CN112314361B - 一种甘蓝富硒栽培的方法 - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/15—Leaf crops, e.g. lettuce or spinach
Abstract
本发明公开了一种甘蓝富硒栽培的方法，即采用不同浓度的硒酵母溶液对甘蓝进行根浇，每隔10天浇灌一次硒酵母溶液，共浇灌4次，实验结果表明，添加浓度为0.4mmol/L时，甘蓝根、叶、球中的总硒含量分别达到25.96mg/kg DW、21.73mg/kg DW、19.09mg/kg DW，且植株生长良好；本申请提供的栽培方法促进了甘蓝的生长、改善了其品质且提高了硒含量，达到了富硒栽培的效果，同时提高了甘蓝中芥子油苷含量，对富硒甘蓝的栽培具有重要的意义。
Description
技术领域
本发明涉及一种富硒甘蓝的培育方法，在甘蓝根部施入一定浓度的硒酵母溶液，使甘蓝吸收、转化硒，并使甘蓝正常生长，从而得到富硒甘蓝。
技术背景
甘蓝（Brassica oleracea L.）是十字花科的一种蔬菜，由于其具有丰富的膳食纤维、矿物质、类胡萝卜素、维生素C、叶酸和硫甙，在世界各地被广泛种植。硒是人和动物必需的微量元素之一，对于人和动物的健康具有重要意义，近年来，通过栽培措施对植物进行硒强化，对于提高蔬菜中的硒含量及改善人体硒不足具有重要的意义。
甘蓝具有一定的硒富集能力，适当浓度的硒处理可以增加甘蓝生长量，丰富营养品质且减轻一些重金属引起的氧化应激。目前对富硒甘蓝的栽培多以硒酸钠或亚硒酸钠等无机硒为硒源，而无机硒毒性大、不利于吸收转化，添加过量的无机硒易导致动植物中毒，且未被吸收转化的无机硒会造成土壤污染。植物吸收的无机硒一般先转化为硒代胱氨酸和硒代蛋氨酸等有机硒形式。
芥子油苷是十字花科植物中的一种主要的次生代谢物，由于其在人类健康及植物防御中的重要作用而受到特别的关注。芥子油苷的降解产物异硫代氰酸盐具有抗癌的作用，同时，芥子油苷其自身的特殊气味能有效地保护植物免受食草动物的侵害。根据现有报道，硒处理一般会降低十字花科植物中芥子油苷含量（Hsu, F.C., Wirtz, M., Heppel,S.C., Bogs, J., Krämer, U., Rausch, T., 2011. Generation of Se-fortifiedbroccoli as functional food: impact of Se fertilization on S metabolism.Plant Cell Environ. 34,192-207. https://doi.org/10.1111/j.1365-3040.2010.02235.x;Sepúlveda. I., Barrientos. H., Mahn. A., Moenne, A., 2013.Changes in SeMSC, glucosinolates and sulforaphane levels, and in proteomeprofile in broccoli (Brassica oleracea var. Italica) fertilized with sodiumselenate. Molecules. 18, 5221-5234; https://doi.org/10.3390/molecules18055221;Ávila, F.W., Yang, Y., Faquin, V., Ramos, S.J., Guilherme,L.R.G., Thannhauser, T.W., Li, L., 2014. Impact of selenium supply on Se-methylselenocysteine and glucosinolate accumulation in selenium-biofortifiedBrassica sprouts. Food Chem. 165, 578-586. https://doi.org/10.1016/j.foodchem.2014.05.134.)植物可以通过氨基酸通透酶直接吸收有机硒，最常见的两种有机硒形式是硒代蛋氨酸和硒代半胱氨酸，研究发现，小麦和春油菜对有机硒（硒代蛋氨酸和硒代半胱氨酸）的吸收速度是无机硒（硒酸盐和亚硒酸盐）的20倍。目前，针对甘蓝的有机硒栽培尚未见报道。本发明首次通过根施硒酵母的方法进行甘蓝的培育，对比于专利CN104584845（硒酵母栽培油麦菜）叶喷的方式具有更高的安全性。硒酵母是通过发酵技术在含有硒元素的培养基上添加酵母菌而获得的一种有机硒源，因此，采用叶喷的方式可能会导致硒酵母溶液附着于植物叶片表面，从而不利于销售。
发明内容
为了实现富硒甘蓝的规模化生产，本发明的目的在于提供一种富硒甘蓝的栽培方法，同时提升甘蓝中芥子油苷含量。
上述目的是通过如下方式实现的：
