Immobilization of Fructooligosaccharides Producing Enzymes for the Continuous/Batch Production of Prebiotics Neokestose and Kestose from an Astaxanthin Over-Production Mutant Xanthophyllomyces Dendrorhous Nchu-Fs701

寡果糖(Fructooligosaccharides, FOS)廣泛存在於天然植物中，亦可藉由微生物之β.fructofuranosidase或果糖轉移酵素(fructosyltransferase)將蔗糖催化而生成。寡果糖除具有利於食品加工特性外，不易為人體酵素所消化，然卻有利於腸道有用菌群及雙叉桿菌(Bifidobacterium)之活化，進而增進人類及動物之健康。目前生產寡果糖之菌株幾乎都是黴菌，如：Aspergillus niger, Aspergillus japonicus, Aureobasidium pullulans, Fusarium oxysporum等。然黴菌生長時會產生大量菌絲，因此商業生產時須消耗較高之能量以維持足夠的攪拌與氧氣供給。經本研究室初步測試發現高蝦紅素生產變異株Xanthophyllomyces dendrorhous NCHU-FS701亦具有生產寡果糖之能力，且由於該菌為酵母菌，可以克服上述黴菌生產寡果糖時之缺點，且該菌所產生之益生物質果寡糖包含neokestose。本研究擬培養X. dendrorhous NCHU-FS701H並經破菌萃取蝦紅素後，將其含有果寡糖生成酵素之粗酵素液，利用酵素固定化之技術固定在幾丁聚醣上，進行批式或連續式生產果寡糖kestose及neokestose。本研究之目的為：1. 建立X. dendrorhous NCHU-FS701同步生產蝦紅素及果寡糖生成酵素之發酵技術2. 分析X. dendrorhous NCHU-FS701所生產之果寡糖種類及含量；3. 該菌果寡糖生成酵素之酵素特性及酵素動力學模式；4. 建立該菌果寡糖生成酵素固定化之最佳條件；5. 經固定化後之酵素與未固定化酵素之特性比較；6. 開發固定化酵素以批式重覆利用或連續式生產果寡糖。X. dendrorhous NCHU-FS701發酵終產物主要為neokestose，很少有微生物或植物細胞可發酵產生該果寡糖，通常以少量副產物之方式存在，若本研究開發成功，則可大量生產neokestose用以其它用途之開發，並藉以提升X. dendrorhous NCHU-FS701的利用價值

Inhibition+of+Malic+Acid+Degradation+in+Malolactic+Bacteria+by+Sulfur+Dioxide

The effect of sulfur dioxide and pH on the L-malate degradation by malolactic bacteriawas examined. Malolactic bacteria, including Leuconostoc oenos PSU-1, CSb5, and a homolactic strain BB were grown in CBB medium at two pHs (pH 3.4 and 5.5). The本研究探討二氧化硫及pH值對蘋果酸乳酸菌分解L-蘋果酸之影響。所採 用之蘋果酸乳酸菌包括Leuconostoc oenos PSU-1.CSb5及一株同型乳酸醱酵菌BB菌 株。菌體濃度及對L-蘋果酸分解能力之線性關係依菌株而異。L. oenos CSb5對二 氧化硫最為敏感;然而其貝有最高之L-蘋酸分解能力。L-蘋果酸分解之最佳pH值 因菌株而異;對L. oenos PSU-1,CSb5及同型乳酸醱酵菌BB而言，其最佳L-蘋果酸分 解之pH值分別為4.0-5.0，4.5及4.0。雖然L-蘋果酸

