CN102132778B - 植物和植物提取物作为饲料添加剂影响瘤胃发酵的用途 - Google Patents
植物和植物提取物作为饲料添加剂影响瘤胃发酵的用途 Download PDFInfo
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- CN102132778B CN102132778B CN201110049502XA CN201110049502A CN102132778B CN 102132778 B CN102132778 B CN 102132778B CN 201110049502X A CN201110049502X A CN 201110049502XA CN 201110049502 A CN201110049502 A CN 201110049502A CN 102132778 B CN102132778 B CN 102132778B
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Abstract
植物和植物提取物作为饲料添加剂影响瘤胃发酵的用途。选自Eugenia caryophyllata(丁香)、Bellis perennis(雏菊)、Olea europaea(带根橄榄)、Symphytum officinale(聚合草)、Carduus pycnocephalus(芸香苷)、Paeoniae alba radix(白芍)、Populus tremula(欧洲山杨)、Prunus avium(甜樱桃)、Salix caprea(黄花柳)、Rheum nobile(高山大黄)、Helianthemum canum、Arctostaphylos uva-ursi(熊果)、Peltiphyllum peltatum(盾叶草)、Epilobium montanum、Knautia arvensis(欧洲山萝卜)、Latuca sativa(莴苣)和Urtica dioica(异株荨麻)及其提取物以及β-香叶烯的植物材料、植物提取物和天然等同组分的用途，用于影响瘤胃发酵以及用于增加反刍动物对能量和氮源的可利用性。
Description
本申请是基于中国专利申请号200580011439.3的分案申请。
技术领域
本发明涉及一种添加剂用于提高瘤胃发酵中能量或氮的保留，或者用于减少乳酸酸中毒的用途，所述添加剂含有来自植物材料、植物提取物或天然等同物的精油化合物中的一种或多种组分。所述化合物适于通过动物饲料或饮用水以口服方式施予反刍动物。
发明背景
瘤胃是反刍动物，例如骆驼、绵羊和山羊的第一个胃。其结构和营养意义如Hungate(1966)所述。瘤胃使这些动物能消化对于大多数非反刍动物来说不适合的高纤维植物材料。瘤胃是很大的器官，奶牛中瘤胃含有超过150kg的消化物，其中所有消化都是通过微生物来进行的。瘤胃以下述方式进化，所述方式使得动物能够从微生物的纤维消化活动获得好处。终产物，挥发性脂肪酸通过瘤胃壁被吸收，用于能量和蛋白合成。瘤胃前肠发酵较之后肠发酵的主要好处是发酵期间形成的微生物细胞转到皱胃，在那里它们会被消化，其氨基酸能被吸收。因此，与后肠发酵不同，微生物的氨基酸可被宿主动物利用。因此瘤胃是生长在贫瘠牧场上的食草动物进化出的高效器官。
瘤胃发酵也带来一些缺点。甲烷作为厌氧发酵的天然后果产生(Hungate，1966)。甲烷的释放还体现了来自动物的能量损失。此外，甲烷是潜在的温室气体，所以，反刍动物会损害环境(Leng，1992)。反刍动物在对膳食蛋白的利用上较之其它物种效率更低。反刍动物的氮损失例外地高，尤其是牧草动物(Nolan，1975)。这是环境问题也是经济问题，因为富含氮的排泄物对环境造成影响。此外，反刍动物还特有作为瘤胃微生物发酵受损的直接后果发生的消化紊乱。