一种甘蓝的富硒栽培方法，其具体栽培步骤如下：
第一步，穴盘基质培育甘蓝幼苗，当幼苗长到4-5片真叶时，将其移栽于塑料温室大棚中；
第二步，配制溶液，以硒酵母和清水为原料，制备0.2 -0.4 mmol/L的硒酵母溶液；
第三步，将硒酵母水溶液施入移栽7天后的甘蓝苗根部；甘蓝生长期间根施共4次，每次间隔10天，每次300 mL/株；
第四步，甘蓝生长期间浇水1周/次，常规施肥2次，除草1次，除虫1次，即实现甘蓝的甘蓝的富硒栽培，并提高甘蓝中芥子油苷的含量。
待甘蓝达到生理成熟，分球、叶、根采集，测定生长量、总硒含量、生理指标及抗氧化酶活。经检测，采用本发明的一种富硒甘蓝的培育方法，甘蓝球中总硒含量分别可达到19.09和33.69 mg/kg DW，
符合DBS42/002 规定的富有机硒食品硒含量要求。
本发明利用根部施硒酵母的方式，促进甘蓝的生长量，提高总硒含量，品质指标明显得到优化，增加了甘蓝的营养价值和商品价值。本发明采用有机硒作为硒源，相较于无机硒具有更高的利用率及吸收率，使甘蓝能最大程度的吸收、转化硒，且不产生硒中毒，并提升了甘蓝中芥子油苷含量。硒酵母主要是通过在富硒培养基上培养特定的酵母菌而产生的，一般含有60%-80%硒代蛋氨酸。本专利采用的硒酵母是通过生物发酵获得的天然有机硒产品，其有机硒含量≥98%，故将硒酵母作为富硒栽培的硒源，更安全有效，对富硒甘蓝的规模化种植有较高的应用价值。
附图说明
图1为不同浓度硒酵母水溶液处理后甘蓝根、叶、球中的总硒含量示意图。
图2为甘蓝球中的叶绿素、类胡萝卜素含量示意图；
其中(A)为叶绿素含量示意图，(B)为类胡萝卜素含量示意图。
图3为甘蓝球中可溶性糖、可溶性蛋白、游离氨基酸、维生素C含量示意图；
其中(A)为橄榄球中可溶性糖含量示意图，(B)为甘蓝球中可溶性蛋白含量示意图，(C)为甘蓝球中游离氨基酸含量示意图，(D)为甘蓝球中维生素C含量示意图。
图4为甘蓝球中总酚酸、总黄酮、总芥子油苷的含量示意图；
其中(A)为甘蓝球中总酚酸含量示意图，(B)为甘蓝球中总黄酮含量示意图，(C)为甘蓝球中总芥子油苷的含量示意图。
图5为甘蓝球中超氧化物歧化酶（SOD）和过氧化物酶（POD）含量示意图；其中(A)为甘蓝球中SOD含量示意图，(B)为甘蓝球中POD含量示意图。
具体实施方式
下面结合具体实施案例，进一步阐述本发明。这些实施案例仅用于说明本发明而不用于限制本发明的范围。
硒酵母购自安琪酵母公司（硒含量为2000 mg/kg，中国宜昌），甘蓝的品种为春丰，购于邢台正一种子公司。
1、甘蓝育苗：甘蓝于2018年9月18日使用32孔穴盘基质育苗，将甘蓝种子撒播于穴盘基质上，基质购自于山东莘县一亩田农业科技有限公司（有机物≥50%），育苗环境温度25℃，相对湿度70-80%，光照100 μmol/m2/s，光周期为14/10小时。
2、移栽：2018年10月17日待甘蓝幼苗生长至四至五片真叶时，将幼苗从穴盘中小心取出，移栽至花盆中，花盆口径23.5cm，深度14cm，每盆装土3.5kg，并放置于温室大棚中（土壤pH为7.5；全氮、全磷含量分别为73.53 mg/kg、23.97 mg/kg）。
3、移栽后7天，根浇硒酵母溶液的浓度梯度分别为0 mmol/L、0.1 mmol/L、0.2mmol/L、0.4 mmol/L、0.8 mmol/L，期间每隔10天浇一次，连续浇4次，每次300 ml。
（硒酵母溶液的配置：分别称取0、23.7、47.4、94.8、189.6 g 硒酵母溶于6L清水，搅拌使其充分溶解，即可得终浓度为0 mmol/L、0.1 mmol/L、0.2 mmol/L、0.4 mmol/L、0.8mmol/L的硒酵母溶液。）
4、甘蓝生长期间浇水1周/次，施肥2次，除草1次，除虫1次。
4个月后，采收植株，称量鲜重，并测定生长量（表1）,重复三次，将植株分部分进行干燥，用于总硒含量的测定（图1）。
表1
| 硒处理 (mmol/L) | 球径 (cm) | 球重 (g) | 株重 (g) |
| 0 | 11.4±0.6b | 303.33±60.82b | 539.47±68.48b |
| 0.1 | 12.23±0.86ab | 356.6±68.25b | 639.67±22.88ab |
| 0.2 | 13.1±0.7ab | 440.8±81.97ab | 723.63±74.23a |
| 0.4 | 12.3±0.44ab | 469.3±44.91ab | 772.74±33.32a |
| 0.8 | 14.3±0.6a | 571.13±44.61a | 800.48±46.69a |
从表1和图1的实验结果可见，甘蓝的球径、球重、株重和硒含量都是随着硒酵母浓度的增加而增加。当硒处理为0.2 mmol/L和0.4 mmol/L时，甘蓝球中的总硒含量分别达到19.09 mg/kg DW和33.69 mg/kg DW, 远远高于常德农学会发布的《结球甘蓝富硒栽培技术规程》中的硒含量0.15 mg/kg- 0.50 mg/kg Se 干重。硒代胱氨酸(SeCys2)和甲基硒代半胱氨酸(MeSeCys)是检测到的主要硒形态。