Funtional Soymilk Fermented by Tremella flava and Innovation of Health Food

本年度之計畫目標在於利用黃白木耳發酵豆奶之發酵進行time course之異黃酮及胞外多醣測定以利此發酵豆奶之產程開發。另外透過發酵技術改良或是添加關鍵因子促進胞外多醣之生產亦是此次研究發酵部分之重點。以期能同時得到具有機能性胞外多醣及異黃酮轉化之發酵豆奶。發酵之產物透過生物分析(bioassay)的細胞實驗進行免疫能力提升之探討及對大腸癌及胃癌細胞抑制活性之評估。最後根據產品特性之分析，如風味、流體特性及黏度等等，進行白木耳發酵豆奶之保健產品研發，並於最終產品之型式評估後進行保存後機能性確認之試驗，以期能兼顧完整的研究結果及實際之產品開發，進行研究發表及成果之技術轉移評估，彰顯本計畫之成果。透過本研究，能更一步了解白木耳發酵豆漿在各種機能性方面的表現，在異黃酮轉化部分預期能促使aglycone形式的異黃酮素含量增加，以利於人體吸收。利用發酵技術的調控使白木耳於發酵豆漿過程中分泌胞外多醣或其他可能的機能性物質，藉由生物分析(bioassay)評估發酵豆奶是否具有免疫提升以及抗腫瘤的能力。最後將白木耳豆漿經由機能性驗證及儲藏活性試驗開發成適合銀髮族之保健產品並建立基礎之生產發酵平台。The objective of this proposal will focus on the soymilk fermented with Tremella flava to investigate the production of exopolysaccharide and the biotransformation of isoflavone in the soybeans. In addition, parameters that enhance the exopolysaccharide production during the fermentation, such as improvement of fermentation techniques and supplementation with key factors will also be investigated. Innovated fermented soymilk with functional exopolysaccharide and isoflavones is the products will be developed in this proposal. Immunity studies and bioassay including various cancerous cell lines will be used to evaluate the functionality of the fermented soymilk. Sensory, rheology and viscosity properties will be investigated before the fermented health soymilk are innovated. We try to learn more about the Tremella flava fermented soymilk, for example, the biotransformation between glycone and aglycone in the fermented soymilk and the fermentation technology of this health food. Through this research, not only scientific data will be published, innovated health food for elderly such as soymilk fermented with fungus Tremella flava will be developed

Functionality of Yoghurt and Soymilk Fermented With Botanical Lactobacillus Plantarum SLC 13 Supplemented with Tremella Flava Chen Polysaccharide and Citrus Peel Fiber

本研究擬以本研究室所篩選之植物源乳酸菌（Lactobacillus plantarum SLC 13）添加不同處理之橘子皮纖維與台灣新種黃白木耳（Tremella flava Chen）多醣，評估其所產生乳酸菌胞外多醣之抗氧化能力及乳酸菌菌液對抗菌活性之提升，並建立其最適添加及發酵條件。L. plantarum SLC 13 為篩選自本土植物發酵產品，具下列特性：耐鹽性（18％ NaCl）；耐酸性佳，其於pH 3，3 hr之存活率高達91.76 ％；耐膽鹽性佳，於0.3 ％膽鹽，24hr之存活率為82.82 ％；對Staphylococcus aureus (BCRC 10908) 與Yersinia enterocolitica (BCRC 10807) 有較佳的抑制效果；對12種抗生素具有耐受性；具有生產多醣之能力，初步產量可達到2.03 ± 0.16 g/L。本研究室對於黃白木耳之研究已完成發酵前後之黃豆奶組成分變化、抗氧化性、RAW 264.7細胞發炎反應之影響、Caco-2 (人體分離之腸道癌細胞) 和AGS (人體胃癌細胞)之抑癌試驗、黃白木耳發酵豆奶（TFFS）的致突變性、血清生化及免疫相關指標的評估及TFS對BALB/c小鼠口服試驗期間發炎媒介物的變化等，結果均顯示TFFS之安全性及作為機能性產品之潛力。本研究擬藉由黃白木耳多醣及柑橘皮纖維素之添加，除探討其對本研究室所分離L. plantarum SLC 13之抗氧化及抗菌能力提升外，並期將來有機會開發富含果皮纖維及菇類多醣之優酪乳與發酵豆奶新型產品。The purpose of this proposal is to evaluate the antioxidative activity of exopolysaccharide produced and the antibacterial activity of by Lactobacillus plantarum SLC 13, isolated from fermented mustard pickle by our laboratory, supplemented with tangerine peel fiber and Tremella flava Chen. The optimization of the fermentation process will be established. L. plantarum SLC13 is a botanical lactic acid bacterium with the following traits: tolerate up to 18％ NaCl, with 91.76％ and 82.82％ survival rate at pH 3 for 3 h and 0.3％ bile salt for 24 h, respectively. This microorganism also showed better inhibition to Staphylococcus aureus (BCRC 10908) and Yersinia enterocolitica (BCRC 10807). SLC13 not only resisted to 12 antibiotics but also had the ability to produce exopolysaccharide (2.03 ± 0.16 g/L). Our laboratory have completed several Tremella flava-related studies including basic composition changes in fermented and nonfermented soymilk, antioxidant properties, RAW 254.7 cell inflammatory effects, Caco-2/AGS inhibitory studies, mutagenicity and inflammatory mediated reactions in BALB/c mice. All the results indicated the safety and potential of products fermented by T. flava. This study will focus on the effect of citrus peel fiber and exopolysaccharide from T. flava on the antioxidative and antimicrobial activity of S. plantarum SLC13. By fermented with L. plantarum SLC 13, yoghurt and soymilk supplemented with tangerine peel fiber and T. flava Chen will be developed in the future

