專業(yè)英語(yǔ)翻譯葡萄籽提取物對(duì)面包的抗氧化活性和質(zhì)量屬性的影響

1、The effects of grape seed extract fortification on the antioxidant activity and quality attributes of bread 葡萄籽提取物對(duì)面包的抗氧化活性和質(zhì)量屬性的影響Abstract: The antioxidant activity change of breads added with grape seed extract (GSE) was investigated. The results showed that bread with the addition of GSE had stro

2、nger antioxidant activity than that of blank bread, and increasing the level of GSE addition further enhanced the antioxidant capacity of the bread. However, thermal processing caused antioxidant activity of GSE added to bread to decrease by around 3040%. We also studied the effect of GSE on theform

3、ation of detrimental Ne-(carboxymethyl) lysine (CML), a famous advanced glycation endproduct in bread. According to the results, GSE could reduce CML in bread and acted in a dose-dependent manner. Meanwhile, except for an acceptable colour change, adding GSE to bread had only little effect on the qu

4、ality attributes of the bread. Altogether, our findings indicate that GSE-fortified bread is promising to be developed as a functional food with relatively lower CML-related health risks, yet a high antioxidant activity.Keywords: Antioxidant activity Grape seed extract Bread Advanced glycation endpr

5、oducts摘要：對(duì)加入葡萄木?提取物(GSE)對(duì)面包的抗氧化活性的變化進(jìn)行了研究。結(jié)果表明，加入 GSE的面包具有 比空白面包(空白對(duì)照)更強(qiáng)的抗氧化活性，并且增加GSE 的添加量會(huì)進(jìn)一步提高面包的抗氧化能力。然而，熱加工造成添加GSE 的面包的抗氧化活性降低大約 3040% 。我們還研究了添加GSE 對(duì)形成有害氖-(羧甲基)賴氨酸( CML )的影響，我們還研究了 GSE 對(duì)有害氖(羧甲基)賴氨酸( CML )的形成的影響，是面包中著名 的晚期糖基化終末產(chǎn)物。根據(jù)研究結(jié)果， GSE 能減少面包中 CML 的形成，并且呈現(xiàn)劑量依賴性。同時(shí)，除了具 有可接受的顏色變化，添加GSE 對(duì)面包質(zhì)量屬

6、性的影響不大?？傊覀兊难芯拷Y(jié)果表明， GSE 強(qiáng)化面包有望被開發(fā)作為功能性食品，有相對(duì)較低的 CML 含量和較低的健康風(fēng)險(xiǎn)，但具有較高的抗氧化活性。 關(guān)鍵詞： 抗氧化活性；葡萄籽提取物；面包；晚期糖基化終末產(chǎn)物1. IntroductionPhenolic compounds are widely distributed in foods, such as fruits (Robards, Prenzler, Tucker, Swatsitang, & Glover, 1999), vegetables (Bonoli, Gallina Toschi, & Lercker,

7、 2005) and cereals (Dykes & Rooney, 2007; Liyana-Pathirana & Shahidi, 2006). As naturally occurring antioxidants, phenolic compounds have been reported to possess diverse beneficial bioactivities, including anti-allergic, antiviral, anti-inflammatory and anti-mutagenic properties (Yao et al.

8、, 2004). Meanwhile, a large number of in vitro and animal studies have also suggested that phenolic compounds may be effective in protecting against cancer, and cardiovascular diseases. The protective effects might be mediated through their action as antioxidants to prevent oxidative damage induced

9、by reactive oxygen species to some important biomolecules (like DNA, lipids andproteins) under pathological conditions (Hollman, 2001; Yao et al.,2004). Recently, phenolic antioxidants have been viewed as an important class of food ingredients either as food additives or as novel ingredients to intr

10、oduce extra health benefits to various food products. Considering the fact that heat treatment is a widespread processing method in the food industry, a salient question is whether these thermal processes would lead to significant alterations in the antioxidant capacities of phenolic additives. It h

