流行病学研究表明,西兰花、萝卜等十字花科蔬菜具有抗炎、抗癌、预防心血管疾病等功能作用,主要是由于这类蔬菜富含硫代葡萄糖苷类化合物,在蔬菜组织结构被破坏后,释放出硫代葡萄糖苷水解酶(黑芥子酶),酶解硫代葡萄糖苷产生葡萄糖、硫酸盐和异硫氰酸酯等水解产物[1],其中的异硫氰酸酯具有抗氧化、抗菌、抗突变和抗癌等生物活性[2-3]。萝卜硫素[1-异硫氰酸基-4-(甲基亚磺酰基)丁烷]和莱菔素[4-异硫氰酸基-1-(甲基亚磺酰基)-1-丁烯]是萝卜、西兰花、花椰菜等十字花科蔬菜及种子中重要的异硫氰酸酯成分,虽然硫代葡萄糖苷可以被肠道菌群水解为萝卜硫素等异硫氰酸酯化合物,但临床试验表明其在人体内的转化效率很低[4],通常采用酶解法或化学合成法制备萝卜硫素和莱菔素,但由于萝卜硫素和莱菔素存在手性异构现象,化学合成成本较高,因此,酶解法是制备萝卜硫素和莱菔素的主要方法。
1 萝卜等蔬菜中萝卜硫素和莱菔素的分布及含量
萝卜根茎主要含有萝卜硫苷(glucoraphanin,C12H23NO10S3)和莱菔苷(glucoraphenin,C12H21NO10S3),其中,萝卜硫苷含量达到74%,而莱菔苷的含量不到10%[5]。萝卜硫素和莱菔素的前体物质分别为萝卜硫苷和莱菔苷这2种硫代葡萄糖苷类化合物,在水溶液中,硫代葡萄糖苷水解酶分别从萝卜硫苷和莱菔苷中分离出一个葡萄糖,生成性质不稳定的磺酸盐中间产物,再经过分子内重排,形成萝卜硫素和莱菔素(图1)[4]。
图1 酶解萝卜硫苷和莱菔苷分别生成萝卜硫素和莱菔素的途径
Fig.1 Enzymatic hydrolysis way of glucoraphanin and glucoraphenin to sulforaphane and sulforaphene respectively
萝卜硫素和莱菔素在癌症预防方面都有很好的效果[2-3]。莱菔素在烷基链上比萝卜硫素多一个不饱和双键,在体外抗诱变实验中,莱菔素活性是萝卜硫素的1.3～1.5倍[6]。在我国民间的饮食习惯中,常把萝卜或萝卜叶等做成凉拌菜或直接食用,生萝卜汁也用于治疗包括百日咳、咳嗽、胃部不适等疾病,萝卜籽可用来治疗哮喘和其他胸部不适。PILIPCZUK等[7]通过C18固相萃取柱及HPLC-DAD-MS分析得到萝卜中莱菔苷含量为0.204 μmol/g干重。萝卜芽中硫代葡萄糖苷含量较高,是成熟萝卜根的3.8倍,萝卜芽中异硫氰酸酯含量是根的8.2倍,其含量与抗氧化活性正相关,萝卜芽中的莱菔素能诱导小鼠肝癌细胞醌还原酶作用,激活抗氧化反应[4]。
在不同的蔬菜和种子中,萝卜硫素和莱菔素的含量存在较大差异。萝卜硫素在萝卜根及芸薹属蔬菜中含量较高[8],甘蓝属蔬菜的主要异硫氰酸酯化合物为萝卜硫素,而莱菔素则在萝卜种子中含量较高,占其异硫氰酸盐总量的60%以上[9],含量可达到7 mg/g[10]。泰国鼠尾萝卜种子含有2 315.6 μg/g莱菔素和3.5 μg/g萝卜硫素[11]。未发芽的西兰花种子中硫代葡萄糖苷和萝卜硫素含量最高,在种子发芽过程中这2种物质含量逐渐降低,而硫代葡萄糖苷水解酶的活性在发芽过程中逐渐增高[12]。LIM等[13]测得Taeback和Chungwoon plus两种萝卜种子的莱菔素含量较高,分别为10.5和5.9 mg/g鲜重,但在种子发芽的3 d内,莱菔素含量迅速降至3.0 mg/g鲜重以下。SIVAKUMAR等[14]对幼苗萌发后第3天和第9天的18种芸薹属蔬菜中萝卜硫素含量分析结果表明,3 d幼苗中萝卜硫素含量最高,其最高的甘蓝San martino幼苗萝卜硫素含量为2.21 mg/g干重,在第9 d降为1.53 mg/g干重。新鲜西兰花的小花、叶、茎中萝卜硫素含量分别为585、420和229 μg/g干重,整棵新鲜西兰花萝卜硫素含量为280 μg/g干重,在冰箱冷藏和冷冻贮藏7 d后整棵西兰花萝卜硫素含量分别降低了14%和29%[5]。萝卜硫素在体内吸收后,与谷胱甘肽结合,通过硫醚氨酸途径代谢,其体内主要代谢产物为N-乙酰半胱氨酸和半胱氨酸复合物,萝卜硫素、萝卜硫素半胱氨酸、萝卜硫素-N-乙酰半胱氨酸,这几种成分可作为西兰花摄入量是否适合的标志化合物[8]。
