Defatted rice bran is an undervalued agricultural by-product which can be hydrolyzed by dilute acids not only to release carbon sources from starch and lignocellulose but also enough nitrogen sources for growing microorganisms. Detoxification of the defatted rice bran hydrolysate is required to reduce the level of inhibitors in the fermentation medium. Detoxified DRBH was found to be an effective medium for cultivating Y. lipolytica Po1g, and high cellular lipid content was obtained. The composition of the neutral lipid obtained is similar to those of most staple vegetable oils indicating its potentials for biodiesel production.
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[33] S. E. Karatay and G. D¨onmez, Improving the lipid accumulation properties of the yeast cells for biodiesel production using molasses, Bioresource Technology, 101(20), pp. 7988–7990, 2010.
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124–130, 2003.
國科會補助專題研究計畫成果報告自評表
請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價 值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性） 、是否適 合在學術期刊發表或申請專利、主要發現或其他有關價值等，作一綜合評估。
1. 請就研究內容與原計畫相符程度、達成預期目標情況作一綜合評估
▓達成目標
□ 未達成目標（請說明，以 100 字為限）
□ 實驗失敗
□ 因故實驗中斷
□ 其他原因 說明：
2. 研究成果在學術期刊發表或申請專利等情形：
論文：▓已發表 □未發表之文稿 □撰寫中 □無 專利：□已獲得 □申請中 ▓無
技轉：□已技轉 □洽談中 ▓無
其他：（以 100 字為限）
3. 請依學術成就、技術創新、社會影響等方面，評估研究成果之學術或應用價 值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）（以 500 字為限）
本計畫利用農業廢棄物米糠為主要營養源來培養解脂耶氏酵母菌(Yarrowia lipolytica)，利 用其所含之油脂以生產生質柴油。先將米糠及甘蔗渣水解，在去除有水解液中之有害物 後，利用硫酸(1–4% v/v)在不同溫度(60–120◦C)下水解一段時間(1–8 h)後，以此水解液 作為主要營養源以培養解脂耶氏酵母菌，目標是達到最大之中性脂質產率。發現產生最大
總醣量之最佳水解條件為以3% 硫酸在 90◦C 下水解 6 小時。葡萄糖為主要所含之糖(43.20
± 0.28 g/L)，其次是木糖(4.93 ± 0.03 g/L)，再其次為阿拉伯糖(2.09 ± 0.01 g/L)。水解液經去
除有害物如醣醛及HMF 後用做培養解脂耶氏酵母菌之培養介質。在最佳條件下可得乾乾
菌重及脂質含量分別為10.75 g/L 及 48.02%。
國科會補助計畫衍生研發成果推廣資料表
日期： 年 月 日
國科會補助計畫
計 畫 名 稱 ： 利 用 農 業 廢 棄 物 水 解 液 作 為 媒 介 培 養 Yarrowia lipolytica Po1g 生產生質柴油
計畫主持人：朱義旭
計畫編號：NSC 101-2221-E-011 -101 領域：再生能源
（中文）
研發成果名稱
（英文）
成果歸屬機構 發明人
(創作人)
技術說明
（中文）
（200-500 字）
（英文）
產業別
技術/產品應用範圍
技術移轉可行性及預期 效益
註：本項研發成果若尚未申請專利，請勿揭露可申請專利之主要內容。
國科會補助專題研究計畫移地研究心得報告
日期： 年 月 日
計畫編號 NSC － － － －
計畫名稱 出國人員
姓名
服務機構 及職稱 出國時間 年 月 日至
年 月 日 出國地點
一、移地研究過程
二、研究成果
三、建議
四、其他
國科會補助專題研究計畫出席國際學術會議心得報告
日期： 年 月 日
一、參加會議經過
The registration started at 08:00 on 2012/11/07. My advisor, My PhD student, Do Quy Diem, and I arrived at the venue, Inna Hotel, Legion, Bali at 08:30. There was an opening ceremony at 09:00 followed by two sessions of keynote speakers (One from Ministry of Research and Technology, Indonesia; and the other from Ministry of Trade, Indonesia) and one session of Keynote speaker (from Japan). This ended the morning sessions. After the lunch,the conference resumed by two keynote sessions (one keynote speaker from Australia and the other from Taiwan). The rest of the afternoon session was devoted to 5 parallel sessions for oral presentation. The oral presentation ended at 17:20, followed by a banquet at 19:00.
