TW201833327A - Method for increasing fructosylated mangiferin content in plant extract wherein a bioconversion reaction is performed in the presence of a substance having the activity of fructosidase - Google Patents
Method for increasing fructosylated mangiferin content in plant extract wherein a bioconversion reaction is performed in the presence of a substance having the activity of fructosidase Download PDFInfo
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Abstract
Description
本發明係關於一種使用保藏號為BCRC 11697乳酸菌(Lactobacillus plantarum)將芒果皮萃取液中的果糖基化芒果苷含量增加的一種方法,以及一種以保藏號為BCRC 11697乳酸菌(Lactobacillus plantarum)進行芒果苷和糖基供體的生物轉化反應生成果糖基化芒果苷的用途。 The present invention relates to a method for increasing the content of fructosylated mangiferin in a mango peel extract using a deposit number BCRC 11697 ( Lactobacillus plantarum ), and a mangiferin using a deposit number BCRC 11697 lactic acid bacteria ( Lactobacillus plantarum ). Use of a biotransformation reaction with a glycosyl donor to produce fructosylated mangiferin.
芒果苷是一種四羥基吡酮的碳酮苷,屬雙苯吡酮類黃酮類化合物,在多種植物中存在,如芒果樹、扁桃樹、東北龍膽及知母等。芒果苷分子式:C19H18O11,分子量:422.3。芒果苷具有多種生物活性和藥理作用,如抗氧化、抗腫瘤、免疫調節、抗糖尿、抗炎等(鄧家剛,蕈驪蘭.長春中醫藥大學學報,2008,24(4):463-464.)。 Mangiferin is a carbanoside of tetrahydroxypyridone, which belongs to flavonoid flavonoids. It exists in a variety of plants, such as mango tree, almond tree, northeast gentian and timidum. Mangiferin molecular formula: C 19 H 18 O 11 , molecular weight: 422.3. Mangiferin has a variety of biological activities and pharmacological effects, such as anti-oxidation, anti-tumor, immunoregulation, anti-diabetes, anti-inflammatory, etc. (Deng Jiagang, Yulan. Journal of Changchun University of Traditional Chinese Medicine, 2008, 24 (4): 463-464. ).
芒果苷雖具有廣泛藥理活性,但其溶解度很差,嚴重限制了其製劑的開發,為方便應用,需要改善芒果苷溶解性和生物利用度。 Although mangiferin has a wide range of pharmacological activities, its solubility is very poor, which severely limits the development of its preparations. In order to facilitate the application, the solubility and bioavailability of mangiferin need to be improved.
對於複雜結構的天然活性成分來說,利用化學合成來進行結構修飾存在著產率低、反應專一性差、副產物多 等缺點,特別是有些反應目前利用化學手段較難實現,生物轉化技術卻可彌補化學合成的不足。Cantagrel等(公開號:FR 2882762 A1)以來源於兩株腸膜狀明串珠菌的葡萄糖基轉移酶,對芒果苷進行糖基化修飾,在芒果苷為糖基受體,蔗糖為糖基供體時,得到葡萄糖基-β-(1,6)-芒果苷(CASRN:908570-23-0)。當Leuconostoc mesenteroides NRRL B-1299菌株作為生物催化劑,在0.4g/L芒果苷,40g/L蔗糖條件下,糖基化反應收率為25%;當Leuconostoc mesenteroides NRRL 521-F菌株作為生物催化劑,在0.4g/L芒果苷,40g/L蔗糖條件下,糖基化反應收率為28%。 For natural active ingredients with complex structures, chemical synthesis for structural modification has the disadvantages of low yield, poor reaction specificity, and many by-products. In particular, some reactions are currently difficult to achieve using chemical means, but biotransformation technology can Make up for the lack of chemical synthesis. Cantagrel et al. (Publication number: FR 2882762 A1) glycosylated the mangiferin with a glucosyltransferase derived from two strains of Leuconostoc mesenteroides, where mangiferin is a glycosyl acceptor and sucrose is a glycosyl In vivo, glucosyl-β- (1,6) -mangiferin (CASRN: 908570-23-0) was obtained. When Leuconostoc mesenteroides NRRL B-1299 strain was used as a biocatalyst, the yield of glycosylation reaction was 25% under the conditions of 0.4g / L mangiferin and 40g / L sucrose; when Leuconostoc mesenteroides NRRL 521-F strain was used as a biocatalyst, Under the condition of 0.4g / L mangiferin and 40g / L sucrose, the yield of glycosylation reaction was 28%.