过去化学和抗生素饲料添加剂已被用于减轻这些问题中的一些。此类物质在食物链中的存在对于调控机构和消费者来说愈发不被接受了。因此，对于反刍牲畜问题的解决方法应当是天然的、可持续的。
进入反刍动物的膳食蛋白以明显不受控制的途径分解，这导致氨的形成以及随后尿中氮的流失(Nolan，1975)。氮保持(retention)的低效率，代表很大的经济损失，导致动物的新陈代谢压力，还通过富含氮的垃圾对环境造成了负担。如果分解过程可被减少，这些问题也将减少。
此外，提高反刍动物中蛋白保持的第二种可能性是抑制反刍动物瘤胃纤毛原生动物的种群。原生动物消耗瘤胃中大量的细菌，它们的分解能导致从瘤胃发酵来的微生物蛋白的净产量降低高达50％(Wallace et al.，1997)。如果原生动物可被抑制，形成的氨将会更少，对膳食蛋白补充的需要也更少。
正常情况下，乳酸仅仅是瘤胃发酵的次要产物。但是，当过快引入迅速降解的饲料时，或者当浓缩物(concentrate)占到膳食中的高比例时，挥发性脂肪酸的产量将超过瘤胃的缓冲能力。这可能导致瘤胃中很低的pH值，这样就仅有产乳酸细菌能够生长了(Russell & Hino，1987)。
因为乳酸比挥发性脂肪酸酸性更强，pH降得甚至更多，所以不可能恢复正常发酵，动物会死亡。但是，更典型地，高浓缩物膳食的反刍动物会遭遇亚临床酸中毒，这是痛苦的，并且会降低生产效率，虽然没有生命威胁。化学缓冲剂可以改善这种情况，但是，如果产乳酸细菌的生长能被抑制将会更好。
公开文本WO 03/094628描述了如下尝试：通过添加一种或多种精油化合物，降低反刍动物消化活动导致的甲烷产生，所述精油化合物选自由柠檬油精、丁香油酚、水杨酸酯、喹啉、香草、百里酚和甲酚所构成的组。同时，瘤胃中的微生物活动和挥发性脂肪酸(例如丙酸)的产生可被保持为可接受的水平。
发明目的和发明内容
本发明的目的是：一般地、集中性地解决瘤胃中的消化紊乱，提高对能量的利用，以及增加反刍动物生长可获得的氮源。该目的可通过下述方法达到：有利于挥发性脂肪酸的形成(所述脂肪酸可经过瘤胃壁被吸收，可用于能量和蛋白合成)，以及减少膳食蛋白和微生物蛋白的分解。其它方法是用于限制甲烷形成以及用于控制或抑制产乳酸细菌的。
根据本发明，可通过下述方法来达到所述的目的，将一种添加剂通过口服施予(适合地，通过动物饲料或饮用水)，所述添加剂含有：选自由Lonicera japonica、Gentiana asclepidea、Gentiana lutea、Eugeniacaryophyllata、Bellis perennis、Olea europaea、Symphytum officinale、Carduus pycnocephalus、Paeoniae alba radix、Populus tremula、Prunusavium、Salix caprea、Rheum nobile、Helianthemum canum、Arctostaphylosuva-ursi、Peltiphyllum peltatum、Epilobium montanum、Knautia arvensis、Latuca sativa和Urtica dioica所构成的组的植物材料及其提取物和β-香叶烯中的一种或多种组分。施予动物的组分的总量适合在每天每kg体重0.02mg至20g之间。
本发明的组分全都显示出如下性质：明显有用于瘤胃发酵效率，而没有任何明显的缺陷。这些组分中的一些会抑制原生动物的活性，其它组分会降低蛋白质水解活性、甲烷形成活性或乳酸的产生。
本发明还涉及含有所定义组分中一种或多种的添加剂和饲料组合物。
发明详述
因为所述组分可以以不同途径影响发酵，因此，根据本发明的目的将特定化合物组合以优化发酵是有利的。