The next day began with talks by two plenary speakers, both of them from Indonesia, which ended at 09:20. The rest of the second was all devoted to 7 parallel sessions for oral presentation. I had my oral presentation in Session IV, my topic of presentation is “Isolation and characterization of starch from Vietnamese Limnophila aromatica”. All sessions ended at 16:45, followed by a closing ceremony.
二、與會心得
計畫編號 NSC 101-2221-E-011 -101 － － － －
計畫名稱 利用農業廢棄物水解液作為媒介培養 Yarrowia lipolytica Po1g 生產 生質柴油
(英文)19
thRegional Symposium on Chemical Engineering
發表題目
(中文)
(英文) Isolation and Physicochemical Properties of Starches from
Vietnamese Limnophila aromatica
區域化工會議(Regional Symposium on Chemical Engineering)為東南亞地區最重
要國際性化工會議之一，每年舉辦一次，輪流由東協會員國主辦。今年由印尼泗水
理工學院(Institut Teknologi Sepuluh Novemper)主辦，地點在風光明媚之 Bali 島 Kuta
市。由於 ITS 化工系與臺灣科技大學化工系有合作關係(雙學位學程)，因此在系主
任帶領下，本系老師及學生共約十人組團前往參加。除東協成員國主要大學化工系
之外，日本、韓國、澳洲及歐美地區也都有代表參加，有機會與這些國家(特別是東
南亞地區)代表交流，是難得之經驗。
三、發表論文全文或摘要
Isolation and Physicochemical Properties of Starches from Vietnamese Limnophila aromatica
Quy Diem Doa, Lien Huong Huynhb and Yi-Hsu Jua
a Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Sec.4, Keelung Road, Taipei 106-07, Taiwan.
b Department of Chemical Engineering, Can Tho University, 3-2 Street, Can Tho City, Vietnam
Abstract
Starch serves as the major source of polysaccharide in plants that provides the bulk nutrient and energy source in human diet. In this study, starch was isolated from L. aromatica by using wet-milling method.
The purity of the starch product was 84.76 % and 71.12% of the total starch was the resistant starch. The amylopectin content of the starch was 88.13%. Starch particle diameter was found to be 6.22 to 110 μm.
Furthermore, physicochemical properties such as swelling power, pasting, gelatinization, retrogradation were determined. The results indicate that starch from L. aromatica possesses good physicochemical properties and can be considered as a prospective renewable raw material in food, phamarceutical and polymer industries with potential to compete with current feedstock such as potato and maize.
Keywords: Limnophila aromatica; starch; resistant starch, renewable material; isolation;
physicochemical properties
1. Introduction
Starch has been known as a biocompatible, biodegradable, non-toxic, eco-friendly and cheap polymer [Rodrigues and Emeje, 2012]. Recently, starch has been extensively used in medical applications. For example as a drug carrier, as hydrogels and partially-degradable bone cements, as materials for bone
replacement/fixation or fillers for bone defects, as scaffolds in tissue engineering of bone and cartilage [Balmayor, et al., 2009]. Thus there is need to search for new cheap source of starch that can replace starch from conventional food source such as corn and rice.
Limnophila aromatica (Lamk.) Merr. (syn. Limnophila grastissima Blume) is belong to Scrophulariaceae (figwort family, snapdragon family) family. Limnophila aromatica is a common plant in Southeast Asia where it is used as spice and medicinal herb. It has been used to treat kidney stone, painful cramp, wound care and ulcer [Do, 1999]. After the removal of lipid, phenolics and flavonoids, the use of the extraction residue has never been reported. In this study, starch was isolated from the extraction residue of L. aromatica by wet milling method. Physicochemical, thermal and structural properties of the isolated starch were investigated.