中國專利申請案公開號CN 102863484 A揭示了一種果糖基化芒果苷的製備方法,該方法包括將具有果糖基化酶活性的物質加入含有芒果苷的轉化液中,進行生物轉化反應,使芒果苷轉化為果糖基化芒果苷,所述轉化液中含有芒果苷和糖基供體,該具有果糖基化酶活性的物質為來源自煙草節桿菌,其保藏號為CCTCC M2010164,的發酵液。 Chinese Patent Application Publication No. CN 102863484 A discloses a method for preparing fructosylized mangiferin, which method comprises adding a substance having fructosylase activity to a transformation solution containing mangiferin, and carrying out a biotransformation reaction to make mangiferin It is transformed into fructosylized mangiferin, and the transformed solution contains mangiferin and a glycosyl donor. The substance having fructosylase activity is a fermentation broth derived from Arthrobacter nicotianae and its deposit number is CCTCC M2010164.
在前述法國公開號FR 2882762 A1及中國公開號CN 102863484 A的專利申請案中均使用了純化的芒果苷來進行芒果苷的果糖基化。 In the aforementioned French Patent Application No. FR 2882762 A1 and Chinese Patent Application No. CN 102863484 A, purified mangiferin was used for fructosylation of mangiferin.
本發明的一主要目的是提供一種直接以含有芒果苷的植物萃取液作為基質進行生物轉化反應,使該芒果苷轉化為果糖基化芒果苷的方法。 A main object of the present invention is to provide a method for directly carrying out a biotransformation reaction using a plant extract containing mangiferin as a substrate to convert the mangiferin into fructosylized mangiferin.
本發明的另一目的是提供一種合適的微生物,來進行直接以含有芒果苷的植物萃取液作為基質的生物轉化反應,使該芒果苷轉化為果糖基化芒果苷。 Another object of the present invention is to provide a suitable microorganism for carrying out a biotransformation reaction directly using a mangiferin-containing plant extract as a substrate to convert the mangiferin into fructosylated mangiferin.
本發明的又一目的是提供一種合適的微生物及方法,將芒果皮萃取液中的果糖基化芒果苷的含量提昇。 Another object of the present invention is to provide a suitable microorganism and method for increasing the content of fructosylated mangiferin in the extract of mango skin.
本發明的一種增加植物萃取液中的果糖基化芒果苷含量的方法,包含下列步驟:提供一含有芒果苷的植物萃取液;及將一糖基供體加入該植物萃取液中,於一具有果糖基化酶活性的物質的存在下進行生物轉化反應,使該芒果苷轉化為果糖基化芒果苷,其中該具有果糖基化酶活性的物質係通過保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的發酵而產生。 A method for increasing the content of fructosylized mangiferin in a plant extract according to the present invention includes the following steps: providing a plant extract containing mangiferin; and adding a sugar-based donor to the plant extract, A biotransformation reaction is performed in the presence of a substance having fructosylase activity to convert the mangiferin to fructosylizing mangiferin, wherein the substance having fructosylase activity is passed through a lactic acid bacteria ( Lactobacillus plantarum ) having the deposit number BCRC 11697 Produced by fermentation.
較佳的,該植物萃取液為芒果皮萃取液。 Preferably, the plant extract is a mango peel extract.
較佳的,該糖基供體為蔗糖或葡萄糖。更佳的,該糖基供體為蔗糖。 Preferably, the glycosyl donor is sucrose or glucose. More preferably, the glycosyl donor is sucrose.
較佳的,該保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)被加入於該植物萃取液中。 Preferably, the lactic acid bacteria ( Lactobacillus plantarum ) with the accession number of BCRC 11697 is added to the plant extract.
較佳的,該保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的一發酵液被加入於該植物萃取液中。 Preferably, a fermentation broth of the Lactobacillus plantarum deposited under the accession number BCRC 11697 is added to the plant extract.
較佳的,該保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的一發酵上清液被加入於該植物萃取液中。 Preferably, a fermentation supernatant of the lactic acid bacteria ( Lactobacillus plantarum ) deposited under the accession number BCRC 11697 is added to the plant extract.
較佳的,純化自該保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的一發酵上清液的果糖基化酶被加入於該植物萃取液中。 Preferably, a fructosylase from a fermentation supernatant of the Lactobacillus plantarum deposited as BCRC 11697 is added to the plant extract.
較佳的,該生物轉化反應係於一乳酸菌培養基中、20-40℃及不攪拌或攪拌速度不大於400rpm下進行0.5至96小時,且以100mL的該培養基為基準,該糖基供體的用量為1-20克,該植物萃取液的用量為1-60mL,及該乳酸菌的用量為105~107CFU/mL。 Preferably, the biotransformation reaction is performed in a lactic acid bacteria culture medium at 20-40 ° C. for 0.5 to 96 hours without stirring or stirring speed not more than 400 rpm, and based on 100 mL of the culture medium, the glycosyl donor in an amount of 1-20 g, the amount of the extract of the plant is 1-60mL, and the amount of the lactic acid bacterium is 10 5 ~ 10 7 CFU / mL .
