• 中国中文核心期刊
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Volume 32 Issue 2
Jul.  2019
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Citation:

Effect of Harvest Time on Forage Quality of Toona sinensis

  • Corresponding author: SU Shang, sssushang@126.com
  • Received Date: 2018-06-29
    Accepted Date: 2018-10-30
  • Objective To reveal and compare the contents of crude protein, flavonoids, nitrates and their antioxidant activity of Toona sinensis harvested in different time, and to find the optimal harvest time for fodder utilize. Method The contents of crude protein, flavonoids and total nitrates of rachises of T. sinensis harvested from May to November were analyzed, and their antioxidant activity were determined by ORAC, DPPH and FRAP. Combining the nutritional and healthy analysis, the optimal harvest time was determined. Result There were significant differences in the crude protein, flavonoids, total nitrates contents and antioxidant activity of T. sinensis harvested in different time(P < 0.05). The crude protein content (varied from 13.17% to 22.65%) presented a downward trend with the maturity of T. sinensis. But except the samples harvested after mid-October, the rest samples showed protein forage advantage with the crude protein contents higher than 15%, which were far higher than the conventional grain feedstuffs. The flavonoids content of T. sinensis harvested at any time were highlighted (15.87 mg·g-1 in average), which was far higher than that of the common fruits and vegetables, such as blueberries, and was persistently accumulated with the maturity of rachises, and reached the highest in October (27.02 mg·g-1). The results of ORAC, DPPH and FRAP assay all showed outstanding antioxidant activities which increased with the maturity of rachises. Meanwhile, very significant positive correlations (P < 0.01) were detected between the flavonoids content and the antioxidant activities. A large amount of nitrates was detected in T. sinensis from different harvest time (2 306.39~7 346.80 mg·kg-1). The accumulation of nitrates in T. sinensis followed a V-shape along with its maturity. The T. sinensis harvested in the mid-July and August, with the average nitrates content of 2 603.78 mg·kg-1, contained the lowest nitrates content. Conclusion The feeding quality of T. sinensis harvested in different time is proved to be significantly different. Comprehensively considering the nutritional value and health care effect of T. sinensis, the mid-July and August are the optimum harvesting time for the forage development of T. sinensis.
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Effect of Harvest Time on Forage Quality of Toona sinensis

    Corresponding author: SU Shang, sssushang@126.com
  • Key Laboratory of Tree Breeding and Cultivation, National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

Abstract:  Objective To reveal and compare the contents of crude protein, flavonoids, nitrates and their antioxidant activity of Toona sinensis harvested in different time, and to find the optimal harvest time for fodder utilize. Method The contents of crude protein, flavonoids and total nitrates of rachises of T. sinensis harvested from May to November were analyzed, and their antioxidant activity were determined by ORAC, DPPH and FRAP. Combining the nutritional and healthy analysis, the optimal harvest time was determined. Result There were significant differences in the crude protein, flavonoids, total nitrates contents and antioxidant activity of T. sinensis harvested in different time(P < 0.05). The crude protein content (varied from 13.17% to 22.65%) presented a downward trend with the maturity of T. sinensis. But except the samples harvested after mid-October, the rest samples showed protein forage advantage with the crude protein contents higher than 15%, which were far higher than the conventional grain feedstuffs. The flavonoids content of T. sinensis harvested at any time were highlighted (15.87 mg·g-1 in average), which was far higher than that of the common fruits and vegetables, such as blueberries, and was persistently accumulated with the maturity of rachises, and reached the highest in October (27.02 mg·g-1). The results of ORAC, DPPH and FRAP assay all showed outstanding antioxidant activities which increased with the maturity of rachises. Meanwhile, very significant positive correlations (P < 0.01) were detected between the flavonoids content and the antioxidant activities. A large amount of nitrates was detected in T. sinensis from different harvest time (2 306.39~7 346.80 mg·kg-1). The accumulation of nitrates in T. sinensis followed a V-shape along with its maturity. The T. sinensis harvested in the mid-July and August, with the average nitrates content of 2 603.78 mg·kg-1, contained the lowest nitrates content. Conclusion The feeding quality of T. sinensis harvested in different time is proved to be significantly different. Comprehensively considering the nutritional value and health care effect of T. sinensis, the mid-July and August are the optimum harvesting time for the forage development of T. sinensis.