11、as been reported that total antioxidant activities of tomatoes and carrots were enhanced with thermal processing (Dewanto, Wu, Adom, & Liu, 2002; Patras, Brunton, Da Pieve, Butler, & Downey, 2009) while antioxidant capacities of soybeans were lowered with similar processing (Xu & Chang,

12、2008).1. 前言酚類化合物廣泛分布于食品中，如水果、蔬菜、谷物等。作為天然存在的抗氧化劑，酚類化合物已被報(bào)道具有多樣有益的生物活性，包括抗過敏，抗病毒，抗炎和抗誘變性等。同時(shí)，大量的體外和動(dòng)物研究還表明，酚類化合物可以有效地防止癌癥和心血管疾病。 其保護(hù)功能可能是通過它們作為抗氧化劑的作用來實(shí)現(xiàn)的， 防止活性氧的氧化損傷從而引起一些重要生物分子(如 DNA 、脂質(zhì)和蛋白質(zhì))處于病理狀態(tài)。最近，酚類抗氧化劑已被視為食品成分的一類重要的或者作為食品添加劑或作為新成分引入到各種食品中有額外的健康益處。 考慮到事實(shí) 情況， 熱處理是在食品工業(yè)中的普遍的處理方法， 一個(gè)突出的問題就是在這些熱加工過

13、程中是否會(huì)導(dǎo)致酚類添加劑抗氧化能力的顯著改變。 據(jù)報(bào)道， 在熱加工過程中番茄和胡蘿卜的抗氧化活性會(huì)增強(qiáng)， 而大豆的抗氧化能力會(huì) 降低。So far, little work has been focused on evaluating the relationship between thermal processing of food and changes in antioxidant capacities of phenolic additives. For this consideration, we investigated the influence of thermal proc

14、essing on antioxidant capacity of grape seed extract (GSE) in the present work. As a well-known nutraceutical product, GSE is an abundant source of catechins and proanthocyanidins with a strong antioxidant and free radical scavenging activity (Liang, Wang, Simon, & Ho, 2004; Wu, Wang, & Simo

15、n, 2005). Moreover, it shows other biological effects as well, such as inhibition of platelet aggregation, anti-inflammation and anti-ulcer activity (Saito, Hosoyama, Ariga, Kataoka, & Yamaji, 1998; Vitseva, Varghese, Chakrabarti, Folts, & Freedman, 2005). In this study, different amounts of

16、 GES were mixed with bread ingredients before starting the bread-making program on bread makers. Comparing the antioxidant activities of the GSE-fortified bread with those of standard GSE solutions would enable estimation of the extent to which the thermal process might affect the antioxidant of GSE

17、. Textural analysis, colour measurement and sensory evaluation were also conducted to investigate whether addition of GSE will affect quality attributes of bread or not.迄今為止， 很少有研究工作集中于評(píng)價(jià)熱加工處理與酚類添加劑抗氧化能力變化之間的關(guān)系。 出于這種考慮，在目前工作中我們研究了熱加工對(duì)葡萄籽提取物( GSE )抗氧化能力的影響。作為一種眾所周知的營(yíng)養(yǎng)保健品，GSE 是兒茶素和花青素的豐富來源，具有很強(qiáng)的抗氧化活性和

18、清除自由基的能力。此外，它也同樣顯示了多種生物學(xué)效應(yīng)，例如抑制血小板聚集，抗炎，抗?jié)兓钚缘?。在本研究中，在面包機(jī)開始面包制作程序之前，在面包配料中加入不同量的 GSE 。 比較 GSE 強(qiáng)化面包與標(biāo)準(zhǔn)GSE 溶液的抗氧化活性， 能夠估計(jì)熱加工過程影響GSE抗氧化活性的程度。進(jìn)行結(jié)構(gòu)分析，顏色測(cè)量和感官評(píng)價(jià)來研究添加GSE 是否會(huì)影響面包的質(zhì)量屬性。In addition, the effect of GSE on the formation of Ne-(carboxymethyl)lysine (CML) in bread was studied. As a well-characteri