2 萝卜硫素和莱菔素的稳定性及影响因素
萝卜硫素和莱菔素受加热、pH和氧化剂的影响,如加热会导致硫代葡萄糖苷水解酶失活,从而影响萝卜硫素和莱菔素稳定性。生食西兰花中异硫氰酸盐含量是煮熟西兰花的3倍[15]。萝卜根茎中的主要硫代葡萄糖苷(萝卜硫苷和莱菔苷)在低温(0～1.5 ℃)贮藏8周后没有显著变化,但是莱菔素含量和硫代葡萄糖苷水解酶活性显著降低,且莱菔素含量和酶活性之间存在正相关[16]。煮沸和热烫会对芥子苷和萝卜硫素造成很大损失,但短时间蒸汽加热和发酵有助于硫代葡萄糖苷向萝卜硫素的生物转化,而短时间微波处理比加热和发酵更能促进萝卜硫素的形成,低温冷冻则可有效避免硫代葡萄糖苷和萝卜硫素的损失[17]。萝卜硫素在水中的主要热降解产物为硫脲化合物,是萝卜硫素的二聚体,而莱菔素(纯度>95%)在水中产生的降解产物也是其二聚体[18]。TIAN等[18]研究表明-20、4和26 ℃条件下贮藏莱菔素会生成二聚体,主要由水中羟基亲核攻击下产生的—NH2和莱菔素的—N
CS相互作用生成。蔬菜中含硫物质(H2S等)能促进莱菔素的降解反应,而低pH和金属离子能有效抑制莱菔素的降解反应。
萝卜硫素在碱性条件下迅速降解,在低pH和低温条件下比较稳定。萝卜硫素在大于60 ℃高温条件下不稳定,在pH 2.2、60 ℃加热6 h后,萝卜硫素的保留率仍可以达到95.1%,而在pH 6.0的相同温度时间条件下,萝卜硫素的保留率仅为32.1%[19]。西兰花提取液中萝卜硫素的降解速率常数低于萝卜硫素标准溶液,说明食品基质对其有一定的保护作用。在50 ℃加热9 h内,萝卜硫素稳定性不受影响[20],60 ℃时,当pH从2.2增至6.0,萝卜硫素的降解速率常数k从0.01 h-1增加到0.10 h-1,在75、82、90 ℃条件下降解系数随pH的变化也出现相似的趋势,相同pH条件下,当温度升高15 ℃时,k值增加2.4～5.0倍[21]。羟丙基环糊精和大豆分离蛋白包被的莱菔素微胶囊在90 ℃处理168 h,含量比没包被的莱菔素高20%,且其降解速率显著降低[22]。
3 萝卜等蔬菜中萝卜硫素和莱菔素的酶法制备途径
植物细胞中硫代葡萄糖苷和硫代葡萄糖苷水解酶是相互分离的,前者存在于细胞液泡中,而后者存在于特定的蛋白体中,当萝卜等十字花科蔬菜采摘后被切碎或粉碎时,自身组织结构被破坏,内源硫代葡萄糖苷水解酶被释放出来,与硫苷接触后会破坏其糖苷键,生成硫酸盐、葡萄糖和各种苷元,苷元再经过非酶的分子内重排,生成异硫氰酸盐、硫氰酸盐或腈等成分,整个水解过程称为硫苷-黑芥子酶系统。
硫代葡萄糖苷水解酶(黑芥子酶)是一种糖蛋白,糖侧链的多样性导致了黑芥子酶具有多种同工酶,维生素C是黑芥子酶的辅因子,有维生素C存在条件下,黑芥子酶的活性显著增加[23]。采用共价交联的方式将萝卜中提取得到的黑芥子酶固定化,酶解效率提高到2.91倍[24]。以最长柔性间隔的癸二胺固定化黑芥子酶用于莱菔素的生产,莱菔苷的转化率达到93.25%,且固定化酶在10 d后依旧保持95%的活性[25]。LX-1000EP树脂带有含环氧丙基官能团,能和黑芥子酶共价结合,提供酶的活性和稳定性,在循环使用10次以后,酶活力仍可达到初始酶活力的80%以上[26]。