广泛的研究已经显示，来自Helianthemum canum、Arctostaphylos uva-ursi、Epilobium montanum、Knautia arvensis和Peltiphyllum peltatum的植物材料及其提取物所构成的亚组的组分会很可观地降低瘤胃的蛋白质水解。此外，这些组分全都能抑制原生动物的活性，它们中的大多数还对其它重要的发酵参数(例如，甲烷产量、挥发性脂肪酸的含量、消化率、微生物生物质和/或发酵效率)有有利影响。最合适的组分是来自Arctostaphylos uva-ursi、Knautia arvensis和Helianthemum canum的植物材料及其提取物。最优选的组分是来自Knautia arvensis的植物材料及其提取物，尤其是甲醇提取物。
来自Lonicera japonica、Gentiana asclepidea、Gentiana lutea、Eugeniacaryophyllata、Bellis perennis、Olea europaea和Symphytum officinale的植物材料及其提取物和β-香叶烯形成了另一亚组，该亚组的组分能抑制瘤胃流体中纤毛原生动物的溶菌活性。Eugenia caryophyllata还能增加微生物生物质，降低甲烷形成。Bellis perennis对甲烷产生有少量降低作用，其还能提高发酵效率以及微生物生物质的产生，而Symphytum officinale能增加发酵效率，降低酸中毒作用。β-香叶烯对原生动物活性具有适当的抑制作用，还发现其具有降低甲烷产生的用途。从Lonicera japonica的花制备的提取物还能抑制纤毛原生动物的溶菌活性。Lonicera japonica的种子制备物也能抑制原生动物活性，但只能到大约60％。从该亚组中优选的组分是来自Bellis perennis、Eugenia caryophyllata、Olea europaea和Symphytumofficinale的植物材料及其提取物，尤其是Bellis perennis和Gentianaasclepidea的甲醇和水提取物。
来自Carduus pycnocephalus、Paeoniae alba radix、Populus tremula、Prunus avium、Salix caprea和Rheum nobile的植物材料及其提取物形成了第三亚组，其组分适合用于降低瘤胃消化物中的甲烷形成活性。对于这些组分而言，没有观察到对其它发酵参数的重要有害作用。优选的组分是来自Carduus pycnocephalus、Populus tremula和Rheum nobile的植物材料及其提取物，尤其优选的是Carduus pycnocephalus和Rheum nobile。
最后，第四亚组由来自Latuca sativa和Urtica dioica的植物材料及其提取物形成。这些组分能降低乳酸的活性和/或形成。Latuca sativa一些时候被称为Latuca virosa，其还能降低甲烷的产生以及原生动物活性，还能增加挥发性脂肪酸的形成。Urtica dioica还有额外的作用，其能抑制蛋白质水解和原生动物活性，并且有利于挥发性脂肪酸的形成。
在本发明的一种合适的实施方式中，添加剂含有来自上文定义的亚组中的不止一组的组分，添加剂含有来自全部四个亚组的组分可能是有好处的。有利地，添加剂含有选自下述每组的至少一种组分，所述的组由下述物质构成：
i)来自Knautia arvensis、Arctostaphylos uva-ursi和Helianthemumcanum的植物材料及其提取物，
ii)来自Bellis perennis、Eugenia caryophyllata、Olea europaea、Lonicera japonica和Symphytum officinale的植物材料及其提取物，
iii)来自Carduus pycnocephalus、Populus tremula和Rheum nobile的植物材料及其提取物，以及
iv)来自Latuca sativa和Urtica dioica的植物材料及其提取物。