本發明提供一種使用保藏號為BCRC 11697乳酸菌(Lactobacillus plantarum)進行芒果苷和糖基供體的生物轉化反應生成果糖基化芒果苷的用途。 The invention provides the use of a lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for carrying out a biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin.
本發明提供一種使用保藏號為BCRC 11697乳酸菌(Lactobacillus plantarum)的一發酵液進行芒果苷和糖基供體的生物轉化反應生成果糖基化芒果苷的用途。 The invention provides the use of a fermentation broth with a deposit number of BCRC 11697 ( Lactobacillus plantarum ) for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin.
本發明提供一種使用保藏號為BCRC 11697乳酸菌(Lactobacillus plantarum)的一發酵上清液進行芒果苷和糖基供體的生物轉化反應生成果糖基化芒果苷的用途。 The invention provides the use of a fermentation supernatant of lactic acid bacteria ( Lactobacillus plantarum ) with a deposit number of BCRC 11697 for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylated mangiferin.
本發明提供一種使用純化自該保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的一發酵上清液的果糖基化酶進行芒果苷和糖基供體的生物轉化反應生成果糖基化芒果苷的用途。 The present invention provides the use of a fructosylase from a fermentation supernatant of a lactic acid bacterium ( Lactobacillus plantarum ) deposited under the deposit number BCRC 11697 for the biotransformation reaction of mangiferin and a glycosyl donor to produce fructosylized mangiferin. .
圖1為果糖基化芒果苷標準品在不同波長下之紫外光吸收光譜。 Figure 1 shows the ultraviolet absorption spectra of fructosylated mangiferin standards at different wavelengths.
圖2為依本發明方法以Lactobacillus plantarum乳酸菌轉化後離心去除菌體後的上清液在不同波長下之紫外光吸收光譜。 FIG. 2 is an ultraviolet light absorption spectrum of a supernatant liquid after centrifugal removal of lactic acid bacteria by Lactobacillus plantarum lactic acid bacteria transformation according to the present invention at different wavelengths.
圖3為果糖基化芒果苷標準品的液相層析質譜(LC-MS)圖譜。 Figure 3 is a liquid chromatography mass spectrometry (LC-MS) spectrum of a fructosylated mangiferin standard.
圖4為依本發明方法以Lactobacillus plantarum乳酸菌轉化後離心去除菌體後的上清液LC-MS圖譜。 FIG. 4 is a LC-MS spectrum of a supernatant liquid after centrifugal removal of Lactobacillus plantarum lactic acid bacteria according to the method of the present invention.
圖5顯示不同糖基供體對本發明菌株將芒果苷轉化成果糖基化芒果苷之影響。 Figure 5 shows the effect of different glycosyl donors on the conversion of mangiferin to the glycosylated mangiferin by the strain of the present invention.
圖6顯示不同用量(比例)的蔗糖糖基對本發明菌株將芒果苷轉化成果糖基化芒果苷之效率的比較結果。 FIG. 6 shows the comparison results of the sucrose glycosyl groups with different dosages (ratio) on the efficiency of glycosylated mangiferin converted from mangiferin by the strain of the present invention.
依本發明內容所完成的一較佳具體實施態樣包括一種將芒果皮萃取液中的果糖基化芒果苷含量增加的方法,主要包含將一糖基供體加入該芒果皮萃取液中,於一具有果糖基轉化酶活性的物質的存在下進行生物轉化反應,使該芒果皮萃取液中所含有的芒果苷轉化為果糖基化芒果苷,其中該具有果糖基轉化酶活性的物質係通過保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的發酵而產生。透過上述方法可以使芒果皮更有效率的被製備成果糖基化芒果苷,不僅解決芒果盛產時將芒果製成果汁或芒果乾或芒果果 肉罐頭時,芒果皮的出處,有利於減少環境汙染,也達到全果利用、提升芒果的經濟價值的目標。 A preferred embodiment according to the present invention includes a method for increasing the content of fructosylated mangiferin in a mango peel extract, which mainly comprises adding a sugar-based donor to the mango peel extract, and A biotransformation reaction is performed in the presence of a substance having fructosyl converting enzyme activity to convert the mangiferin contained in the mango skin extract to fructosylized mangoside, wherein the substance having fructosyl converting enzyme activity is deposited It is produced by fermentation of Lactobacillus plantarum BCRC 11697. Through the above method, the mango skin can be more efficiently prepared into glycosylated mangiferin, which not only solves the source of mango skin when mango is made into fruit juice or dried mango or canned mango flesh when mango is in full production, which is beneficial to reducing environmental pollution. It also achieves the goal of using the whole fruit and improving the economic value of mango.