19、sed detrimental advanced glycation endproduct (AGE) (Sebekova & Somoza, 2007; Tessier & Niquet, 2007), CML was widely found in a range of foods including bread (Charissou, Ait-Ameur, & BirlouezAragon, 2007; Hartkopf & Erbersdobler, 1994) and its content in crust was reported to be mu

20、ch higher than in crumb (Assar, Moloney, Lima, Magee, & Ames, 2009). Currently it is viewed as a potential toxicant in food. Moreover, it has become a biomarker associated with oxidative stress, atherosclerosis and diabetes in humans (Nerlich & Schleicher, 1999; Schleicher, Wagner, & Ner

21、lich, 1997). Natural antioxidants like bean extracts (Madhujith, Amarowicz, & Shahidi, 2004), peanut skin and some flavonoids have been proven to possess strong inhibitory effects on the formation of AGEs in vitro, which are mainly attributed to their potent antioxidant activities (Lou, Yuan, Ya

22、mazaki, Sasaki, & Oka, 2001; Peng et al., 2007; Wu & Yen, 2005). In addition, capability of certain phenolics such as tea catechins and proanthocyanidins to scavenge reactive carbonyl species (such as glyoxal, methylglyoxal) in glycation process has also been proposed to contribute to inhibi

23、tion of AGE formation (Lo et al., 2006; Peng et al., 2008). Therefore, it is of great interest to investigate whether GSE fortification could reduce CML content in bread. In this regard, influence of different levels of GSE addition on CML content of bread was examined.此外，研究了 GSE 對(duì)面包中氖-(羧甲基)賴氨酸(CML

24、)形成的影響。作為一個(gè)特征明顯的有害的晚期糖基化終末產(chǎn)物( AGE ) ， CML 廣泛存在于各種食品中包括面包，并且其含量在面包皮中比在面包心中高得多。目前， 它被看作是食品的潛在毒物。 而且， 它已成為與人類氧化應(yīng)激， 動(dòng)脈粥樣硬化和糖尿病相關(guān)聯(lián)的生物標(biāo)志物。在體外實(shí)驗(yàn)中，天然抗氧化劑像豆子提取物，花生皮和一些黃酮類化合物已被證明對(duì)AGE 的形成有很強(qiáng)的抑制作用， 這主要?dú)w功于其強(qiáng)大的抗氧化活性。 此外， 某些酚類物質(zhì)如茶葉中的兒茶素和原花青素清除活性羰基物質(zhì)(如乙二醛，甲基乙二醛)的能力，也已經(jīng)被提出在糖化過程中有助于抑制 AGE 形成。因此，研究GSE 強(qiáng)化能否降低面包中的 CML 含

25、量是非常有意義的。就這一點(diǎn)而言，研究了不同水平的 GSE 添加量對(duì)面包中 CML 含量 的影響。2. Materials and methods2.1. ChemicalsIngredients for bread-making were bought from a local supermarket in Hong Kong. Grape seed extract with 95% proanthocyandins including catechin and epicatechin was a gift from Shenzhen BannerBio Inc. (Shenzhen, PR

26、China). The compound Ne-(carboxy methyl) (CML) was purchased from NeoMPS (Strasbourg, France). Ortho-phthalaldehyde(OPA), 2-mercaptoethanol, sodium borohydride, boric acid, sodium tetraborate decahydrate, sodium hydroxide, hydrochloric acid,2,2 0-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (AB

27、TS), potassium peroxodisulphate and trolox were all obtained from Sigma 力Idrich Company (St. Louis, MO, USA). All analytical and HPLC grade solvents used were obtained from BDH Laboratory Supplies (Poole, UK).2.材料與方法2.1. 化學(xué)藥品面包制作的材料購(gòu)買于香港當(dāng)?shù)氐某小Ｆ咸炎烟崛∥镉?5 原花青素包括兒茶素和表兒茶素，由深圳Banner 生物公司提供。化合物氖- (羧甲基)( CM