3.1 酶解温度的选择
黑芥子酶的热稳定性较差,在50 ℃左右性质比较稳定。西兰花中的黑芥子酶最适pH为6.2,最适温度为60 ℃左右[23],生西兰花在组织结构破坏后,异硫氰酸酯的产生量显著增加,但1 000 W微波加热1 min后,萝卜硫素损失了65.9%[27]。在家用微波炉烹调1 min后,产生异硫氰酸酯的量下降了近40%,而外源硫代葡萄糖苷水解酶的加入使得熟西兰花异硫氰酸酯的含量提高了38%[28],950 W微波加热19 min可完全灭活西兰花和红色卷心菜中的黑芥子酶[3],但低功率短时间微波处理有助于萝卜硫素的释放[29-30]。在低微波功率(180 W,24 min)和中等微波功率(540 W,8 min)条件下,红球甘蓝的硫代葡萄糖苷水解酶活性损失很小,而在900 W下加热4.8 min,红球甘蓝的硫代葡萄糖苷酶完全失活[31]。这是由于上皮硫特异蛋白(epithiospecifier protein,ESP)在60～70 ℃加热5～10 min会变性失活,但100 ℃加热5 min以上才能灭活黑芥子酶[32]。
西兰花中ESP的存在会导致大量的硫苷转化成有害的腈类物质,影响萝卜硫苷向萝卜硫素的转化,但是ESP较黑芥子酶的热稳定性差,整个酶解体系加热到50～60 ℃会导致ESP变性失活,而硫代葡萄糖苷水解酶的活性得到保留[33]。在均质前将西兰花的小花和芽加热到60 ℃,ESP活性显著降低,萝卜硫素生成量增加,而腈类物质产生量降低,但加热到70 ℃后,西兰花小花中萝卜硫素和腈类物质的生成量均降低,说明黑芥子酶活性也受到了影响。姜睿等[34]采用预加热的方式钝化ESP,提高了莱菔素的提取效率。低功率微波加热能有效钝化ESP蛋白,避免低活性腈类物质生成[35]。
PONGMALAI等[36]以水为提取溶剂,超声波和微波协同提取卷心菜中芥子苷,在超声波提取过程中,水温从48 ℃升到61 ℃,黑芥子酶一直保持较高活性,芥子苷的提取率相对较低,在超声波作用之后将水温降至48 ℃开始微波辅助提取,并在微波辅助提取4 min后,将水温升高到96 ℃,芥子酶活性损失了86%[36]。总体来说,低功率短时间微波处理对黑芥子酶活性影响较小,可以促进萝卜硫素的形成[29-30, 35]。相比于生西兰花和100 ℃加热组,600 MPa高静水压处理显著增加了西兰花异硫氰酸酯的生成量,芥子苷的转化率达到70.4%,这是由于高压对叶片的挤压损伤激活了黑芥子酶-芥子苷-异硫氰酸酯反应体系[37]。4 ℃贮藏4个月的2个品种萝卜根中黑芥子酶活性分别从0.31和0.32 U/g鲜重降到了0.10和0.06 U/g鲜重,而莱菔素的提取量也分别由66.0和69.2 μg/g分别降到了12.7和41.2 μg/g,说明低温长时间贮藏也会影响萝卜黑芥子酶活性,从而降低莱菔素的生成量[38]。
表1为近年来从十字花科蔬菜及其种子中提取萝卜硫素和莱菔素的酶解条件,大部分研究者采用的酶解方式为物料粉碎后,室温条件下内源葡萄糖苷水解酶自然水解,所需时间20 min～24 h不等,为提高酶解效率,也可将酶解温度设定在35～55 ℃[5,39-40]。而额外添加硫代葡萄糖苷水解酶则只需酶解20 min左右即可达到最佳酶解效果[31, 41]。
表1 不同蔬菜来源萝卜硫素和莱菔素的酶解条件
Table 1 Enzymatic hydrolyzing conditions of sulforaphane and sulforaphene from different vegetables
