Production and Mechanism of Glucose Tolerance Factor of Yeast (III)

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Project title

Code/計畫編號

NSC92-2313-B019-040

Translated Name/計畫中文名

酵母菌葡萄糖耐量因子的生產及其作用機制探討(III)

Project Coordinator/計畫主持人

Guo-Jane Tsai

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=850972

Year

2003

Start date/計畫起

01-08-2003

Expected Completion/計畫迄

01-07-2004

Bugetid/研究經費

710千元

ResearchField/研究領域

食品科技(農)

早期由啤酒酵母菌萃取出含鉻之物質，因可改善餵食低鉻飼料之大白鼠所產生之葡萄糖不耐症（glucose intolerance），稱之為葡萄糖耐量因子（glucose intolerance factor, GTF）。傳統 GTF 活性以 SD 大白鼠副睪丸脂肪細胞進行分析（SD 模式），非常受限於動物原料，且飼養與犧牲過程非常耗時與不便，因此本研究利用分化 3T3-L1 細胞株來分析酵母所產 GTF 活性與探討其最適生產條件。本研究自各酒廠、葡萄園及葡萄表面分離得 74 株酵母菌，自菌種中心購得 14 株標準酵母菌，二者分別有 58 株及 12 株可在含啤酒花的麥汁中生長，以此 70 株供作測試菌株。分別以 SD 模式及分化 3T3-L1 細胞（3T3-L1 模式）分析其 GTF 活性，二種模式均以 Saccharomyces cerevisiae No.1 及 Saccharomyces sp. No.54 二株菌 GTF 活性最高。二種模式分析之最適 insulin 濃度均為 10 nM， SD 模式在 0~1000 .mu.g/mL 的 GTF 濃度時活性呈直線關係， 3T3-L1 模式則在 0~250 .mu.g/mL。 3T3-L1 模式探討二株菌所產 GTF 活性之最適培養液為馬鈴薯浸出液（PIB），菌株 No.1 所需最佳濃度（以還原糖含量計算）為 3.48 g/L，菌株 No. 54 則為 1.74 g/L。以 PIB 為各菌最適濃度之基礎培養基，添加 1% sucrose、 0.1% (NH4)2SO4及 1 ppm nicotinic acid 可增加菌體 GTF 活性。 0.1 ppm 微量金屬添加反而不利 No.54 菌株 GTF 活性。高轉速（150 rpm）有利溶氧量增加、菌體生長及 GTF 活性產生。高接菌量（10/sup 5/ CFU/mL）有利菌體 GTF 活性， 20℃ 培養有利菌量增加，但對 GTF 活性則與 25 或 30℃ 培養所得相似。 GTF 乃菌之二級代謝產物，主要在穩定期持續產生， 20℃ 至少需 4 天以上，以得足夠 GTF 活性，當到達 8 天時菌株 No.1 與 No.54 之 GTF 活性分別為 94.17%及 145.78%。將 No.1 與 No.54 餵食經 STZ 誘發糖尿病之老鼠 42 天後，餵食此二株菌可使老鼠血糖由控制組之 310 mg/dL 分別降至 135 mg/dL 左右。菌株 No.54 之 GTF 活性物質經初步分離純化顯示其為不帶電荷、不含鉻、分子量介於 28-44 kDa 之蛋白分子。此蛋白 GTF 活性在 40~80℃範圍隨著溫度上升 GTF 活性隨之下降，在中性環境下 GTF 具有最大活性。 No.54 菌株之 GTF 活性物質促進葡萄糖代謝之作用機制可能由於其可促進細胞膜上 PTP 酵素之活化，再藉由 p21ras 路徑活化 p38 MAPK 系統，使 GLUT4 活化而增加對葡萄糖的吸收。 The chromium(III)-containing extract from Brewer's yeast first demonstrated to improve the phenomenon of impaired glucose tolerance, which was observed in rat fed with low-chromium diet and was defined as glucose tolerance factor (GTF). Traditionally the GTF activity was analyzed by using the epididymal adipocyte of Sprague-Dawley rat fed low-chromium diet (SD model). The restricts on animal source and time-consuming on feeding for SD model. The aims of this research are to establish the analytical model of the differentiated 3T3-L1 cells(3T3-L1 model) for measuring the GTF activity of yeasts from various source and to investigate the cultural conditions for the production of GTF from yeasts. Among 70 strains, including 58 strains isolated from winery, grape and vineyard and 12 strains from Culture Collection Research Center (CCRC), the strains of Saccharomyces cerevisiae No.1 and Saccharomyces sp. No. 54 showed the highest GTF activity analyzed by both SD model and 3T3-L1 model. The optimal concentration of insulin used in these 2 models is 10 nM. The linear relation of dose-response curve is obtained between 0 ~ 1000 .mu.g/mL in SD model, and 0 ~ 250 .mu.g/mL in 3T3-L1 model. The medium of potato infusion broth (PIB) was chosen as the basal cultural medium for the GTF production, with optimal concentration (based on reducing sugar content) of 3.48 g/L for No.1 and 1.74 g/L for strain No. 54 isolated. The addition of 1% sucrose, 0.1% (NH4)2SO4 and 1 ppm nicotinic acid increased the GTF activity of both strains. However the addition of 0.1 ppm various metal elements tested decreased the GTF activity of No. 54 isolate. High shaking rate (150 rpm) raises the dissolved oxygen (DO) in medium, which favors the cell proliferation and GTF production . High inoculum (10/sup 5/ cells/mL) increases the GTF activity of yeasts. The highest amount of yeast dry weight was obtained at 20.degree.C incubation; while the GTF activity of yeast cultured at between 20 ~ 30.degree.C was similar. GTF demonstrated to be a secondary metabolite, which is mainly produced in the stationary phase of yeast. At 20.degree.C, the incubation time of 4 days at least needed before the yeasts have enough GTF activity. The GTF activities for No.1 and No. 54 isolate were 94.17% and 145.78%, respectively, after incubation at 20.degree.C for 8 day. After feeding the STZ-induced hyperglycemia mice with the yeast powder of strain No.1 and No.54 for 42 days, the concentration of blood sugar was decreased from 310 mg/dL for the control group to 135 mg/dL for the experiment groups. After column chromatography and SDS-PAGE analysis, the GTF from strain No.54 was demonstrated to be a protein with a molecular mass of 28~44 kDa. This protein does not contain chromium. The protein has the highest GTF activity at neutral pH and its GTF activity decreased with the increasing temperature from 40.degree.C to 80.degree.C. The proposed mechanism of the GTF from strain No.54 maybe due to the activation of a membrane phosphotyrosine phosphatase (PTP), which then activates the p38 MAPK via p21ras pathway and finally the transporter of GLUT4 is activated to increase the transportation of glucose in cell.

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Production and Mechanism of Glucose Tolerance Factor of Yeast (III)

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