总硒含量的测定：称取0.5 g干燥的样品至于消化管中，加入10 mL HNO3和2 mLH2O2于微波消解仪中消化（YMK40，永乐康仪器有限公司，中国长沙）。消解程序为：120℃下2分钟, 150℃下5分钟和180℃下20分钟。微波消解结束后，将样品转移到加热板上，200℃加热至有白烟冒出，然后加入5ml的6 mol/L HCl，继续加热至溶液澄清。消解好的样品加水稀释至合适的浓度，过滤，备测。采用液相色谱-原子荧光光谱法(LC-AFS8510，北京海光，中国)测定硒的含量。Se标准样品(100ug/mL)购自中国计量科学院，绘制标准曲线。LC-AFS仪器测定程序参考Sun and Feng(Sun, H., Feng, B., 2011. Speciation of organic andinorganic selenium in selenium-enriched eggs by hydride generation atomicfluorescence spectrometry. Food Anal. Method. 4, 240-244. )。
硒形态测定方法：将干燥粉碎后的甘蓝球（0.3 g）与5 mL蛋白酶XIV（4 mg mL-1）混合，并在37°C，200 rpm下过夜震荡。水解后的样品在4°C下以5000 g离心10分钟。使用0.45μm水系滤膜过滤上清液，并通过液相色谱-原子荧光光谱法（LC-AFS8510，中国北京海光）进行硒形态分析。根据峰面积和标准化合物的保留时间，鉴定出的硒种类及含量。甘蓝球硒形态测定结果如表2所示，所有结果均三次重复取平均值。
表2
| 处理 (mg) | 硒代胱氨酸 (μg/ g) | 甲基硒代半胱氨酸 (μg/g) | 硒代蛋氨酸 (μg/g) | 亚硒酸根 (μg/g) | 硒酸根(μg/g) |
| 0 | ND | ND | ND | ND | ND |
| 0.1 | ND | ND | ND | ND | ND |
| 0.2 | ND | 0.360±0.017c | ND | ND | ND |
| 0.4 | ND | 0.992±0.066b | ND | ND | ND |
| 0.8 | 1.380±0.029a | 2.694±0.189a | ND | ND | ND |
硒含量测定完成后，测定甘蓝球中的叶绿素、类胡萝卜素（图2）可溶性糖、可溶性蛋白、游离氨基酸、维生素C含量（图3）和总酚酸、总黄酮、总芥子油苷的含量（图4）。如图2中(A)和(B)所示，与对照相比，甘蓝的叶绿素和类胡萝卜素较对照先增加后下降；如图3中(A)-(D)所示，甘蓝叶球中的抗坏血酸、可溶性糖和游离氨基酸都是随着酵母硒处理的增加而增加，在0.2-0.4 mmol/L处理时，可溶性蛋白含量有下降的趋势，但与对照无显著性差异。
如图4所示，硒酵母处理促进了甘蓝球部总芥子油苷的合成，但总酚酸和总黄酮与对照无显著性差异。现有的研究表明，适量的硒可以促进蔬菜一些营养指标的合成，如游离氨基酸、可溶性糖、色素和黄酮等，且促进了芥子油苷的合成，这证明了硒酵母更适合植物的生长。
叶绿素和类胡萝卜素含量的测定参照Molnárová and Fargasová（Molnárová,M., Fargasová, A., 2009. Se (IV) phytotoxicity for monocotyledonae cereals(Hordeum vulgare L., Triticum aestivum L.) and dicotyledonae crops (Sinapis alba L., Brassica napus L.). J. Hazard. Mater. 172, 854-61.）。抗坏血酸，可溶性糖，可溶性蛋白和游离氨基酸含量的测定分别通过Guo等（Guo, L., Yang, R., Wang, Z.,Guo, Q., Gu, Z., 2014. Effect of NaCl stress on health-promoting compoundsand antioxidant activity in the sprouts of three broccoli cultivars. Int. J.Food Sci. Nutr. 65, 476-481.），Turakainen等（Turakainen, M., Hartikainen, H.,Seppänen, M.M., 2004. Effects of selenium treatments on potato (Solanum tuberosum L.) growth and