一適合製備用於本發明的芒果皮萃取液的方法包括(但不限於)下列步驟:將芒果皮清洗及攪碎;接著以纖維酵素(Cellulase)於室溫至50℃進行均質處理60~180分鐘;於60℃烘乾去除水分,再粉碎成粉末;將該粉末以1:5~1:20g/mL的比例與一極性溶劑混合,且於室溫至50℃及較佳地於超音波振盪例如125~500W/L功率下,於50℃萃取60分鐘,移除該極性溶劑,可得到含芒果苷之果皮萃取液。 A method suitable for preparing a mango peel extract for use in the present invention includes (but is not limited to) the following steps: washing and pulverizing the mango peel; and then homogenizing the cellulase at room temperature to 50 ° C for 60-180 Minutes; drying at 60 ° C to remove moisture, and pulverizing into powder; mixing the powder with a polar solvent at a ratio of 1: 5 to 1: 20g / mL, and at room temperature to 50 ° C and preferably on ultrasonic With shaking, for example, at a power of 125 to 500 W / L, extraction is performed at 50 ° C. for 60 minutes, and the polar solvent is removed to obtain a peel extract containing mangiferin.
合適的極性溶劑例如水、C1-C3的醇及其等之混合液,較佳地為乙醇與水的混合,例如50%(v/v)乙醇。 Suitable polar solvents such as water, C1-C3 alcohols, and mixtures thereof are preferably a mixture of ethanol and water, such as 50% (v / v) ethanol.
前述芒果皮萃取液為本發明應用微生物轉化芒果苷的基質材料來源,本發明應用纖維酵素(Cellulase)或其它類似能分解植物細胞璧之酵素來促進細胞內物質之萃取效益。另外,萃取時輔以超音波振盪,能進一步提升芒果皮中類黃酮物質之萃取效果。本發明經過探討溶劑比例、超音波功率與萃取時間等參數,得到以50%(v/v)乙醇水溶液,超音波功率500W/L,於50℃萃取60分鐘,可得到較高含量之芒果多酚及富含芒果苷之芒果皮萃取液。於一較佳具體實施例中,經HPLC定量分析芒果皮萃取液中的果糖基化芒果苷含量為18μg/mL。 The aforementioned mango skin extract is a source of matrix material for transforming mangiferin by microorganisms according to the present invention, and the present invention uses cellulase or other similar enzymes capable of decomposing plant cell ridges to promote the extraction efficiency of intracellular materials. In addition, the extraction is supplemented by ultrasonic oscillation, which can further improve the extraction effect of flavonoids in mango skin. In the present invention, parameters such as the solvent ratio, ultrasonic power, and extraction time are studied to obtain a 50% (v / v) ethanol aqueous solution, ultrasonic power of 500 W / L, and extraction at 50 ° C for 60 minutes. A higher content of mango can be obtained. Mango peel extract rich in phenol and mangoside. In a preferred embodiment, the content of fructosylated mangiferin in the mango skin extract was quantitatively analyzed by HPLC as 18 μg / mL.
一適合用於本發明的糖基供體包括(但不限於)蔗糖、葡萄糖及果糖,以蔗糖或葡萄糖為較佳,而以蔗糖為 最佳。 A sugar-based donor suitable for use in the present invention includes, but is not limited to, sucrose, glucose, and fructose. Sucrose or glucose is preferred, and sucrose is most preferred.
較佳地,於本發明方法的生物轉化反應中該保藏號為BCRC 11697的乳酸菌(Lactobacillus plantarum)的一發酵上清液被加入於該芒果皮萃取液。一適合用於該BCRC 11697的乳酸菌的培養基配方包含:葡萄糖2~80g/L,蛋白腖5~50g/L,KH2PO4 0.4~4g/L,MgSO4.7H2O 0.05~1.0g/L,MnSO4 H2O 0.01~0.1g/L,pH 6~8的乳酸菌培養基。 Preferably, in the biotransformation reaction of the method of the present invention, a fermentation supernatant of the lactic acid bacteria ( Lactobacillus plantarum ) with the accession number of BCRC 11697 is added to the mango skin extract. A suitable medium formula for lactic acid bacteria used for the BCRC 11697 includes: glucose 2 ~ 80g / L, peptone 5 ~ 50g / L, KH 2 PO 4 0.4 ~ 4g / L, MgSO 4 . 7H 2 O 0.05 ~ 1.0g / L, MnSO 4 H 2 O 0.01 ~ 0.1g / L, lactic acid bacteria culture medium with pH 6 ~ 8.