原料前处理酶解pH酶解温度酶解时间/min异硫氰酸酯种类参考文献萝卜种子磨碎未调节50^55 ℃60莱菔素[42]西兰花副产物未处理6.045 ℃210萝卜硫素[5]萝卜种子研磨成粉7.0室温加黑芥子酶, 20 min莱菔素[41]萝卜种子油粕提取后剩余的籽粕4.535 ℃540莱菔素[39]西兰花种子加水均质未调节45 ℃180萝卜硫素[40]西兰花磨碎5.825 ℃240 萝卜硫素[43]西兰花微波烹调后,立即冰浴降温7.0室温240萝卜硫素[3]
3.2 物料粒度的影响
一般通过粉碎或研磨的方式释放黑芥子酶,种子加水高速均质或蔬菜直接高速均质方法也经常被采用[39,43-44],55 MPa高压均质使物料细胞被破坏,原有光滑的表面变为小片状结构和碎片,传质效率提高,有利于硫苷和黑芥子酶接触,小于8 000 psi的均质压力可取得很好的提取效果[45]。但均质压力、研磨频率或时间的增加并不能持续提高萝卜硫素得率,甚至降低了萝卜硫素的得率,可能是由于高频高压导致物料细胞破碎并聚集,另一个可能的原因是黑芥子酶的活性受到影响[29, 45]。综上所述,粉碎或研磨可促进黑芥子酶的充分释放,高压均质可提高酶解效率从而提高异硫氰酸酯的提取效率,但均质或研磨的物料过细会影响提取效率。
3.3 酶解pH的影响
硫代葡萄糖苷在不同pH条件下生成的水解产物不同,在中性和偏酸性(pH 3～8)条件下,水解产物以异硫氰酸酯为主。因此,大部分异硫氰酸酯提取的前处理工艺中未调节pH[46]或加入pH 7.0的PBS[10,41],也有一些将pH调节为偏酸性,如加入pH 4.5柠檬酸缓冲液[39],但pH 3.0是已有报道中较低的水解pH条件,与自然pH水解相比,pH 3.0酸性条件下水解则能增强硫代葡萄糖苷酶水解能力,形成更多的异硫氰酸酯成分[47]。也有研究表明,当水解pH为5.0左右时,莱菔素得率较高,而硫代葡萄糖苷水解生成萝卜硫素的最适pH略高,为pH 6～7[5, 48],ZHANG等[48]考察了pH(2.0、3.0、4.0、5.0、6.0、7.0、8.0)和温度(45、55、65、75、85 ℃)对西兰花黑芥子酶活性的影响,结果表明,在pH 4～8和30～75 ℃范围内黑芥子酶活性都保持的很好,在pH 4～6和温度30～60 ℃范围内酶稳定性良好,但在65 ℃加热60 min后,西兰花黑芥子酶已经失活。这与LOMELINO等[49]和SANGTHONG等[50]对黑芥子酶活性适宜温度的研究结果是一致的,酶解温度不能超过65 ℃。另外,不同部位蔬菜的基质不同,水解生成萝卜硫素的最适温度和pH条件也有所差异。样品水解后,一般采用调节pH为1～2去除水解液中蛋白质[10, 39, 41],酶解后降低pH来提高莱菔素等异硫氰酸酯化合物的生成量。
4 总结与展望
随着营养健康研究的深入,富含抗癌成分——萝卜硫素和莱菔素的萝卜属、芸薹属等十字花科蔬菜越来越受到人们的关注,如何在加工过程中减少其活性成分的损失,成为众多研究者和消费者关注的重点。萝卜硫素和莱菔素不耐热,但60 ℃以下温度的加热烹调对萝卜硫素和莱菔素稳定性没有显著影响,改变萝卜、西兰花等富含异硫氰酸酯化合物的烹调方式,采用短时间快炒及速烫等对芥子酶活性影响较小的加工方式,以摄入更多对身体健康有益的萝卜硫素和莱菔素等功效因子,以及开发高催化活性的硫代葡萄糖苷水解酶(黑芥子酶)生产技术,是有必要的。减少化学试剂的使用,建立绿色环保的萝卜硫素和莱菔素制备方法是今后的研究方向和趋势。
[1] HOUGHTON C A, FASSETT R G, COOMBES J S.Sulforaphane:Translational research from laboratory bench to clinic[J].Nutrition Reviews, 2013, 71(11):709-726.