28、L )購(gòu)買自 NeoMPS 。正 -鄰苯二醛(OPA) ， 2-巰基乙醇，硼氫化鈉，硼酸，四硼酸鈉十水合物，氫氧化鈉，鹽酸， 2,2 -連-二( 3-乙基苯并噻唑-6-磺酸)( ABTS ) ，過氧化鉀和生育酚均全部購(gòu)自 Sigma-Aldrich 公司。所有分析和 HPLC 級(jí)溶劑由 BDH 實(shí)驗(yàn)室提供。2.2. Preparation of breadBread was made using a bread maker (Moulinex, Ecully Cedex, France) bought from a local supermarket. The program 2 for fa

29、st basic white bread (500 g/each) was chosen for bread-making.Briefly, this program adopts the following sequential process: first kneading (5 min), rest (5 min), second kneading (20 min), first rising (15 min), third kneading (10 s), second rising (8 min and 50 s), fourth kneading (10 s), third ris

30、ing (29 min and 50 s) and baking (43 min). The recipe for bread includes canola oil (3.5 tea spoon), water (190 mL), salt (1 tea spoon, about 5.7 g), sugar (2.5 tea spoon, about 8.88 g), milk powder (1.5 table spoon, about 10.28 g), white bread flour (350 g) and flaked dried yeast (1 tea spoon, abou

31、t 2.85 g). GSE with different levels (300 mg, 600 mg and 1 g) was added into breads (500 g/each), respectively. All breads were made in three batches. Bread crusts were carefully sliced off and ground for later determination of CML.2.2. 面包的準(zhǔn)備面包是用從當(dāng)?shù)爻匈?gòu)買的面包機(jī)制作的。選擇程序 2 為快速基本白面包(500 克/個(gè))制作。簡(jiǎn)單地說，這個(gè)程序采用以

32、下順序加工：首先揉制( 5 分鐘) ，靜置( 5 分鐘) ，第二揉制( 20 分) ，第一次上升( 15 分鐘) ，第 三次揉制( 10 秒) ，第二次上升( 8 分鐘 50 秒) ，第四次揉制( 10 秒) ，第三次上升( 29 分鐘 50 秒) ，然后烘烤(43 分鐘) 。面包配方包括低芥酸菜子油( 3.5茶匙) ，水( 190ml) ，鹽(1 茶匙，約 5.7 克) ，糖( 2.5茶匙，約8.88 克) ，奶粉( 1.5湯匙，約 10.28 克) ，白面包粉( 350克)和片狀干酵母( 1 茶匙，約為 28.5 克) 。 GSE 以不 同水平( 300 毫克， 600 毫克和 1 克)分

33、別加入到面包( 500 克/個(gè))中。所有的面包都平行制作三組。將面包皮 仔細(xì)切掉以便隨后測(cè)定CML 含量。2.3. Antioxidant activity measurementGround bread powders were vortex-dispersed (5 s), respectively, into water, 30%, 50% and 70% ethanol with a finalconcentration of 100 mg/mL. The samples were extracted by sonication for 30 min and then centrifu

34、ged (3233g) for 20 min. The supernatants were used for subsequent determination of antioxidant capacity. Meanwhile, 0.06, 0.12 and 0.2 mg/mL GSE solutions were prepared as controls corresponding to different concentrations of GSE added to bread. Total antioxidant capacity was determined using the tr

35、olox equivalent antioxidant capacity (TEAC) assay according to the literature (Cheng, Chen, & Wang, 2007) with minor modifications. In brief, 7 mM ABTS salt solution was reacted with 2.45 mM potassium peroxodisulphate solution and the reaction mixture was allowed to stand in the dark for 16 h at

36、 room temperature (25C)and was used in 2 days. The resultant radical solution was diluted with deionized water to anabsorbance of 0.70.05± at 734 nm. Fifty microlitres of bread sample solution or standard (different concentrations oftrolox) and 50 pL of water were added to 1.8 mL of diluted ABT