本發明方法較佳地進一步包含將該生物轉化反應得到的果糖基化芒果苷含量增加的產物純化,例如藉由樹脂純化的手段得到其中的水相部份。該水相部份可以直接或濃縮後被使用作為抗氧化劑,添加於防曬乳等保養品或化妝品。選擇性,也可通過已知純化方法將果糖基化芒果苷從該液體部份分離出來而得到純化的果糖基化芒果苷。 The method of the present invention preferably further comprises purifying a product having an increased fructosylized mangiferin content obtained from the biotransformation reaction, for example, obtaining a water phase portion thereof by means of resin purification. This aqueous phase can be used directly or after concentration as an antioxidant, and it can be added to skin care products such as sunscreen or cosmetics. Alternatively, the fructosylized mangiferin can also be separated from the liquid portion by known purification methods to obtain a purified fructosylized mangiferin.
1.培養基配方及菌株培養 1. Medium formula and strain culture
(1)乳酸菌培養基(MRS培養基) (1) Lactic acid bacteria medium (MRS medium)
MRS培養基配方:依序加入1g月示蛋白腖(Proteose peptone)No.3、1g的牛肉汁(Beef extract)、0.5g的酵母菌萃取物(Yeast extract)、2g的葡萄糖(Dextrose)、0.1g Tween 80界面活性劑、0.2g檸檬酸銨(Ammonium citrate)、0.5g的CH3COONa、10mg的MgSO4.7H2O、 5mg MnSO4.H2O、0.2g K2HPO4,隨後補水使其體積達到100mL,調pH至6.5後進行高壓滅菌。菌株於培養基活化後,菌株接種量為培養基體積的10%。生長溫度為37℃、pH 6.5、靜置培養(0rpm)培養,菌液之OD600達到10時離心去除菌體,得到上清液。 MRS medium formulation: 1g Proteose peptone No. 3, 1g Beef extract, 0.5g Yeast extract, 2g glucose, 0.1g Tween 80 surfactant, 0.2 g of Ammonium citrate, 0.5 g of CH 3 COONa, 10 mg of MgSO 4 . 7H 2 O, 5mg MnSO 4 . H 2 O, 0.2 g K 2 HPO 4 , and then make up the volume to 100 mL with water, adjust the pH to 6.5 and then autoclave. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. The growth temperature was 37 ° C, pH 6.5, and the culture was allowed to stand (0 rpm). When the OD 600 of the bacterial solution reached 10, the bacterial cells were removed by centrifugation to obtain a supernatant.
(2)細菌培養基(Nutrient培養基) (2) Bacterial medium (Nutrient medium)
依序加入0.3g牛肉汁(Beef extract)、0.5g蛋白腖(peptone),隨後補水使其體積達到100mL,調pH至7.0後進行高壓滅菌。菌株於培養基活化後,菌株接種量為培養基體積的10%。生長溫度為26℃、pH 7.0、100rpm培養,菌液之OD600達到10時離心去除菌體,得到上清液。 Add 0.3 g of Beef extract and 0.5 g of peptone in sequence, then add water to make the volume reach 100 mL, adjust the pH to 7.0, and then autoclave. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. The growth temperature was 26 ° C., pH 7.0, and 100 rpm. When the OD 600 of the bacterial solution reached 10, the bacterial cells were removed by centrifugation to obtain a supernatant.
(3)酵母菌培養基(YPD培養基) (3) Yeast medium (YPD medium)
依序加入2g蛋白腖(peptone)、2g葡萄糖(Dextrose)和1g酵母菌萃取物(Yeast extract),隨後補水使其體積達到100mL後進行高壓滅菌。菌株於培養基活化後,菌株接種量為培養基體積的10%。生長溫度為25℃、pH 7.0、100rpm培養,菌液之OD600達到10時離心去除菌體,得到上清液。 2g of peptone, 2g of glucose (Dextrose), and 1g of yeast extract (Yeast extract) were sequentially added, and then the water was replenished to a volume of 100 mL and then autoclaved. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. The growth temperature was 25 ° C., pH 7.0, and 100 rpm. When the OD 600 of the bacterial solution reached 10, the bacterial cells were removed by centrifugation to obtain a supernatant.
(4)絲狀真菌培養基(麥芽萃取物) (4) Filamentous fungal medium (malt extract)
依序加入2.0g葡萄糖(Glucose)、1.0g蛋白腖(peptone)和2.0g麥芽汁(Malt extract),隨後補水使其體積達到100mL後進行高壓滅菌。菌株於培養基活化後,菌株接種量為培養基體積的10%。生長溫度為24-30 ℃、pH 7.0、100rpm培養,菌液之OD600達到10時離心去除菌體,得到上清液。 2.0 g of glucose (Glucose), 1.0 g of peptone, and 2.0 g of malt extract were sequentially added, followed by rehydration to make the volume reach 100 mL, and then autoclaving. After the strain was activated in the culture medium, the inoculation amount of the strain was 10% of the volume of the culture medium. When the growth temperature is 24-30 ° C, pH 7.0, and 100 rpm, when the OD 600 of the bacterial solution reaches 10, the bacterial cells are removed by centrifugation to obtain a supernatant.