  • 香椿(Toona sinensis (A. Juss.) Roem)是楝科(Meliaceae)香椿属(Toona)多年生落叶乔木,广泛分布于黄河至长江流域,在我国已有2 000多年的栽培历史,种植面积已超6万hm2。目前,香椿的开发利用及相关研究多集中在其嫩芽的营养、保健功能测评、加工工艺开发以及逆境生理等方面[1-2],而大量的成熟复叶仍处于浪费状态,鲜有研究。本团队前期研究表明,香椿的成熟复叶年产量可达66~104 t·hm-2,其粗蛋白含量均值约17%,富含氨基酸、矿物质等营养成分,且羊喜食,饲用效果良好,属高蛋白型木本饲料原料,具有较高的饲用开发价值[2]。同时,香椿成熟复叶富含类黄酮,在常见56种果蔬中抗氧化活性最高,并具有突出的抑菌、消炎、抗癌等活性功效[3-6]。可见,香椿成熟复叶的饲用开发除避免资源浪费外,还有助于减少养殖环节的抗生素用量。

    研究表明不同采收期获得的香椿复叶,其营养品质差异极大,如粗蛋白、可溶性糖、维生素C等,都呈现出生长初期较低,随复叶成熟度增加而不断增加,到衰老期前后开始下降的趋势[7]。同时,不同采收期香椿类黄酮等次生代谢物的积累量也差异显著(P < 0.01),其11月的积累量是5月的7.25倍[8]。此外,杨玉珍等[9]研究发现,香椿可大量富集硝酸盐,采收期跨度仅4月1日到13日间,其硝酸盐积累量即可从438.58 μg·g-1增加至2 950.93 μg·g-1,入列蔬菜卫生标准中硝酸盐严重污染范畴[10]。通常,硝酸盐本身不具毒性,少量摄入有利于NO信号转导,有益心血管健康[11];但由于其可在体内代谢过程中形成有毒的亚硝酸盐和致癌物质亚硝胺等,大量摄入则会严重威胁人畜健康。综上,本研究拟比较不同采收时期的香椿复叶中粗蛋白、总黄酮、硝酸盐含量及其抗氧化活性的差异,并综合考虑其生物量,筛选饲用香椿的最佳采收期,为其产业化开发提供依据。

1.   材料与方法
  • 供试香椿种植于北京阳坊镇金太阳农场,为北京门头沟同年同株种子种植所得的4年生苗木。试验所用复叶均源自农场内随机标定的3株香椿,于5月15日至11月1日间,每月15日采收一次香椿复叶。每次均从已标定的3株香椿的相同部位采收无病虫害、长势一致的复叶,将所得样品分别标记后,置于冰盒中,尽快运回实验室,置于-20℃冰箱中保存,后续分别对其进行测评。

  • 参照国家标准GB5009.5-2010《食品中蛋白质的测定—凯氏定氮法》。

  • 类黄酮提取:液氮研磨香椿复叶,取约0.5 g粉末,置于离心管中,加5 mL 0.2%甲酸甲醇(v:v),震荡混匀,室温超声20 min,4℃ 10 000 r·min-1离心5 min,取上清液。此后,依次向沉淀中分别加入3、2、1 mL 0.2%甲酸甲醇,重复超声提取至沉淀无色。合并上清,加入占其总体积30%的超纯水,混合均匀,于-20℃冰箱静置过夜(>12 h)。取出样品,4℃ 10 000 r·min-1离心10 min,留上清液备用。

    类黄酮含量测定:参考陈丛瑾等[12]的方法。取1 mL类黄酮抽提液于离心管中,加入50 g·L-1 NaNO2溶液0.7 mL,混合均匀,静置5 min;加入100 g·L-1 Al(NO3)3·9H2O 0.7 mL,混匀,静置6 min;加入10 mL添加了30%超纯水的0.2%甲酸甲醇(v:v),混匀后,加40 g·L-1 NaOH溶液5 mL,并用添加了30%超纯水的0.2%甲酸甲醇定容至25 mL,静置5 min;超声振荡2 min,此后测定其在510 nm处的吸光度。

    制作标准曲线:用提取液稀释出终浓度为12.5、25、50、100、150、200、250、300 mg·L-1的芦丁标准溶液,用上述方法测定,并绘制标准曲线。本研究所得标准曲线为:y=0.000 4 x+0.006 3(r2=0.999 7),测定结果以每g样品中所含芦丁当量的mg数表示(n=3)。