[2] MART
NEZ-ZAMORA L, CASTILLEJO N, ARTÉS-HERN
NDEZ F.Postharvest UV-B and UV-C radiation enhanced the biosynthesis of glucosinolates and isothiocyanates in Brassicaceae sprouts[J].Postharvest Biology and Technology, 2021, 181:111650.
[3] ZHANG J, LI X Q, GE P, et al.Sulforaphene:Formation, stability, separation, purification, determination and biological activities[J].Separation &Purification Reviews, 2022, 51(3):330-339.
[4] SHAPIRO T A, FAHEY J W, DINKOVA-KOSTOVA A T, et al.Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates:A clinical phase I study[J].Nutrition and Cancer, 2006, 55(1):53-62.
[5] CAMPAS-BAYPOLI O N, SNCHEZ-MACHADO D I, BUENO-SOLANO C, et al.HPLC method validation for measurement of sulforaphane level in broccoli by-products[J].Biomedical Chromatography, 2009,24:387-392.
[6] SHISHU, KAUR I P.Inhibition of cooked food-induced mutagenesis by dietary constituents:Comparison of two natural isothiocyanates[J].Food Chemistry, 2009, 112(4):977-981.
[7] PILIPCZUK T, KUSZNIEREWICZ B, CHMIEL T, et al.Simultaneous determination of individual isothiocyanates in plant samples by HPLC-DAD-MS following SPE and derivatization with N-acetyl-l-cysteine[J].Food Chemistry, 2017, 214:587-596.
[8] SUN J H, CHARRON C S, NOVOTNY J A, et al.Profiling glucosinolate metabolites in human urine and plasma after broccoli consumption using non-targeted and targeted metabolomic analyses[J].Food Chemistry, 2020, 309:125660.
[9] 娄艳坤, 杜宣利, 张羽霄, 等.冷榨萝卜籽油生产的工艺技术实践[J].现代食品, 2018(16):138-141.
LOU Y K, DU X L, ZHANG Y X, et al.Technology and practice of the production of cold pressed radish seed oil[J].Modern Food, 2018(16):138-141.
[10] KUANG P Q, SONG D, YUAN Q P, et al.Preparative separation and purification of sulforaphene from radish seeds by high-speed countercurrent chromatography[J].Food Chemistry, 2013, 136(2):309-315.
[11] SONGSAK T, LOCKWOOD G B.Glucosinolates of seven medicinal plants from Thailand[J].Fitoterapia, 2002, 73(3):209-216.
[12] BARILLARI J, CERVELLATI R, PAOLINI M, et al.Isolation of 4-methylthio-3-butenyl glucosinolate from Raphanus sativus sprouts (kaiware daikon) and its redox properties[J].Journal of Agricultural and Food Chemistry, 2005, 53(26):9890-9896.
[13] LIM S, LEE J, KIM J K.Analysis of isothiocyanates in newly generated vegetables, Baemuchae (×Brassicoraphanus) as affected by growth[J].International Journal of Food Science &Technology, 2009, 44(7):1401-1407.
[14] SIVAKUMAR G, ALIBONI A, BACCHETTA L.HPLC screening of anti-cancer sulforaphane from important European Brassica species[J].Food Chemistry, 2007, 104(4):1761-1764.
[15] CONAWAY C C, GETAHUN S M, LIEBES L L, et al.Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli[J].Nutrition and Cancer, 2000, 38(2):168-178.
[16] LEE J G, LIM S, KIM J, et al.The mechanism of deterioration of the glucosinolate-myrosynase system in radish roots during cold storage after harvest[J].Food Chemistry, 2017, 233:60-68.
