番荔枝分子分類、乳酸菌發酵飲品開發及果酸酯酶抑制劑之研究

Molecular classification, development of lactic-acid fermented product and pectinesterase inhibitor investigation of sugar apple.

田永彥(Yung-Yen Tien)

指導教授 ： 徐源泰

共同指導教授 ： 吳瑞碧(Swi-Bea Wu)

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01608

摘要

番荔枝為台灣省重要的經濟果樹之一。本研究之第一部份係以十三種台灣有種植的番荔枝品種(系)為材料，以 RAPD 及各種不同核酸序列分析，找尋可鑑定不同品種(系)之番荔枝的分子分類方法。對台灣栽培之十三種番荔枝品種(系)之核糖體 DNA 上 SSU 區域之序列進行分析，發現所得序列大致相同，無法將十三種番荔枝分類。而利用葉綠體 DNA 上 matK 區域序列比對結果可得知台灣栽培之十三種番荔枝之親緣關係，核酸序列以軟體進行 UPGMA、NJ、PARS及ML樹狀親緣關係圖的分析，均大致相同，將各個種分開，但同種則無法鑑別。在利用 RAPD 針對台灣所栽培之番荔枝作鑑別分析，發現確實能清楚鑑別台灣栽培之十三種番荔枝，而且各個品種(系)均能鑑別。
　　番荔枝多以鮮食的型態消費，在近年有產量過剩的趨勢。第二部份研究之目的即以盛產的番荔枝打成果泥後，利用不同的乳酸菌以單一或混合的組合進行乳酸發酵，乳酸發酵後具有特殊之香氣和質地。番荔枝果泥具有高可溶性固形物（20.5 ± 4.33 °Brix），極適合作為發酵的基質。乳酸發酵後經由對其食品組成分和抗氧化特性進行分析顯示，其清除 DPPH 之能力，由 92% 降至 78%。發酵果泥和鮮果汁以 2:8 的比例調配的成品具有高 DPPH 清除能力（88%）和鐵離子螯合活性（49%），顯示其具有發展成為健康食品且有助解決本省番荔枝生產過剩之問題。
　　番荔枝是一種很典型的的更年期水果，果實質地會隨著後熟作用的進行而軟化，其中最主要先經過果膠酯酶(pectinesterase，PE)作用，再經聚乳半糖醛酸酶(polygalacturonase，PG)及纖維素酶(cellulase)的作用達到軟化的目的。PE在室溫貯存下於第7天達到最高活性，而PEI活性趨勢則與PE活性相反，與日遞減。已有多位學者發現，在數種蔬果植物體中具有抑制果膠酯酶的物質存在，稱之果膠酯酶抑制劑(pectinesterase inhibitor，PEI)。第三部份實驗以 Sepharose 4B CN-PE 親合性膠體來純化番荔枝中的PEI，並搭配西方免疫及免疫染色。可顯示出一條PEI的專一性蛋白質色帶，其分子量 66 KDa。
關鍵字: SSU區域、matK區域、DPPH清除、乳酸桿菌屬、果膠酯酶、果膠酯酶抑制劑、親和性層析

關鍵字

SSU區域 ； matK區域 ； DPPH清除 ； 乳酸桿菌屬 ； 果膠酯酶果膠酯酶抑制劑 ； 親和性層析

並列摘要

Annona squamosa L. (Sugar apple) is an economic important fruit tree in Taiwan. In the first part of this research, a total 13 strains of Annonaceae plants in Taiwan were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of SSU gene region in rDNA, matK(maturase K) gene region in cpDNA, and RAPD(random amplified polymorphic DNA).The SSU(small subunit) region of ribosomal DNA was used to estimate molecular diversity of 13 Annona species cultivated in Taiwan. There were no discrimination due to the sequences of 13 Annona species have high similarity.Analysis of matK region of chloroplast DNA tells us the phenetic relationship between 13 Annona species. Same results of phonetic similarity dendrogram were obtained by UPGMA(unweighted pair group method with arithmetic average)、NJ(neighbor-joining method)、PARS(parsimony) and ML(maximun likelihood). The interspecies of 13 Annona species were distinct but the intraspecies were not. However, a cluster analysis based on RAPD markers was performed to build a phonetic similarity dendrogram. It did confirmed the discrimination of 13 Annona species.
Sugar apple is a fruit primarily consumed fresh and overproduced in recent years.In second part presents an exploration of lactic-fermented sugar apple products. The product has a unique sweet aroma and a test panel found its texture to be appealing. For this study, sugar apple puree was used as the substrate for fermentation using mixed starters. The high total soluble solid nature (20.5 ± 4.33 °Brix) of sugar apple puree as compared with that of mixed fruit juice (lower than 10 °Brix) indicates its potential to be used in fermentation. Following fermentation, the properties and effects of different starter inoculations were recorded and discussed. Fermentation achieved a pH value of 3.8 after 60 hr. The performance of fermented product in the DPPH (α,α-diphenyl-β- picrylhydrazyl) decreased from 92% to 78% after 48 hrs, followed by a stationary state. Fermented sugar apple puree and fresh sugar apple juice blended in a ratio of 2:8 delivered the highest DPPH scavenging efficiency (88%) and iron chelating ability (49%). The relatively high values of these properties offer the potential for sugar apple juice to be further developed as a novel functional food. Such a development would surely help to ease recent overproduction problems.
Annona squamosa L. is a very typical climacteric fruit. It softens during ripening due to the first pectinesterase (PE) reaction and the subsequent polygalacturonase (PG) and cellulose actions. PE activity reached a maximum on the 7th day during storage at room temperature, but PEI activity decreased with the increasing storage days. Many substances exhibiting PE inhibitory activity, PE inhibitor (PEI), in fruits and vegetables have been reported. In the third part research, isolation and purification of Annona squamosa L PEI using Sepharose 4B CN-PE affinity gel chromatographics, and collocating Western blotting analysis. We acquire one single protein band with a molecular weight is 66 KDa.
Key words: SSU region, matK region, DPPH scavenging, Lactobacillus, pectinesterase, pectinesterase inhibitor, affinity chromatography

並列關鍵字

SSU region ； matK region ； DPPH scavenging ； Lactobacillus ； pectinesterase ； pectinesterase inhibitor ； affinity chromatography

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