37、S+ solution and absorbance was taken at 734 nm on a UV-1206 Spectrophotometer (Shimadzu, Kyoto, Japan) after 6-min incubation. Results were expressed as TEAC values (nmol trolox/mg bread sample). Triplicate analyses were performed.2.4. 抗氧化活性測(cè)定研磨過得面包粉呈渦流分散( 5 秒) ，分別地加入水、 30、50和70的乙醇，最終濃度為 100mg/ml 。將

38、樣品超聲處理提取30 分鐘，然后離心( 3233g) 20 分鐘。上清液用于后續(xù)測(cè)定抗氧化能力。同時(shí)，制備 0.06、0.12 和 0.2mg/ml 的 GSE 溶液作為相應(yīng)不同濃度的 GSE 加入到面包中。 總抗氧化能力的測(cè)定根據(jù)文獻(xiàn)稍作修改， 使用 Trolox (生育酚)當(dāng)量抗氧化能力( TEAC )法測(cè)定。簡(jiǎn)言之， 7mM ABTS 鹽溶液與 2.45mM 過氧化鉀溶液 反應(yīng)，然后將反應(yīng)混合物于室溫下(25)避光靜置(在黑暗中)16 個(gè)小時(shí)，在兩天之內(nèi)使用。所得自由基溶液用去離子水稀釋至在734nm 處吸光度為0.7 ± 0.05。 五十微升的面包樣品溶液或標(biāo)準(zhǔn)的 (不同濃度

39、的生育酚) 和50 dL水加入到1.8ml稀釋的ABTS溶液中，靜置6min后，使用UV-1206分光光度計(jì)(島津，京都，日本)在 734nm 處測(cè)定吸光度。結(jié)果以 TEAC 值表示( nmol 生育酚 /mg 面包樣品) 。測(cè)定平行進(jìn)行三次。2.5. Textural analysisAfter cooling for 1 h, breads were cut into slices of 25 mm thickness with a bread knife. The central two slices were used to perform textural analysis on a

40、 TA-XT2 texture analyzer (Stable Micro System, Surrey, UK) equipped with a cylindrical probe of 20 mm in diameter. Based on texture profile analysis (TPA), hardness was calculated. Textural analysis was performed in the mode of Measure Force in Compression ” , while trigger type waaustos-5etgaast th

41、e speed of 2 mm/s followed by a 10 mm compression distance.2.6. Measurement of colour changesColour changes of bread slices with or without the addition of different concentrations of GSE was measured with a tristimulus reflectance colourimeter (Minolta CR-400 Chroma Meter, Konica Minolta, NJ, USA),

42、 and readings were expressed as L* , a*and b* values, where L * indicates whiteness (value 100) or blackness (value 0),a* indicates red (positive value) or green (negative value), and b* indicates yellow (positive value) or blue (negative value).2.7. Sensory evaluationTen untrained panelists engaged

43、 in the sensory evaluation based on scoring various bread samples according to five attributes: sweetness, porosity, astringency, stickness and colour. In terms of corresponding intensity, each attribute was ranked as low, medium and high grades gaining 1, 2, 3 score, respectively. Sensory evaluatio

44、n was conducted on three batches of bread. The mean scores and corresponding standard deviation values for each attribute were calculated for comparison.2.8. 織構(gòu)分析冷卻 1 小時(shí)后，用面包刀將面包切成25mm 的厚度。采用裝配有直徑20 毫米圓柱形探針的 TA-XT2 質(zhì)構(gòu)儀（穩(wěn)定微系統(tǒng)，薩里，英國(guó)）對(duì)中間兩片面包進(jìn)行織構(gòu)分析。根據(jù)紋理剖面分析（ TPA ） ，計(jì)算硬度?？棙?gòu)分析在 “測(cè)量壓縮力量”模式上進(jìn)行， 觸發(fā)器類型被設(shè)定為自動(dòng)5