Functional Properties of Exopolysaccharides from Native Tremella in Taiwan---Purification, Structural Identification, Anti-tumor and Anti-diabetics

抗腫瘤、抗病毒、抗氧化、抗感染、降血糖和降血脂等。具有免疫和抗腫瘤活性之多醣結構，目前之研究歸納出包含同質葡萄聚糖（Homoglucans）、異質葡萄聚糖（Heteroglucans）、異質半乳聚糖（Heterogalactans）和其他的異質多醣（Heteroglycans）等，其中又以β-(1,3)-葡萄聚醣被廣為研究，然因多醣的結構複雜且分子性質不易被鑑定，因此其活性機制與活性結構及分子性質間之關係僅少數被釐清。本研究室曾研究台灣原生種黃白木耳（Tremella flava Chen）之機能性，發現其有清除自由基及抑制癌細胞生長之潛力，值得進一步研究。本研究擬歸納已被報導具潛在免疫活性、抗腫瘤多醣(包括菇類真菌多醣和食品多醣)共通之結構特性後，再深入探討白木耳屬真菌多醣其結構和分子性質與免疫調節和抗腫瘤活性間之關聯性。本計畫擬以三年的時間，針對包含台灣原生種之不同木耳屬真菌（Tremellaflava Chen, Tremella fuciformis, Tremella aurantia, Tremella encephala, Tremellaneofoliacea Chen, Tremella nivalis Chen, Tremella resupinata Chen, Tremellataiwanensis Chen and Tremella tropica Chen）以液態培養方式生產胞外多醣，解析其單醣組成、結構特性、分子性質、免疫調節以及抗腫瘤有關的生物活性，進一步篩選出具有較佳生理活性之木耳屬菌種，並以動物實驗進行降血糖功效評估，藉此瞭解其活性醣類的結構和分子量區間與機能性之關聯性，及利用染劑作為定量活性多醣方法，希望綜合以上結果以為未來深入研究其提升免疫力、抗感染或抗腫瘤機轉和開發高保健性的機能性多醣素材與培養微生物發酵生物技術之基礎。Mushrooms have been widely used as edible and medicinal resources. Variousbioactivities of polysaccharide from mushroom have been extensively studied, such asimmunomodulating, antiflammatory, antitumor, antiviral, antioxidation,hyperglycemia, and hyperlipidemia. Several structural characterizations which relatedto immunomodulating and antitumor activities have been classified, includinghomoglucans, heteroglucans, heterogalacans, and heteroglycans. Among them, β- (1,3)-glucan was the thoroughly studies than others. However, polysaccharides are verycomplicated; the relationship between the bioactivity and the chemical composition aswell as the high order structure of their active components structure characterization isyet to be established. In our previous study, the results showed that the nativeTremella flava Chen in Taiwan has the antioxidation and antitumor activity in cellmodel and worth further investigation. In this proposal, various Tremella species willbe selected as the samples for elucidating the relationships between the structure,molecular property, immunostimulating effects, antitumor activity, as well asanti-diabetic of the polysaccharides.Nine Tremella spp., including native species found in Taiwan, will be collected.They are Tremella flava Chen, Tremella fuciformis, Tremella aurantia, Tremellaencephala, Tremella neofoliacea Chen, Tremella nivalis Chen, Tremella resupinataChen, Tremella taiwanensis Chen and Tremella tropica Chen. The experiments inevery year will start with the submerge incubation to produce tremella speciesexopolysaccharide. Then the polysaccharides will be isolated and fractionated.Immunomodulating and antitumor activities will be examined with the test samples asprepared. The monosaccharide composition, acidic functional groups, molecularproperty and structural character of the polysaccharides are then identified, using highperformance liquid chromatography techniques and nuclear magnetic resonanceanalysis. From the results of this study, the structural and molecular property of thepolysaccharides with high immunostimulating effects, antitumor and anti-diabeticactivities are expected to be understood, which provide the base for their applicationto functional food in the future