2.芒果皮萃取液之製備 2. Preparation of Mango Peel Extract
將芒果皮洗淨攪碎,以2%(w/w)比例加入纖維酵素(Cellulase)於50℃進行水解反應180分鐘,後於60℃烘箱內烘乾去除水分,均質粉碎成芒果皮粉末後,進行超音波輔助萃取製程,萃取條件為50%(v/v)乙醇、料液比1:5、超音波功率500W/L、超音波頻率40Hz、萃取溫度50℃,萃取60分鐘後,於60℃真空烘箱去除乙醇,得到含芒果苷之果皮萃取液。 Wash and crush the mango skin, add cellulase at 2% (w / w), and hydrolyze at 50 ° C for 180 minutes, then dry it in an oven at 60 ° C to remove water, homogenize and pulverize it into mango skin powder The ultrasonic-assisted extraction process was carried out. The extraction conditions were 50% (v / v) ethanol, a material-liquid ratio of 1: 5, an ultrasonic power of 500 W / L, an ultrasonic frequency of 40 Hz, and an extraction temperature of 50 ° C. After extraction for 60 minutes, The ethanol was removed in a vacuum oven at 60 ° C to obtain a peel extract containing mangiferin.
3.果糖基化芒果生物轉化反應: 3. Biotransformation reaction of fructosylated mango:
將上述菌液、上述芒果皮萃取液、及一糖基供體的水溶液添加於水或磷酸鹽緩衝鹽水(PBS)中,於37℃進行生物轉化反應,使芒果苷轉化成為果糖基化芒果苷,反應時間為96小時。該菌液的添加為全部轉化反應液體積之10%,該芒果皮萃取液的添加量為全部轉化反應液體積之10%,而糖基供體的水溶液的添加量為每100mL的轉化反應液含0~20克的糖基(0~20 w/v %)。以高效液相層析法來分析果糖基化芒果苷含量。 The bacterial solution, the mango peel extract, and an aqueous solution of a glycosyl donor were added to water or phosphate buffered saline (PBS), and a bioconversion reaction was performed at 37 ° C to convert mangiferin to fructosylated mangiferin The reaction time is 96 hours. The bacterial solution was added at 10% of the total conversion reaction solution volume, the mango peel extract was added at 10% of the total conversion reaction solution volume, and the glycosyl donor aqueous solution was added at 100 mL of the conversion reaction solution. Contains 0 to 20 grams of glycosyl (0 to 20 w / v%). The content of fructosylated mangiferin was analyzed by high performance liquid chromatography.
除了不添加前述菌液外重覆上述生物轉化反應的步驟,而得到作為對照之用的一控制組的數據。 The above-mentioned steps of the biotransformation reaction are repeated except that the aforementioned bacterial solution is not added, and data of a control group used as a control is obtained.
4.果糖基化芒果苷之高效液相層析法(HPLC)定量 4. Quantification of fructosylated mangiferin by high performance liquid chromatography (HPLC)
精密稱取1mg果糖基化芒果苷(Fructosylated mangiferin)標準品,用50%乙醇溶解,製成1mL中含0.01~1mg的對照品溶液。測試樣品為轉化反應完成後的液體,以8000rpm離心5分鐘,取上清液,將1mL的樣品溶液,利用HPLC Atlantis T3管柱(C18,4.6mm內徑×250mm,5μm粒徑),移動相溶液為乙腈:1%冰醋酸溶液(15:85),反應溫度25℃,流速設定為1.0mL/min,偵測器波長設定為254nm,注射樣品體積為20μl。用針筒吸取出樣品,再以0.22um PVDF過濾後,做HPLC定量分析(郭伶伶、張諱、劉二偉、韓立峰、王濤。2013。芒果葉中芒果苷含量的測定。天津中醫藥大學學報,32(1):43-45)。 1 mg of fructosylated mangiferin standard was accurately weighed and dissolved with 50% ethanol to prepare a reference solution containing 0.01 to 1 mg in 1 mL. The test sample is the liquid after the completion of the conversion reaction. Centrifuge at 8000 rpm for 5 minutes, take the supernatant, and apply 1 mL of the sample solution to a HPLC Atlantis T3 column (C18, 4.6 mm inner diameter × 250 mm, 5 μm particle size). The solution was acetonitrile: 1% glacial acetic acid solution (15:85), the reaction temperature was 25 ° C, the flow rate was set to 1.0 mL / min, the detector wavelength was set to 254 nm, and the injection sample volume was 20 μl. Aspirate the sample with a syringe and filter it through 0.22um PVDF for quantitative HPLC analysis (Guo Lingling, Zhang Tab, Liu Erwei, Han Lifeng, Wang Tao. 2013. Determination of mangiferin content in mango leaves. Journal of Tianjin University of Traditional Chinese Medicine, 32 ( 1): 43-45).