45、g ， 以 2mm /秒的速度， 接著通過 10mm 的壓縮距離。2.9. 顏色變化測(cè)定采用三色反射色度計(jì)（美能達(dá) CR-400色度儀，柯尼卡美能達(dá)， NJ,美國(guó)）測(cè)定加入或不加入不同濃度GSE后的面包片的顏色變化，并且讀數(shù)表示為 L *, a *和b*值，其中L*表示白度（值100）或黑度（值0）, a*表示 紅色（正值）或綠色（負(fù)值），b*表示黃色（正值）或藍(lán)色（負(fù)值）。2.10. 官評(píng)定10 名未經(jīng)訓(xùn)練的小組成員參與感官評(píng)定，根據(jù)他們對(duì)不同面包樣品的打分來評(píng)定，根據(jù)五種特性：甜度，孔隙率，收斂性（澀味） ，粘性和顏色。依照相對(duì)強(qiáng)度，每種特性分為低、中和高三個(gè)等級(jí)，分別獲得 1、 2、 3

46、 分。感官評(píng)價(jià)對(duì)三組面包進(jìn)行。計(jì)算每種特性的平均得分和相應(yīng)的標(biāo)準(zhǔn)偏差值用于比較。2.11. Determination of CML in bread crustHPLC analysis was adopted to determine CML content in bread crust with reference to the reported method (Drusch, Faist, & Erbersdobler, 1999) with some modifications. The sample preparation procedures include defatt

47、ing, reduction, hydrolysis and chemical derivatization.2.11.1. DefattingBread crusts (1 g) were defatted using two successive extractions in 9 mL of a chloroform/methanol (2:1, v/v) solution followed by centrifugation (3233g at room temperature) for 20 min. Then, the defatted bread crusts were dried

48、 completely at 50C.2.11.2. ReductionDefatted bread crusts (100 mg) were reduced with 4 mL sodium borate buffer (0.2 M, pH 9.4) and 2 mL sodium borohydride (1 M in 0.1 M NaOH) and then incubated for 4 h at room temperature 2.7.3. HydrolysisSubsequently, hydrochloric acid (HCl) was added to the reduce

49、d samples to achieve a final concentration of 6 MHCl. In order to avoid any oxidative process, samples were degassed with a stream of nitrogen for 5 min. Then samples were hydrolysed at 110C for 20 h. After hydrolysis, samples were dried by rotary evaporation and then mixed with 10 mL of water befor

50、e filtration. Two millilitres of the filtrates were concentrated by rotary evaporation and re-dissolved in sodium borate buffer (0.2 M, pH 9.4) for later derivatization.2.11.4. DerivatizationOPA (10 mg) was dissolved in 2 mL of methanol to obtain an OPA stock solution. The derivatization reagent was

51、 composed of 1 mL of OPA stock solution, 8lL of 2-mercaptoethanol and 3.992 mL borate buffer (consisting of 0.2 M boric acid and 0.2 M NaOH, pH 9.9) (Hanczko & Molnar-Perl, 2003). The reagent was prepared at least 90 min before use for derivatization and was disposed after 2 days. Hydrolysates (

52、200lL) were mixed with 100lL of derivatization reagent and incubated for 3 min before subjecting to HPLC analysis.2.11.5. HPLC analysisAnalytical HPLC was carried out using a Waters 2695 Separation Module equipped with a Waters 2475 Multikfluorescence detector. A pre-packed ODS-A column (1504.6 mm,

53、5lm, YMC Co. Ltd., Kyoto, Japan) was selected for HPLC analysis and detection was at 340 nm (excitation) and 455 nm (emission). The flow rate was 1.0 mL/min and the injection volume was 10lL. The mobile phases were: (solvent A) sodium acetate buffer (pH 6.7, 20 mM) acetonitrile (90/10, v/v) and (sol

54、vent B) acetonitrile. The elution started with 5% B and hold for 9 min, and then it was linear gradient to 70% B in 5 min and kept at 70% B till 17 min. The gradient was subsequently set back to 95% B within 1 min and the post running time was 10 min. Peaks for CML-derivatives in bread samples were

