔素之 (a)化學結構圖 (b)拉曼光譜 110

吳翊暐。2015。壓榨酵母利用甘蔗糖蜜為基質之菌株篩選、饋料批次培養及儲藏安定性。國立中興大學食品暨應用生物科技學系碩士論文，台中市。
周博俊。2015。普魯蘭多醣生產菌株 Aureobasidium pullulans 之篩選、培養條件與普魯蘭多醣純化及特性探討。國立中興大學食品暨應用生物科技學系碩士論文，台中市。
黃詩淳。2012。半纖維素酶生產菌株之篩選、培養條件與Aureobasidium pullulans NCH-218聚木糖酶酵素特性探討。國立中興大學食品暨應用生物科技學系碩士論文，台中市。
裘娟萍、沈寅初。2001。紅發夫酵母的生物學特性。工業微生物，31(3)，6-8。
黨建章。2005。發酵技術概論。新文京出版社，台北市。
超音波Qsonica型錄。(最後瀏覽日：2020.06.30)
https://www.sonicator.com/pages/cell-disruption-homogenization
衛生福利部食品藥物管理署。(最後瀏覽日：2020.07.02)
https://consumer.fda.gov.tw/Food/InfoHealthFoodDetail.aspx?nodeID=162＆id=1449
Aguilar-Uscanga, B., and J. M. Francois. 2003. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Letters in Applied Microbiology, 37: 268-274.
Borchani, C., F. Fonteyn, G. Jamin, M. Paquot, C. Blecker, and P. Thonart. 2014. Enzymatic process for the fractionation of baker''s yeast cell wall (Saccharomyces cerevisiae). Food Chemistry, 163: 108-113.
Borchani, C, F. Fonteyn, G. Jamin, M. Paquot, P. Thonart, and C. Blecker. 2016 Physical, functional and structural characterization of the cell wall fractions from baker''s yeast Saccharomyces cerevisiae. Food Chemistry, 194: 1149‐1155.
Bzducha-Wróbel, A., S. Błażejak, A. Kawarska, L. Stasiak-Różańska, I. Gientka, and E. Majewska. 2014. Evaluation of the efficiency of different disruption methods on yeast cell wall preparation for β-glucan isolation. Molecules, 19: 20941-12961.
Chen, J., and R. Seviour. 2007. Medicinal importance of fungal β-(1→3), (1→6)-glucans. Mycological Research, 111: 635-652.
Chi, Z., F. Wang, Z. Chi, L. Yue, G. Liu, and T. Zhang. 2009. Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast. Applied Microbiology and Biotechnology, 82(5): 793–804.
Du, B., Z. Bian, and B. Xu. 2014. Skin health promotion effects of natural beta-glucan derived from cereals and microorganisms: a review. Phytotherapy research : PTR, 28(2), 159–166.
Du, B., M. Meenu, H. Liu, and B. Xu. 2019. A concise review on the molecular structure and function relationship of β-glucan. International Journal of Molecular Sciences, 20(16): 4032.
Ene, I. V., L. A. Walker, M. Schiavone, K. K. Lee, H. Martin-Yken, E. Dague, N. A. Gow, C. A. Munro, and A. J. Brown. 2015. Cell wall remodeling enzymes modulate fungal cell wall elasticity and osmotic stress resistance. mBio, 6(4): e00986-15.
Fernandes, L., B. Maia, G. Fleury, B. G. Lages, J. C. Creed, L. F. C. de Oliveira. 2014. New strategies for identifying natural products of ecological significance from corals: nondestructive raman spectroscopy analysis. Studies in Natural Products Chemistry, 43: 313-349.
Fesel, P. H., and A. Zuccaro. 2016. β-glucan: Crucial component of the fungal cell wall and elusive MAMP in plants. Fungal Genetics and Biology, 90: 53-60.
Galichet, A., G. Sockalingum, A. Belarbi, and M. Manfait. 2001. FTIR spectroscopic analysis of Saccharomyces cerevisiae cell walls: Study of an anomalous strain exhibiting a pink-colored cell phenotype. FEMS Microbiology Letters, 197(2):179 - 186.
Hallfrisch, J., D. J. Scholfield, and K. M. Behall. 2003. Physiological responses of men and women to barley and oat extracts (Nu-trimX). II. Comparison of glucose and insulin responses. Cereal Chemistry, 80(1): 80-83.
Hamada, T., S. Kanno, and E. Kano. 1982. Stationary stage structure of yeast population with stage dependent generation time. Journal of Theoretical Biology, 97: 393-414.
Hlebowicz, J., G. Darwiche, O. Bjorgell, and L. O. Almer. 2008. Effect of muesli with 4 g oat beta-glucan on postprandial blood glucose, gastric emptying and satiety in healthy subjects: a randomized crossover trial. Journal of the American College of Nutrition, 27(4): 470-475.