5.果糖基化芒果苷之液相層析質譜(LC-MS)鑑定 5. Identification of fructosylated mangiferin by liquid chromatography mass spectrometry (LC-MS)
使用ZORBAX Eclipse XDB-C18 Narrow Bore RR管柱(2.1 x150mm,3.5μm)。流動相分別為0.02%甲酸水溶液及0.02%甲酸甲醇溶液,流速設定為0.2ml/min。溶離過程如下:
使用Micromass ZQ質譜儀以電灑游離質譜儀 (eletrospray Ionozation,ESI-MS)得到圖譜,其中Cone電壓為10V,15V,20V,35V,50V。 The spectra were obtained using a Micromass ZQ mass spectrometer with an eletrospray ionization (ESI-MS), where the Cone voltage was 10V, 15V, 20V, 35V, 50V.
1.具將芒果苷轉化為果糖基化芒果苷的潛力菌株之篩選 1. Screening of potential strains that convert mangiferin to fructosylized mangiferin
從財團法人食品工業發展研究所(台灣,新竹市)的生物資源保存及研究中心(BCRC)生物資源線上目錄中的乳酸菌、細菌、酵母菌與絲狀真菌中初步篩選出具有果糖基化酵素活性(β-fructofuranosidase、fructosyl transferase)或是具有酪氨酸酶(Tyrosinase)抑制活性之潛力菌株,分別有2株細菌、1株酵母菌、10株乳酸菌以及6株絲狀真菌共19株潛力菌。依前述方法將各菌株活化,進行生物轉化反應,及以高效液相層析法來分析果糖基化芒果苷含量,列果被列於表一。由表一結果可看出其中四株具有潛力,分別是2株細菌Arthrobacter nicotiande BCRC 11219、Arthrobacter globiforms BCRC 10598、1株酵母菌Saccharomyces cerevisiae BCRC 20855、以及1株乳酸菌Lactobacillus plantarum BCRC 11697。基於產業之應用性,以GRAS菌株以及果糖基化芒果苷含量高者作為標的,因此篩選乳酸菌Lactobacillus plantarum作為用於本發明的潛力菌株。 Preliminary screening of fructosylase activity from lactic acid bacteria, bacteria, yeasts and filamentous fungi in the online catalog of biological resources of the Center for Biological Resources Conservation and Research (BCRC) of the Food Industry Development Research Institute (Hsinchu City, Taiwan) (β-fructofuranosidase, fructosyl transferase) or potential strains with tyrosinase inhibitory activity, there are 2 strains of bacteria, 1 strain of yeast, 10 strains of lactic acid bacteria, and 6 strains of filamentous fungi, 19 potential bacteria. The various strains were activated according to the aforementioned methods, biotransformation reactions were performed, and the fructosylated mangiferin content was analyzed by high performance liquid chromatography. The fruits are listed in Table 1. From the results in Table 1, we can see that four of them have potential, namely 2 bacteria Arthrobacter nicotiande BCRC 11219, Arthrobacter globiforms BCRC 10598, 1 yeast Saccharomyces cerevisiae BCRC 20855, and 1 lactic acid bacteria Lactobacillus plantarum BCRC 11697. Based on industrial applicability, GRAS strains and those with high fructosylated mangiferin content were used as targets. Therefore, Lactobacillus plantarum was selected as a potential strain for use in the present invention.
2. 不同之Lactobacillus plantarum菌株轉化生成果糖基化芒果苷之能力分析 2. Analysis of the ability of different Lactobacillus plantarum strains to produce fructosylated mangiferin
收集不同之L.plantarum菌株,分別是L.plantarum BCRC 11697、BCRC 12327、BCRC 14059、BCRC 15478、BCRC 17178、BCRC 17638,依前述方法將各菌株活化,進行生物轉化反應,及以高效液相層析法來分析果糖基 化芒果苷含量,列果被列於表二。如表二所示,Lactobacillus plantarum BCRC 11697轉化生成果糖基化芒果苷之能力最好。 Except L. plantarum strains were collected, respectively L.plantarum BCRC 11697, BCRC 12327, BCRC 14059, BCRC 15478, BCRC 17178, BCRC 17638, according to the method of activated each strain, biotransformation reactions, and high-performance liquid layer Analytical method to analyze the content of fructosylated mangiferin, the fruit is listed in Table II. As shown in Table 2, Lactobacillus plantarum BCRC 11697 has the best ability to transform to fructosylated mangiferin.