[17] SUN J, WANG Y F, PANG X Y, et al.The effect of processing and cooking on glucoraphanin and sulforaphane in brassica vegetables[J].Food Chemistry, 2021, 360:130007.
[18] TIAN G F, LI Y, CHENG L, et al.The mechanism of sulforaphene degradation to different water contents[J].Food Chemistry, 2016, 194:1022-1027.
[19] WU Y F, ZOU L G, MAO J W, et al.Stability and encapsulation efficiency of sulforaphane microencapsulated by spray drying[J].Carbohydrate Polymers, 2014, 102:497-503.
[20] 肖倩, 梁浩, 袁其朋.温度、pH和光照对莱菔硫烷水溶液稳定性的影响[J].中国药学杂志, 2007, 42(3):193-196.
XIAO Q, LIANG H, YUAN Q P.Effect of temperature, pH and light on the stability of sulforaphane solution[J].Chinese Pharmaceutical Journal, 2007, 42(3):193-196.
[21] WU Y F, MAO J W, MEI L H, et al.Kinetic studies of the thermal degradation of sulforaphane and its hydroxypropyl-β-cyclodextrin inclusion complex[J].Food Research International, 2013, 53(1):529-533.
[22] SONG D, LIANG H, KUANG P Q, et al.Instability and structural change of 4-methylsulfinyl-3-butenyl isothiocyanate in the hydrolytic process[J].Journal of Agricultural and Food Chemistry, 2013, 61(21):5097-5102.
[23] TIAN G F, LI Y A, YUAN Q P, et al.The stability and degradation kinetics of Sulforaphene in microcapsules based on several biopolymers via spray drying[J].Carbohydrate Polymers, 2015, 122:5-10.
[24] 张静. 西蓝花中萝卜硫素的提取工艺优化及其活性功能研究[D].哈尔滨:哈尔滨商业大学, 2021.
ZHANG J.Optimization of extraction process and activity function of sulforaphane from broccoli[D].Harbin:Harbin University of Commerce, 2021.
[25] YANG Y, YU H M, ZHOU X H.Covalent immobilization of thioglucosidase from radish seeds for continuous preparation of sulforaphene[J].Chemical Engineering Research and Design, 2020, 155:146-155.
[26] 程立, 李思彤, 袁其朋.黑芥子酶固定化在制备莱菔素中的应用[J].中国科学(化学), 2018, 48(6):676-682.
CHENG L, LI S T, YUAN Q P.The application of myrosinase immobilized in the production of sulforaphene[J].Scientia Sinica Chimica, 2018, 48(6):676-682.
[27] ZHANG J E, FENG C R, TAN X M, et al.Effect of aliphatic diamine spacer length on enzymatic performance of myrosinase immobilized on chitosan microsphere and its application for sulforaphene production[J].Journal of Biotechnology, 2019, 299:79-85.
[28] HWANG E S, KIM G H.Effects of various heating methods on glucosinolate, carotenoid and tocopherol concentrations in broccoli[J].International Journal of Food Sciences and Nutrition, 2013, 64(1):103-111.
[29] TABART J, PINCEMAIL J, KEVERS C, et al.Processing effects on antioxidant, glucosinolate, and sulforaphane contents in broccoli and red cabbage[J].European Food Research and Technology, 2018, 244(12):2085-2094.
[30] LIU Y B, ZHANG D, LI X D, et al.Enhancement of ultrasound-assisted extraction of sulforaphane from broccoli seeds via the application of microwave pretreatment[J].Ultrasonics Sonochemistry, 2022, 87:106061.
[31] LU Y J, PANG X Y, YANG T B.Microwave cooking increases sulforaphane level in broccoli[J].Food Science &Nutrition, 2020, 8(4):2052-2058.
[32] VERKERK R, DEKKER M.Glucosinolates and myrosinase activity in red cabbage (Brassica oleracea L.var.Capitata f.rubra DC.) after various microwave treatments[J].Journal of Agricultural and Food Chemistry, 2004, 52(24):7318-7323.
[33] WU Y F, LV C Z, ZOU L G, et al.Approaches for enhancing the stability and formation of sulforaphane[J].Food Chemistry, 2021, 345:128771.
[34] 姜睿, 李红燕, 薛长湖.不同处理方法对莱菔子中莱菔素和萝卜硫苷的影响[J].食品科技, 2019, 44(11):84-90.