2. Experimental 2.1 Materials
n-Hexane (95% purity) and ethanol (95% purity) were purchased from Tedia (OH, USA) and Echo Chemical (Miao Li, Taiwan), respectively. The following chemicals were obtained from Sigma–Aldrich (St.
Louis, MO): glucose standard, amylose standard (from potato), α-amylase (EC 3.2.1.1), protease (EC 3.4.23.18) and amyloglucosidase (EC 3.2.1.3). L. aromatica was obtained from a Vietnamese store in Taiwan. The dried sample was ground and passed through a 60-mesh sieve. The powder was then defatted using n-hexane as the solvent. The solid residue was collected and dried. This dried residue (DFF) was extracted first using ethanol (6 h) and then 75% aqueous ethanol (6 h) to remove phenolic compounds with a DFF to solvent ratio of 1:20 (g/ml). After phenolic extraction, the residue DFF obtained (hereafter referred to as DFF) was air-dried and stored for further analyses.
2.2 Methods
DFF and isolated starch were monitored for their total starch and resistant starch contents. The modified method of AOAC Official Method 996.11 [1996] was used to analyze total starch content. DNS (3,5-dinitrosalicylic acid) was used for quantifying starch instead of the glucose oxidase–peroxidase–aminoantipyrine buffer mentioned in the official method.
Resistant starch analysis was carried out according to the method of Goñi, et al. [1996] with slight modification. Protein and ash contents were determined by AOCS Official Method Ba 4a-38 [1997] and AOCS Official Method Ba 5a-49 [1997], respectively. Total dietary fiber (TDF) was analyzed by using the modified method of AOAC Officical Method 985.29 [1990]. Amylose content in the isolated starch was
determined by using the colorimetrical method of Sadasivam and Manickam [1996]. Swelling power of the isolated starch was determined by using the 40 mg test of Konik-Rose, et al. [2001] with modifications. For determining starch solubility, the whole supernatant was put in a pre-weighed micro-tube (1.5 ml) and freeze dried. The dried soluble starch was weighed and the solubility of starch was calculated as ratio of the weight of soluble starch to the initial dry starch weight. Pasting profile of starch was analyzed by using a Rheometer Anton Paar model MCR. Gelatinization temperature and enthalpy of starch were determined by using a differential scanning calorimeter (DSC Jade, Perkin Elmer, Japan) according to the method of Wang, et al. [2011]. Morphology of the starch sample was characterized by using a Cambridge SEM S-360 with an accelerating voltage of 20 kV.
3. Results and Discussion
3.1 Composition of DFF and isolated starch
Total starch, resistant starch, ash, protein, TDF and lipid contents of the DFF and the isolated starch as well as the amylose content of the isolated starch were presented in Table 1. DFF has high starch content (51.77 wt %), resistant starch (34.83 wt.%) and TDF content (23.37% wt). These initial analyses pointed out that DFF can be considered as a new source of starch. As shown in Table 1, 71.22% of the isolated starch is the resistant starch. Considerable amount of total dietary fiber was detected in the isolated starch as compared to starch from potato, maize and other cereals. The amylopectin content of the isolated starch was 88.13%.
3.2 Swelling and solubility
The swelling power of starch increases from 50 oC (7.84 g/g) to 70 oC (9.16 g/g) where the swelling power reached maximum, and then gradually decreased to 8.62 g/g at 92.5 oC. The starch solubility increased from 1.13 wt % at 50 oC to 1.88 wt % at 92.25 oC. Generally, DFF starch exhibits lower swelling power and solubility compare with some tuber and root starches [Hoover, 2001].
3.3 Pasting properties
Pasting profile of starch is a starch property which usually is determined by heating starch in the presence of moisture under shear, followed by cooling. During heating, starch granules are gelatinized, losing their crystallinity and structural organization, while in the cooling step, the disaggregated starch molecules first form gel and then retrograde gradually into semicrystalline aggregates that differ from the native granules. Pasting profile of the DFF starch was monitored by using Rheometer and the result is presented in Figure 1. The viscosity of starch increased slightly when temperature was raised from 50 oC (15.4 cP) to 95 oC (28.3 cP). As temperature started to decrease, the viscosity increased rapidly until it reached a final value of 926 cP. This indicates retrogradation of the DFF starch happened immediately as temperature was decreased.