属于i)-iv)组的组分的总量适合为：按重量计，根据本发明存在的组分的总量的20-100％，优选地，40-100％。
根据本发明的植物材料的提取物包括但不限于，浸膏(concrete)、精油、树脂质物(resinoid)、酊剂、净油(absolute)和净油(absoluteoil)。当生产植物或植物部分的提取物时，水、有机溶剂及其混合物可以根据传统方法使用，也可以应用干馏。合适的有机溶剂的例子是甲醇、乙醇、丙醇、丁醇、乙酸乙酯、甲乙醚、二乙醚、二氯甲烷、氯仿、四氯化碳、苯、甲苯、石油醚和丙酮。
施予动物的组分的总量取决于组分是精油化合物、植物材料提取物还是植物材料。当组分都是β-香叶烯和/或提取物的时候，施予的量通常在每天每kg体重0.1-50mg，而当组分仅由植物材料构成时，施予的量通常在每天每kg体重0.02-10g。
本发明的添加剂可以含有除本发明组分外的其它成分。合适地，添加剂含有：按重量计，0.1-100％的所述组分，优选地，0.2-90％，以及还含有生长促进剂、香料、吸收支持物和/或其它饲料成分。优选地，生长促进剂、香料、吸收支持物和/或其它饲料成分的总量为：按添加剂重量计10至75％。在任何组分是植物材料的情况下，它们适合被干燥，以及在其与添加剂中其它成分(如果有的话)混合之前被碾为颗粒或粉末。
合适的生长促进剂和香料的例子是甲酚、茴香脑(anethol)、十、十一和/或十二内酯、紫罗兰酮、鸢尾酮、姜酚、哌啶、亚丙基苯酞(phatalide)、亚丁基苯酞、辣椒素和/或丹宁酸。支持物可以含有，例如，按重量计40-50％的木纤维、按重量计8-10％的硬脂酸、按重量计4-5％的姜黄粉、按重量计4-5％的迷迭香粉、按重量计22-28％的石灰石、按重量计1-3％的树胶(例如阿拉伯胶)、按重量计5-50％的糖和/或淀粉以及按重量计5-15％的水。其它饲料成分适合选自由维生素、酶、矿物盐、磨碎的谷物、含有蛋白的组分、含有碳水化合物的组分、小麦粗粉和/或麦麸所构成的组。添加剂合适以下述量加入到根据本发明的饲料组合物中，所述量为：饲料通常将含有按重量计0.4ppm-80％的所述组分。
根据本发明的饲料组合物通常含有下述成分(含量是基于饲料干重计算的)：
a)按干重计0-80％的谷物，
b)按干重计0-30％的脂肪，
c)按干重计0-85％的、不同于谷物的类型的含蛋白营养物，以及
d)按干重计10ppm-40％的本发明的组分。
按干重计，a)-d)的总量优选为至少50％。
当制备所述饲料组合物时，添加剂可与由谷物(例如，碾碎或压碎的小麦、燕麦、大麦、玉米和水稻)、植物蛋白来源(基于，例如，油菜籽、大豆和向日葵籽)、动物蛋白来源、磨拉石和奶制品(例如，多种奶粉或乳清粉)所构成的干燥成分混合。混合所有干燥成分之后，可以加入液体成分以及加热后会成为液体的成分。液体成分可由脂类(诸如脂肪，例如，植物脂肪，可选地，加热后可被液化的)和/或羧酸(例如脂肪酸)构成。充分混合之后，得到粉状或颗粒状的一致性物质，这取决于对成分进行碾磨的程度。为防止贮存期间的分离，优选地，向动物饲料中加入水，然后进行传统的制粒、膨胀或挤出工艺。可以通过干燥除去任何过量的水。如果需要的话，可将得到的颗粒状动物饲料压碎为更小的颗粒大小。所描述的饲料组合物通常与干燥的绿色草料和/或青贮饲料组合施予。
饮用水补充剂可以含有按干重计至少1％的所述组分，适合地，按干重计1-99％，优选地，按干重计10-50％。除一种或多种所述组分之外，所述补充剂还可以含有按干重计1-99％的大量其它成分。其它成分的合适例子是，矿物盐、维生素、增强健康和生长的添加剂、香料、水溶性的或水可分散的载体(例如，糖、奶粉、奶副产品和纤维素衍生物)、分散剂和稳定剂(例如，水溶性的或水可分散的聚合物)和其混合物。当制备饮用水时，补充剂通常以如下量添加，所述量使得所述组分的浓度为按重量计1ppm-10％。
在本发明的范围内，还可以生产饲料组合物的悬浮液。当饲料被制备来用于即时消耗时，这是尤其优选的。