Hopke, A., A. J. P. Brown, R. A. Hall, and R. T. Wheeler. 2018. Dynamic fungal cell wall architecture in stress adaptation and immune evasion. Trends in Microbiology, 26(4): 284-295.
Hromádková, Z., A. Ebringerová, V. Sasinková, J. Šandula, V. Hřı́balová, and J. Omelková. 2003. Influence of the drying method on the physical properties and immunomodulatory activity of the particulate (1→3)-β-d-glucan from Saccharomyces cerevisiae. Carbohydrate Polymers, 51: 9-15.
Hunter, K. W. Jr., R. A. Gault, and M. D. Berner. 2002. Preparation of microparticulate beta-glucan from Saccharomyces cerevisiae for use in immune potentiation. Letter in Applied Microbiology, 35(4): 267-271.
Ibsen, K. H. 1961. Crabtree effect - A review. Cancer Research, 21: 829-841.
Javmen, A., S. Grigiškis, and R. Gliebutė. 2012. β-Glucan extraction from Saccharomyces cerevisiae yeast using Actinomyces rutgersensis 88 yeast lysing enzymatic complex. Biologija, 58(2): 51-59.
Jacob, Friedrich, L. Striegel, M. Rychlik, M. Hutzler, and F.-J. Methner. 2019. Yeast extract production using spent yeast from beer manufacture: influence of industrially applicable disruption methods on selected substance groups with biotechnological relevance. European Food Research and Technology, 245: 1169-1182.
Kakko, N., I. Nicoletta, and R. Anssi. 2018. Cell disruption methods Group 1. CHEM-E3140 Bioprocess Technology II.
Kaur, R., M. Sharma, D. Ji, E. Xu, and D. Agyei. 2019. Structural features, modification, and functionalities of beta-glucan. Fibers, 8(1): 1.
Kirk, T. V., and N. Szita. 2013. Oxygen transfer characteristics of miniaturized bioreactor systems. Biotechnology and bioengineering, 110(4), 1005–1019.
Koobs, D. H. 1972. Phosphate mediation of the Crabtree and Pasteur effects. Science, 178(4057): 127-133.
Krpan, V., V. Petravić-Tominac, I. Krbavčić, S. Grba, and K. Berković. 2009. Potential application of yeast β-glucans in food industry. Agriculturae Conspectus Scientificus, 74: 277-282.
Lachance, M.‐A., and G. M. Walker. 2020. Yeasts. In eLS , John Wiley ＆ Sons, Ltd (Ed.).
Lemus, M. R., E. Roussarie, N. Hammad, A. Mougeolle, S. Ransac, R. Issa, J. P. Mazat, S. Uribe-Carvajal, M. Rigoulet, and A. Devin. 2018. The role of glycolysis-derived hexose phosphates in the induction of the Crabtree effect. Journal of Biological Chemistry, 293: 12843-12854.
Magnani, M., C. M. Calliari, F. Macedo, M. P. Mori, I. M. Cólus, and R. J. Castro-Gómez. 2009. Optimized methodology for extraction of (1→3)(1→6)-β-d-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative. Carbohydrate Polymers, 78: 658-665.
Mantovani, M. S., M. F. Bellini, J. P. Angeli, R. J. Oliveira, A. F. Silva, and L. R. Ribeiro. 2008. β-Glucans in promoting health: prevention against mutation and cancer. Mutation research, 658(3): 154–161.
Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31: 426-428.
Parapouli, M, A. Vasileiadis, A.-S. Afendra, and E. Hatziloukas. 2020. Saccharomyces cerevisiae and its industrial applications. AIMS Microbiology, 6(1): 1‐31.
Peltzer, M., J. F. Delgado, A. G. Salvay, and J. R. Wagner. 2018. β-Glucan, a promising polysaccharide for bio-based films developments for food contact materials and medical applications. Current Organic Chemistry, 22: 1249-1254.
Pengkumsri, N., B. S. Sivamaruthi, S. Sirilun, S. Peerajan, P. Kesika, K. Chaiyasut, and C. Chaiyasut. 2016. Extraction of β-glucan from Saccharomyces cerevisiae: Comparison of different extraction methods and in vivo assessment of immunomodulatory effect in mice. Food Science and Technology, 37: 124-130.
Rezaei, M. N., E. Aslankoohi, K. J. Verstrepen, and C. M. Courtin. 2015. Contribution of the tricarboxylic acid (TCA) cycle and the glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during dough fermentation. International Journal of Food Microbiology, 204: 24-32.
Rodrigues, F., P. Ludovico, C. Leão. 2006. Sugar metabolism in yeasts: an overview of aerobic and anaerobic glucose catabolism. Péter G., Rosa C. (eds) Biodiversity and Ecophysiology of Yeasts. The Yeast Handbook. Springer, Berlin, Heidelberg.
Rodríguez-Sáiz, M., J.L. de la Fuente, and J. L. Barredo. 2010 Xanthophyllomyces dendrorhous for the industrial production of astaxanthin. Applied Microbiology and Biotechnology, 88: 645–658.