concentrations of soluble sugars and starch. J.Agric Food Chem. 52, 5378-5382），Zhang等（ Zhang, M., Hu, C., Zhao, X., Tan,Q., Sun, X., Cao, A., Zhang, Y., 2012. Molybdenum improves antioxidant andosmotic-adjustment ability against salt stress in Chinese cabbage (Brassica campestris L. ssp. Pekinensis). Plant Soil. 355, 375-383.）和Hajiboland（Hajiboland, R., 2012. Selenium supplementation stimulates vegetative andreproductive growth in canola (Brassica napus L.) plants. Acta Agr. Slov. 99,13-19.）。甘蓝球中总酚酸、总黄酮和总芥子油苷的含量分别根据Margraf等（Margraf, T.,Karnopp, A.R., Rosso, N.D., Granato, D., 2015. Comparison between Folin-Ciocalteu and Prussian Blue Assays to Estimate The Total Phenolic Content ofJuices and Teas Using 96-Well Microplates. J. Food Sci. 80, C2397-403.）、Li等（Li, Y., Liu, Y., Ma, A., Bao, Y., Wang, M., Sun, Z., 2017. In vitroantiviral, anti-inflammatory, and antioxidant activities of the ethanolextract of Mentha piperita L. Food Sci. Biotechnol. 26, 1675-1683.）和Hsu等（Hsu, F.C., Wirtz, M., Heppel, S.C., Bogs, J., Krämer, U., Khan, M.S., Bub,A., Hell, R., Rausch, T., 2011. Generation of Se-fortified broccoli asfunctional food: impact of Se fertilization on S metabolism. Plant CellEnviron. 34, 192-207.）方法测定。
品质指标测定后，对甘蓝球中超氧化物歧化酶（SOD）和过氧化物酶（POD）进行测定（图5），这些指标反映的是样品的抗氧化能力。由图5（A）可以看出，随着硒处理的增加，甘蓝球中的SOD先增加后下降，说明低浓度的硒处理可以抗氧化，高浓度的硒处理可能会促氧化。图5（B）中发现，硒处理没有促进POD活性的增加，但0.2-0.4 mmol/L处理时，可使其维持在一个相对较高的水平。
从实验结果可见，硒酵母处理能有效促进了甘蓝的生长，提高甘蓝的硒含量。在酵母处理下，甘蓝中可溶性糖、游离氨基酸、抗坏血酸、总酚酸和总芥子油苷的含量增加。大于0.2 mmol/L处理时，抗坏血酸含量、POD和SOD可维持在相对较高的水平。故认为0.2-0.4mmol/L 硒酵母可用于甘蓝的富硒栽培。
本发明通过硒酵母作为硒源，结合根部浇灌的方式培育出来的富硒甘蓝，不仅生产管理工艺简单，容易实施，同时天然安全。与无机硒处理相比，本发明克服了无机硒吸收率低、转化率低、且容易造成土壤硒污染的缺点，通过硒酵母栽培甘蓝，在保证其正常生长的同时，可使其硒含量达到可观的程度，可增加其生物量，提高营养品质，可用于规模化生产富硒甘蓝。
Claims (2)
1.一种富硒甘蓝的栽培方法，其特征在于，具体步骤如下：
1）育苗：穴盘播种，当甘蓝幼苗长到4-5片真叶时，将甘蓝幼苗移栽于大棚；
2）向移栽后的甘蓝幼苗根施硒酵母水溶液，即将0.2 -0.4mmol/L的硒酵母水溶液施入甘蓝幼苗根部，300mL/株；根施共进行4次，每次间隔10天；
3）甘蓝生长期间进行常规的浇水、施肥、除草、除虫管理，即实现甘蓝的富硒栽培；
通过上述步骤，提高甘蓝的球径、球重、株重、可溶性糖、维生素C、游离氨基酸、总酚酸、芥子油苷、超氧化物歧化酶的含量。
2.根据权利要求1所述富硒甘蓝的栽培方法，其特征在于，步骤2）所述移栽后的甘蓝幼苗是指移栽7天后的甘蓝幼苗。
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