55、confirmed by comparison with an authentic compound. CML contents of the bread samples were calculated based on the peak areas of the corresponding CML derivatives.2.7. 測(cè)定面包皮的 CML 值根據(jù)文獻(xiàn)報(bào)告的方法稍作修改， 采用 HPLC 分析法測(cè)定面包皮的 CML 含量。 樣品制備程序包括脫脂、 還原、 水解和化學(xué)衍生化。2.7.1 脫脂取面包皮(1克)利用9ml氯仿/甲醇(2:1, v/v)溶液連續(xù)兩次萃取脫脂，接著離心分離(

56、室溫 3233g) 20 分鐘。然后將脫脂面包皮在50下完全干燥。2.7.2 還原脫脂面包皮(100毫克) 分別用 4mL 硼酸鈉緩沖液(0.2M ， pH 值 9.4) 和 2ml 硼氫化鈉 (1M ， 在 0.1M NaOH中)還原，然后在室溫下培養(yǎng)4 小時(shí)。2.7.3 水解隨后，還原樣品中加入鹽酸( HCl)以達(dá)到最終濃度6 M HCl。為了避免任何的氧化過程，將樣品用氮?dú)鈿?0ml 水后過流脫氣 5 分鐘。然后將樣品在 110下水解 20 小時(shí)。水解后，將樣品通過旋轉(zhuǎn)蒸發(fā)干燥，然后加入濾。取 2ml 濾液通過旋轉(zhuǎn)蒸發(fā)濃縮，并且重新溶解在硼酸鈉緩沖液（0.2M ， pH 值 9.4 ）中

57、以便隨后的衍生化。2.7.4 衍生化取OPA （10mg）溶解于2ml甲醇中，得到 OPA儲(chǔ)備液。衍生化試劑是由 1ml OPA儲(chǔ)備?夜,8 d 2-疏基乙 醇和 3.992ml 硼酸鹽緩沖液（由 0.2M 硼酸和 0.2M NaOH 組成， pH 值 9.9 ）組成。試劑至少在衍生前90min 制備，并且可放置 2天。水解產(chǎn)物（200由與100 口衍生化試劑混合，靜置 3分鐘后進(jìn)行HPLC分析。2.7.5 HPLC 分析HPLC 分析是采用配備有Waters2475 多熒光檢測(cè)器的 Waters2695 分離模塊進(jìn)行。選擇一種預(yù)包裝的 ODS-A柱（ 150 4.6mm, 5 dm, YMC

58、有限公司，京都，日本）用于 HPLC分析，檢測(cè)是在 340nm （激發(fā)）和455nm （發(fā) 射）下進(jìn)行。流速為 1.0ml/分鐘，注射體積為 10口。流動(dòng)相為：（溶劑A）乙酸鈉緩沖液（pH6.7, 20mM）-乙 腈（ 90/10 ， v/v ）和（溶劑 B ）乙腈。洗脫開始用5的 B 并保持 9 分鐘，然后在 5min 內(nèi)以線性梯度升至70的 B ，并且在70 B 保持 17 分鐘。隨后梯度被設(shè)置回至95 B 1 分鐘內(nèi)，然后運(yùn)行10 分鐘。面包樣品的 CML衍生物的峰與真實(shí)化合物的峰進(jìn)行對(duì)比。根據(jù)相對(duì)應(yīng)的 CML 衍生物的峰面積來計(jì)算面包樣品中的 CML 含量。2.12. Statisti

59、cal analysisStatistical analyses were performed using the SPSS statistical package （SPSS Inc., Chicago, IL）. Paired samples Ttest was applied to determine whether a particular treatment of the sample would result in a significant difference compared with the corresponding control.P< 0.05 was selected as the level decision for significant differences. 2.8.統(tǒng)計(jì)分許統(tǒng)計(jì)學(xué)分析使用 SPSS軟件進(jìn)行。配對(duì)樣本t檢驗(yàn)用于測(cè)定特殊處理的樣品是否具有顯著性差異。選擇水平 P<0.05 認(rèn)為具有顯著差異。3.