  • ORAC法:以生育酚为标准品,参考Su等[13]方法进行。本研究所得标准曲线为y = - 3.389 x2 + 20.341 x - 0.098 5 (r2 = 0.999 8),测定结果以每g样品中所含生育酚当量(TE)的μmol·L-1数表示(n = 3)。DPPH法:以没食子酸(GA)为标准品,参考Wang等[14]方法进行。本研究所得标准曲线为y = 1.689 5 x + 0.038 8 (r2 = 0.999),测定结果以每g样品中所含没食子酸当量(GAE)的mg数表示(n= 3)。FRAP法:以GA为标准品,具体测定方法参考Wang等[14]。本研究所得标准曲线为y = 3.034 3 x - 0.066(r2 = 0.999 4),测定结果以每g样品中所含GAE的mg数表示(n=3)。

  • 硝酸盐提取:液氮研磨香椿复叶,取约1 g粉末于50 mL离心管中,依次加入煮沸冷却后的超纯水20 mL,1 mol·L-1氢氧化钾溶液0.25 mL,振荡混匀,超声萃取30 min,于75℃水浴5 min,将溶液移至容量瓶中并定容至25 mL。10 000 r·min-1离心20 min,留上清,用孔径为0.22 μm的滤膜过滤,备用。

    含量测定:用Dionex ICS-3000型离子色谱检测。色谱条件:色谱柱型号SP6949,抑制器为Dionex ASRS 300,淋洗液A为超纯水,淋洗液B为200 mmol·L-1氢氧化钠。淋洗程序:85% A和15% B,在30℃柱温下,以1.0 mL·min-1流速,等梯度淋洗10 min。

    制作标准曲线:取终浓度为6.25、12.5、25、50、100、200、400 mg·L-1的亚硝酸盐和硝酸盐系列混合标准溶液,以上述方法测定,并绘制标准曲线。本研究所得硝酸盐标准曲线为:y = 0.045 3x - 0.051 4(r2 = 0.999 8);亚硝酸盐标准曲线为:y = 0.053 1x - 0.101 7(r2 = 0.999 9)。样品总硝酸盐含量测定结果以每kg样品中所含硝酸盐及亚硝酸盐的总mg数表示(n = 3)。

  • 试验数据用Excel、SPSS 21.0软件进行分析处理,用Origin 9.0软件制图。

2.   结果与分析
  • 图 1可知,不同采收期的香椿复叶粗蛋白含量约13.17%~22.65%,各采收期间差异显著(P < 0.05),且随复叶成熟度增加而呈下降趋势。其中,5月15日采收的复叶粗蛋白含量最高,为22.65%;11月1日所得样品中含量最低,为13.17%。不同采收期的香椿复叶中粗蛋白含量均值为17.30%±0.94%,除10月后的样品外,其余采收期香椿复叶的粗蛋白含量均大于15%,远高于饲料数据库中的粮食饲料,如玉米(9.4%)、高粱(8.7%)、小麦(13.4%),与优质牧草苜蓿(17.2%)的粗蛋白含量相近[13]。可见,除处于衰老期(10月15日及之后采收)的香椿复叶外,其余采收期获得的香椿叶片均具有高蛋白的饲用优势。

    Figure 1.  The crude protein contents of Toona sinensis from different harvest time(Means ± SE, n=3)

  • 不同采收期的香椿复叶类黄酮含量差异极显著(P < 0.01),且随其不断发育成熟而持续积累,至落叶期稍有下降(图 2)。香椿复叶类黄酮积累量在其发育前期(5—7月)增长缓慢,在生长旺盛期(7月后)积累量显著增加(P < 0.05),并于10月达到最高,为27.02 mg·g-1,此后在落叶期(11月)开始下降,降至22.93 mg·g-1

    Figure 2.  Total flavonoid contents of Toona sinensis from different harvest time(Means ± SE, n=3)

  • 图 3可知,ORAC、DPPH、FRAP法测定结果一致,5月15日样品抗氧化活性最低,为205.46 μmol·L-1·g-1(ORAC)、2.35 mg·g-1(DPPH)和1.79 mg·g-1(FRAP),此后其抗氧化活性随复叶不断成熟而持续增加。其中,ORAC和FRAP法测定显示9、10月较其它采收期(5—8月)样品差异显著(P < 0.05),而DPPH法测定7—11月样品抗氧化活性无显著差异。