3.4 Thermal analysis
DSC was used to analyze thermal properties of the DFF starch in this work. Gelatinization and retrogradation of the DFF starch were determined with original moisture content (5.18 wt.%) and DFF starch mixed with DI water (1:4, w/w) and the results are shown in Table 2. The heat flow profile of gelatinization of the DFF starch mixed with water has two enthothermic peaks (131.01 o C and 164.24 oC) with corresponding gelatinization enthalpies of 2.83 J/g and 1383.73 J/g, respectively. DFF starch with original moisture has three enthothermic peaks at 140.01 oC, 148 oC and 182 oC with corresponding gelatinization enthalpies of 1.5 J/g, 12.61 J/g and 79.47 J/g, respectively. These transition temperatures are higher than 100 o C. According to Flipse, et al. [1996], amylopectin plays major role in starch granule crystallinity. Higher amylopectin content resulted in an increase in structural stability, resistance towards gelatinization and energy for starting starch gelatinization, thus leads to an increase in transition temperature and enthalpy of gelatinization Barichello, et al. [1990]. This result is in agreement with the high content of amylopectin (88.13 wt.%) found in the DFF starch of this study.
The retrograded starch was obtained by storing the gelatinized starch for 7 days at 4 oC. The heat flow profile of gelatinization process of the retrograded starch that contains no water has one endothermic peak at 139.25 oC with a retrogradation enthalpy of 18.21 J/g. The heat profile of the retrograded starch that contains 75 wt.% water has two endothermic peaks at 42.78 o C and 124.50 oC with corresponding retrogradation enthalpy of 4.74 J/g and 20.35 J/g, respectively. The retrogradation temperature and enthalpy are lower than those of the gelatinization (main peak).
3.5 Morphology
Morphology of the DFF starch was obtained by using the scanning electron micrograph (SEM). Fig. 2 shows the shape and size of the DFF starch granules. The starch granule sizes range from about 6 to 110 μm. Starch granule size plays an important role in affecting the physical properties of starch, especially its tensile strength. Tensile strength increases as the particle size decreases [Moura, 2006]. The DFF starch granules are irregular with rough and multiple-layer surface. This may be caused by twice milling (one dry,
one wet) during starch extraction. Rough and cracked surface of granules was
also observed in corn and barley starch after ball-milling [Stark and Yin, 1986, Tester, et al., 1994].
4. Conclusion
DFF starch was isolated from L. aromatica by using the wet-milling process. The DFF starch product contained 84.76% starch, 0.67% protein, 10.67% total dietary fiber and 3.66% ash. The resistant starch content of the DFF starch is 71.12%. The amylopectin content of the DFF starch is 83.13%. The starch grains are irregular, rough and have cracks on surfaces with sizes from 6 to 110 μm. Two peaks (at 131.01
oC and 2.83 J/g, 164.24 oC and 1383.73 J/g) were observed during gelatinization analysis (starch:water
=1:4 w/w), and three peaks (140.01 oC and 1.50J/g, 148.57 oC and 12.14J/g, 182.14 oC and 79.47 J/g) were observed during gelatinization analysis of starch with native moisture. The retrogradation of starch (starch:water = 1:4, w/w) happened at 124.05 oC with an enthalpy of 11.9 J/g, 84.07% lower than that of gelatinization and the retrogradation of starch with native moisture happens at 139.25 oC with an enthalpy of 12.19 J/g, 99.12% lower than that of gelatinization. Major degradation of DFF starch occurred at 319 oC.
Due to high total dietary fiber and resistant starch contents in the isolated starch, this starch can be a new source of health food. The DFF starch also possesses low swelling power and low retrogradation and maybe suitable for application in pharmaceutical industry as diluents, adhesive agents or distintegrating agents, and as carrier for drug delivery.
Acknowledgement(s)
This work was supported by a grant (NSC101-2811-E-011 -001 -ET) provided by the National Science council of Taiwan.
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