3. 紫外光吸收光譜分析結果 3. UV light absorption spectrum analysis results
圖1及圖2分別是50μg/mL果糖基化芒果苷標準品溶液以及以Lactobacillus plantarum BCRC 11697進行生物轉化反應後得到的液體產品在200.0~400.0nm的紫外光吸收光譜圖。由圖1及圖2可知,果糖基化芒果苷標準品與以Lactobacillus plantarum BCRC 11697進行生物轉化反應後得到的液體產品的吸收曲線峰形一致,且分別在238、254、317和367nm波長處均有吸收峰,其中以254nm處的吸收峰最強。因此,在本發明中以254nm為果糖基化芒果苷標準品和進行生物轉化反應後得到的液體產品的果糖基化芒果苷含量 的測定波長。 Figure 1 and Figure 2 are UV absorption spectra of 500.0 g / mL standard solution of fructosylated mangiferin and liquid products obtained after bioconversion reaction with Lactobacillus plantarum BCRC 11697 at 200.0-400.0 nm. From Figs. 1 and 2, it can be seen that the peak shape of the absorption curve of the fructosylated mangiferin standard is the same as that of the liquid product obtained after the bioconversion reaction with Lactobacillus plantarum BCRC 11697, and all of them are at the wavelengths of 238, 254, 317, and 367 nm, respectively. There are absorption peaks, and the absorption peak at 254 nm is the strongest. Therefore, in the present invention, the measurement wavelength of the fructosylized mangiferin content is 254nm as the standard of fructosylized mangiferin and the liquid product obtained after the bioconversion reaction is performed.
4. LC-MS分析結果 4. LC-MS analysis results
圖3及圖4分別是果糖基化芒果苷標準品及以Lactobacillus plantarum BCRC 11697進行生物轉化反應後離心菌體得到的上清液的LC-MS圖譜。比較圖3及圖4的LC-MS圖譜,可以看出以本發明方法進行生物轉化反應後得到的轉化物中確實含有果糖基化芒果苷。 3 and 4 are LC-MS spectra of a fructosylated mangiferin standard and a supernatant obtained by centrifuging a bacterial cell after bioconversion reaction with Lactobacillus plantarum BCRC 11697, respectively. Comparing the LC-MS spectra of FIG. 3 and FIG. 4, it can be seen that the transformant obtained after carrying out the biotransformation reaction by the method of the present invention does indeed contain fructosylated mangiferin.
5. 比較不同糖基供體對菌株轉化生成果糖基化芒果苷之影響 5. Compare the effects of different glycosyl donors on the transformation of strains to fructosylated mangiferin
於前述方法中使用Lactobacillus plantarum BCRC 11697及不同糖基供體(蔗糖、葡萄糖、果糖)進行生物轉化反應的結果被示於圖5,從其中可以看出蔗糖具有最高的果糖基化芒果苷轉化效率,當轉化時間在24小時內,果糖基化芒果苷含量可達相對高點35μg/mL。 The results of the bioconversion reaction using Lactobacillus plantarum BCRC 11697 and different glycosyl donors (sucrose, glucose, fructose) in the aforementioned method are shown in FIG. 5. It can be seen that sucrose has the highest fructosylated mangiferin conversion efficiency. When the conversion time is within 24 hours, the fructosylated mangiferin content can reach a relatively high point of 35 μg / mL.
6. 探討不同比例蔗糖糖基對果糖基化芒果苷轉化效率之比較 6. Explore the comparison of conversion efficiency of fructosylated mangiferin with different ratios of sucrose sugar groups
圖6是比較在添加0.5%~20%(w/v)的蔗糖糖基下,對果糖基化芒果苷轉化效率之影響。控制組是不加Lactobacillus plantarum BCRC 11697菌液,只添加0.5%糖基進行轉化之結果。果糖基化芒果苷含量在0小時為25μg/mL。由圖6結果可得知,在蔗糖糖基高濃度(20%)與低濃度(0.5%)之間,進行生物轉化反應的時間在6小時時,果糖基化芒果苷的含量可以達到相對高值。另外在0.5%蔗糖糖基這組其平均轉化效率優於其他高濃度組。圖6中0.5%糖基及6小時轉化的條件具有最高的果糖基化芒果苷含量,可達57μg/mL比控制組25μg/mL提升1倍以上。 Figure 6 compares the effect of fructosylated mangiferin conversion efficiency with the addition of sucrose sugar groups at 0.5% to 20% (w / v). The control group was the result of transformation without the addition of Lactobacillus plantarum BCRC 11697 and the addition of only 0.5% glycosyl groups. The fructosylated mangiferin content was 25 μg / mL at 0 hours. From the results in FIG. 6, it can be known that when the sucrose sugar concentration is high (20%) and the low concentration (0.5%), the bioconversion reaction time is 6 hours, and the content of fructosylated mangiferin can reach a relatively high level. value. In addition, the average conversion efficiency of the 0.5% sucrose glycosyl group was better than other high-concentration groups. The conditions of 0.5% glycosyl and 6-hour transformation in Fig. 6 have the highest fructosylized mangiferin content, which can reach 57 μg / mL more than double the control group's 25 μg / mL.
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