    Figure 3.  Antioxidant activity of Toona sinensis from different harvest time(Means ± SE, n=3)

  • 表 1可知,香椿复叶抗氧化活性与其类黄酮含量呈极显著(P < 0.01)正相关,ORAC、DPPH及FRAP与类黄酮含量的相关系数分别为0.894、0.891和0.979。可见,类黄酮是香椿抗氧化活性的重要贡献因子,对其保健活性强弱有重要影响。

    项目Items ORAC DPPH FRAP
    类黄酮Flavonoids 0.894** 0.891** 0.979**
    PP value 0.007 0.007 0.000
    **在0.01水平上显著相关。** Correlation is significant at the 0.01 level.

    Table 1.  Correlation coefficients of total flavonoid content and each antioxidant activity assay of Toona sinensis

  • 图 4可知,香椿大量富集硝酸盐,且随复叶不断成熟呈“V”形积累,7、8月样品硝酸盐积累处于峰谷,含量明显低于其它阶段。其中,7月15日采收的复叶中硝酸盐积累量最少,为2 306.39 mg·kg-1,8月中旬后硝酸盐积累量迅速增加,并于10月15日达最大值,为7 346.80 mg·kg-1

    Figure 4.  Total nitrate contents of Toona sinensis from different harvest time(Means ± SE, n=3)

3.   讨论
  • 本研究中各采收期香椿复叶粗蛋白含量均值达17.30%±3.53%,远高于常见粮食饲料[15],略高于常规木本饲料Morus alba(粗蛋白含量15.43%)、Acacia nilotica(11.81%)、Melia azedarach(14.09%)等[16],及新型木本饲料Brosimum alicastrum(14.1%±0.04%)、Guazuma ulmifolia(12.9%±0.32%)、Casimiroa edulis(15.4%)等,与优质豆科木本饲料Acacia angustissima(22.7%)Acacia pennatula(19.6%)等相近[17-18],具有较高的饲用开发价值。

    同时,本研究表明不同采收期香椿复叶粗蛋白含量差异显著(P < 0.05),随复叶不断发育成熟呈下降趋势。Azim等[19]研究同样表明,刺槐、桑树、杨树叶片的蛋白含量分别自春季的23.9%、17.6%、11.2%下降至冬季的14.5%、13.7%及10.0%。Parlak等[20]分析Quercus infectoria Oliv及Paliurus spina-cristi Mill.全季饲用品质时也发现,二者粗蛋白含量分别从4—5月的180.0、220.4 g·kg-1下降至10—11月的67.9、84.7 g·kg-1。因此,从粗蛋白含量考虑,木本饲料的采收不宜选其衰老期,香椿复叶的饲用采收则不宜选在10月之后。

  • 香椿复叶类黄酮含量丰富(5.76~27.02 mg·g-1),且随复叶成熟而持续增加,全季均值达15.87 mg·g-1,远高于常见果蔬,也远高于以类黄酮含量高而闻名的蓝莓(约1.69 mg·g-1)、蔓越橘(约1.39 mg·g-1)等有色浆果[21]。同时,丰富的类黄酮含量赋予了香椿复叶突出的抗氧化活性,其ORAC、DPPH及FRAP抗氧化活性均值分别为666.28 μmol·L-1·g-1、4.45 mg·g-1及3.96 mg·g-1,远高于常见果蔬[3],也远高于公认抗氧化活性优异的蓝莓及银杏等[13, 22]。此外,由于类黄酮具有消炎、抑菌等保健功效,Ma等[23]研究证明,在日粮中添加2 g·只-1·天-1桑叶类黄酮提取物,可发挥其抑菌活性,能够显著(P < 0.01)提高杜泊羊的消化性能并减少甲烷排放。Crespo等[24]研究同样表明类黄酮能够增强瘤胃发酵的稳定性,具有开发为抗生素替代品的潜力。可见,香椿成熟复叶的饲用开发对践行绿色低抗养殖具有重要意义。