JIANG R, LI H Y, XUE C H.Effects of different methods on glucoraphanin and sulforaphane contents in radish seed[J].Food Science and Technology, 2019, 44(11):84-90.
[35] WANG G C, FARNHAM M, JEFFERY E H.Impact of thermal processing on sulforaphane yield from broccoli (Brassica oleracea L.ssp.italica)[J].Journal of Agricultural and Food Chemistry, 2012, 60(27):6743-6748.
[36] PONGMALAI P, DEVAHASTIN S, CHIEWCHAN N, et al.Enhancing the recovery of cabbage glucoraphanin through the monitoring of sulforaphane content and myrosinase activity during extraction by different methods[J].Separation and Purification Technology, 2017, 174:338-344.
[37] WU Y H, LIN Y H, WANG C Y.High hydrostatic pressure treatment induced microstructure changes and isothiocyanates biosynthesis in kale[J].Food Chemistry, 2022, 383:132423.
[38] LIM S, LEE E J, KIM J.Decreased sulforaphene concentration and reduced myrosinase activity of radish (Raphanus sativus L.) root during cold storage[J].Postharvest Biology and Technology, 2015, 100:219-225.
[39] ZHANG J E, ZHOU X, FU M.Integrated utilization of red radish seeds for the efficient production of seed oil and sulforaphene[J].Food Chemistry, 2016, 192:541-547.
[40] GARCA-SALDA
A J S, CAMPAS-BAYPOLI O N, SNCHEZ-MACHADO D I, et al.Separation and purification of sulforaphane (1-isothiocyanato-4-(methylsulfinyl) butane) from broccoli seeds by consecutive steps of adsorption-desorption-bleaching[J].Journal of Food Engineering, 2018, 237:162-170.
[41] KUANG P Q, SONG D, YUAN Q P, et al.Separation and purification of sulforaphene from radish seeds using macroporous resin and preparative high-performance liquid chromatography[J].Food Chemistry, 2013, 136(2):342-347.
[42] MUTO M, HUANG J W, TAKAHASHI H.Effect of water-soluble extracts of radish seed meal on control of lettuce brown leaf spot (Acremonium lactucae Lin et al.)[J].Plant Pathology Bulletin, 2004, 13:275-282.
[43] ZHANG Y, LV C Z, SUN J A, et al.Protective effects of broccoli extracts and sulforaphane against hydrogen peroxide induced oxidative stress in B16 cells[J].Journal of Functional Foods, 2021, 87:104833.
[44] 冯俊杰, 蒋海强, 董梅月, 等.超高效液相色谱-四极杆静电场轨道阱高分辨质谱法分析莱菔子化学成分[J].化学分析计量, 2021, 30(11):14-22.
FENG J J, JIANG H Q, DONG M Y, et al.Analysis of compositions of Raphani Semen by UPLC-Q-orbitrap-MS[J].Chemical Analysis and Meterage, 2021, 30(11):14-22.
[45] XING J J, CHENG Y L, CHEN P, et al.Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica oleracea) seeds[J].Powder Technology, 2019, 358:103-109.
[46] LIANG H, LI C F, YUAN Q P, et al.Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography[J].Journal of Agricultural and Food Chemistry, 2007, 55(20):8047-8053.
[47] 孔凡华, 杨春雪, 方从容, 等.高效液相色谱法测定十字花科蔬菜中萝卜硫素的含量[J].食品与发酵工业, 2021, 47(8):218-223.
KONG F H, YANG C X, FANG C R, et al.Determination of sulforaphane in cruciferous vegetables by HPLC[J].Food and Fermentation Industries, 2021, 47(8):218-223.
[48] ZHANG Y, MAKAZA N, JIANG C M, et al.Supplementation of cooked broccoli with exogenous moringa myrosinase enhanced isothiocyanate formation[J].Food Chemistry, 2022, 395:133651.
[49] LOMELINO C L, ANDRING J T, MCKENNA R, et al.Asparagine synthetase:Function, structure, and role in disease[J].Journal of Biological Chemistry, 2017, 292(49):19952-19958.
[50] SANGTHONG S, WEERAPREEYAKUL N.Simultaneous quantification of sulforaphene and sulforaphane by reverse phase HPLC and their content in Raphanus sativus L.var.caudatus Alef extracts[J].Food Chemistry, 2016, 201:139-144.