Saha, N., A. Samanta, S. Chaudhuri, and D. Dutta. 2015. Characterization and antioxidant potential of a carotenoid from a newly isolated yeast. Food Science and Biotechnology, 24(1) 117-124.
Šandula, J., G. Kogan, M. Kačuráková, and E. Machová. 1999. Microbial (1→3)-β-D-glucans, their preparation, physico-chemical characterization and immunomodulatory activity. Carbohydrate Polymers, 38: 247-253.
Savelkoul, H. F. J., W. Chanput, and H. J. Wichers. 2013. Immunomodulatory effects of mushroom β-glucans, Diet. Immunity and Inflammation, 232:416-434.
Schmidt, F. 2005. Optimization and scale up of industrial fermentation processes. Applied Microbiology and Biotechnology. 68: 425-435.
Sebald, M., and D. Hauser. 1995. Pasteur, oxygen and the anaerobes revisited. Anaerobe, 1: 11-16.
Sreekrishna, K., and R. C. Dickson. 1986. Construction of strains of Saccharomyces cervisiae that grow on lactose. Proceedings of the National Academy of Sciences of the United States of America, 82: 7909-7913.
Stahl, G., S. N. Salem, L. Chen, B. Zhao, and P. J. Farabaugh. .2004. Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae. Eukaryotic Cell, 3(2), 331–338.
Sun, C., M. Lin, D. Fu, J. Yang, Y. Huang, X. Zheng, and T. Yu. 2018. Yeast cell wall induces disease resistance against Penicillium expansum in pear fruit and the possible mechanisms involved. Food Chemistry, 241: 301-307.
Takalloo, Z., M. Nikkhah, R. Nemati, N. Jalilian, and R. H. Sajedi. 2020. Autolysis, plasmolysis and enzymatic hydrolysis of baker''s yeast (Saccharomyces cerevisiae): a comparative study. World Journal of Microbiology and biotechnology, 36(5): 68.
Tan, K. L. and S. H. Yeo. 2016. Cavitation erosion study in deionized water containing abrasive particles. Annals of DAAAM and Proceedings, 26(1): 818-824.
Thiry, M. and D. Cingolani. 2002. Optimizing scale-up fermentation processes. Trends in Biotechnology, 20(3): 103-105.
Tian, X., P. Yang, and W. Jiang. 2017. Effect of alkali treatment combined with high pressure on extraction efficiency of β-d-glucan from spent brewer’s yeast. Waste and Biomass Valorization, 10: 1131-1140.
Varelas, V., M. Liouni, A. C. Calokerinos, and E. T. Nerantzis. 2016a. An evaluation study of different methods for the production of beta-D-glucan from yeast biomass. Drug Test and Analysis, 8(1): 46-55.
Varelas V., P. Tataridis, M. Liouni, and E. T. Nerantzis. 2016b. Valorization of winery spent yeast waste biomass as a new source for the production of β-glucan. Waste and Biomass Valorization, 7: 807-817.
Varelas V.. 2016. Application of different methods for the extraction of yeast β-glucan. e-Journal of Science and Technology, 11: 15.
Varelas V., E. Sotiropoulou, X. Karambini, M. Liouni, and E. T. Nerantzis. 2017. Impact of glucose concentration and NaCl osmotic stress on yeast cell wall β-d-glucan formation during anaerobic fermentation process. Fermentation, 3: 44.
Vetvicka, V. 2011. Glucan-immunostimulant, adjuvant, potential drug. World Journal of Clinical Oncology, 2(2): 115-119.
Volman, J. J., J. D. Ramakers, and J. Plat. 2008. Dietary modulation of immune function by β-glucans. Physiology and Behavior, 94: 276-284.
Wang, J., M. Li, F. Zheng, C. Niu, C. Liu, Q. Li, and J. Sun. 2018. Cell wall polysaccharides: before and after autolysis of brewer''s yeast. World Journal of Microbiology Biotechnology, 34(9): 137.
Wang, Q., X. Sheng, A. Shi, H. Hu, Y. Yang, L. Liu, L. Fei, and H. Liu. 2017. Beta-glucans: relationships between modification, conformation and functional activities. Molecules, 22(2): 257.
Waszkiewicz-Robak, B. 2013. Spent brewer''s yeast and beta-glucans isolated from them as diet components modifying blood lipid metabolism disturbed by an atherogenic diet. Lipid Metabolism, 1: 12.
Zhao, R. Y. 2017. Yeast for virus research. Microbial Cell, 4(10): 311-330.
Zhu, Fengmei, B. Du, and B. Xu. 2016. A critical review on production and industrial applications of beta-glucans. Food Hydrocolloids, 52: 275-288.
Zhu, Q., and S. Wu. 2019. Water-soluble β-1,3-glucan prepared by degradation of curdlan with hydrogen peroxide. Food Chemistry, 283: 302-304.