    此外,前人研究表明植物次生代谢物,如烯萜类化合物等,能够从日粮中转移到动物产品,特别是奶制品(牛奶、乳酪等)中,直接对产品成分组成、风味特性及功能活性造成影响[25-27]。De Feo等[28]研究证实,以类黄酮含量丰富的Borago officinalis L.和Crataegus oxyacantha L.为山羊饲料,饲料中的芦丁等类黄酮成分可转移至羊奶中,提升羊奶品质,且芦丁自身结构保持不变。由此可见,香椿复叶不仅可作为高蛋白饲料原料,还具有开发为低抗保健、风味改良等功能型饲料的潜在价值。结合本研究数据,从香椿复叶类黄酮含量及抗氧化活性考虑,其饲用采收不应早于7月。

  • 全生长季香椿复叶中总硝酸盐含量均值达4 941.83 mg·kg-1,最高值为7 346.80 mg·kg-1,远高于蔬菜食用安全限量3 100 mg·kg-1[10],且远高于常见果蔬,如Lactuca sativa L.(硝酸盐含量33~2 304 mg·kg-1)、Spinacia oleracea L.(545~3 760 mg·kg-1)、Solanum lycopersicum L.(190~347 mg·kg-1)等;与Allium tuberosum L.(3 016~9 638 mg·kg-1)、Ocimum basilicum L.(4 040~5 350 mg·kg-1)、Anethum graveolens L.(2 670~5 290 mg·kg-1)相当[29]。就其饲用开发而言,近年研究表明,适量硝酸盐摄入不仅可作为非蛋白氮为反刍动物提供补给,还可促进反刍动物瘤胃发酵,提高饲料利用率及动物生产性能,减少温室气体甲烷、N2O等排放[30]。前人在牛、羊日粮中添加2.1%~3.04%硝酸盐,均未发现其采食量、增重及产奶量受到负面影响,而其甲烷排放可降低16.5%~35%[31-33]。但是,不同动物的硝酸盐中毒剂量尚未明确,单胃动物对硝酸盐的耐受性远不及反刍动物。

    一般认为日粮中添加超过2.5%的硝酸盐即可能对动物采食及生产性能造成负面影响,但预饲过程及饲喂方式能在很大程度上提高动物对硝酸盐的耐受性[30]。此外,饲料本身的遗传背景、生长环境、农艺管理措施、采收期、采后处理等同样会影响其硝酸盐积累量[29]。综上,香椿复叶中丰富的硝酸盐积累并不可否定其饲用价值,特别是其对于反刍动物的饲用潜力,但其中硝酸盐对其饲用效果的具体影响仍需进一步研究验证。结合本研究结果,7—8月中旬的香椿复叶硝酸盐含量(均值2 603.78 mg·kg-1)明显低于其它时期,相对更适于进行饲用采收。

    此外,资料显示不同遗传背景的香椿,其营养品质存在较大差异。何丹[7]分析发现,不同种源的香椿蛋白含量差异较大。刘常金等[8]分析显示,北方种源的香椿类黄酮含量明显高于南方种源,如郑州种源是镇江种源的两倍多。杨玉珍等[9]研究表明,不同种源香椿硝酸盐含量相差可达6倍,差异极显著(P < 0.01)。除种源间差异外,梁有旺等[35]发现,同一种源香椿的不同单株间同样存在品质差异。可见,香椿良种选育及种质改良均具有较大的可操作空间,且在进行种源比对的同时,有必要针对性地对香椿进行优株筛查,以充分挖掘和利用种质优势。因此,本团队后续研究将把香椿饲用良种筛选及农艺管护措施探索相结合,为香椿产业化饲用开发夯实基础。

4.   结论
  • (1) 香椿复叶粗蛋白含量突出(13.17%~22.65%),衰老期前(10月中旬前)其粗蛋白含量均大于15%,具有开发为高蛋白木本饲料的优势。

    (2) 香椿复叶类黄酮含量丰富(5.76~27.02 mg·g-1),抗氧化活性突出,在落叶前(11月初),随香椿不断成熟,其类黄酮的积累量及抗氧化活性均持续增加,且二者极显著(P < 0.01)正相关。

    (3) 香椿复叶硝酸盐含量较高(2 306.39~7 346.80 mg·kg-1),不同采收期差异显著(P < 0.05),生长全期呈“V”形积累,7—8月间硝酸盐积累量最低(均值2 603.78 mg·kg-1),相对更适于饲用采收。

    (4) 基于香椿粗蛋白、类黄酮、硝酸盐含量及其抗氧化活性分析,综合考虑营养价值及保健功效,认为7—8月中旬是香椿饲用开发的最适采收期。

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