下述实施例将进一步阐述本发明。
实施例
在本发明的实施例中，用植物材料、植物材料提取物和精油化合物的样品进行了大量的试验。植物材料样品被冻干，碾磨，用1mm的筛过筛。样品如下所示。
在下述方面对根据本发明的样品1-22对瘤胃消化物的影响进行了检测，所述方面为：瘤胃纤毛化原生动物的溶菌活性，瘤胃消化物的蛋白质水解活性，瘤胃消化导致的甲烷形成，乳酸酸中毒和挥发性脂肪酸的形成以及对发酵的其它影响。在检验中应用了下述方法。
瘤胃纤毛原生动物的溶菌活性
通过下述方法来测定[14C]亮氨酸标记的Selenomonas ruminantium的分解比例，按照前人所述(Wallace & McPherson，1987)，在存在5mM未标记的L-亮氨酸的条件下，将经过滤的(strained)瘤胃流体与经过标记的S.ruminantium一起体外孵育。在上述条件下，细菌蛋白分解的很大部分是由纤毛原生动物对细菌的摄取和消化导致的。如无特别指明，以5mg/ml的浓度添加样品。绵羊获得混合的干草：浓缩物膳食(Frumholtz etal.，1989)。
抗原生动物活性的持续性
将样品与Coleman’s缓冲液、瘤胃流体或水预孵育24小时，然后在按照上文所述的细菌分解试验中将其与新鲜的瘤胃流体一起孵育。
瘤胃消化物的蛋白质水解活性
方法1.使用Wallace(1983)的14C-酪蛋白方法，用来自相同的绵羊的经过滤的瘤胃流体来测定蛋白质水解活性。加入样品至终浓度为2.5mg/ml，使用1小时的孵育时间。
方法2.基于Hoffman et al.(2003a，b)。五只插管(fistulated)泌乳Holstain奶牛被喂饲典型的乳牛膳食，其被用作为供体动物。在早晨的喂饲之前，从喂饲挡板(feed mat)人工取出瘤胃流体。瘤胃流体经100μm的尼龙网过滤，其与孵育缓冲液以10％(v/v)混合。将体积为75ml的经缓冲瘤胃流体分散到(RPT)孵育瓶中，所述瓶中含有450mg玉米青贮饲料、225mg大麦谷粒(都被碾磨以通过1mm筛)以及蛋白补充剂的底物混合物，所述蛋白补充剂由150mg脱脂粗大豆粗粉和10mg牛血清清蛋白组成。将不加入测试植物的该底物作为阴性对照。当测试植物被加入时，它们代替了青贮饲料。用作为阳性对照的莫能菌素被溶于乙醇中(14mg/ml)，填装至3μM的莫能菌素终浓度之后立即将11.25μl的该贮液加入到每只瓶中。所有的瓶都在39℃孵育长达12小时。以有规律的间隔对每种处理的两份重复样品进行取样，第三份亚重复被留下来用于气体读数。在每次取样时间，在剧烈搅拌下从孵育瓶中取2x900μl，将它们用于测定SCFA、氨和蛋白浓度。通过Laemmli方法来进行SDS-PAGE，根据Hoffman et al.(2002)的点杂交印迹来对总蛋白进行定量。
对瘤胃发酵的一般影响
针对发酵一般影响的Hohenheim方法
实验设计
孵育系统：读取式压力技术(RPT)，75ml，总运行时间24小时
供体动物：3只非泌乳Holstein奶牛
Basal底物：草青贮饲料，0.750g(对照)
测试植物的内含水平：10％(代替青贮饲料)
气体读数：2、4、6、8、10、12、14、16、24小时
取样：对SCFA和TD(NDS处理，在尼龙袋中进行)而言，24小时对SCFA(900μl)和RNA(300μl，被分析的最大值)而言，6、8、10小时
平行：3次独立运行(3只不同的供体动物)，两份重复
RPT缓冲液：
方法详细描述
体外孵育
从插入导管的奶牛收集瘤胃流体，所述奶牛获得8:00和16:00喂饲的两份等量的草青贮饲料干草混合物用于维持。在热水瓶喂饲之前收集瘤胃流体，经过100μm尼龙网过滤，将其加入到被还原的缓冲矿物质溶液中。所有步骤都在39℃于CO2下进行，以保持厌氧条件。
按照10mg/ml培养基的比例来孵育底物，即，750mg底物与75ml经过缓冲的瘤胃流体(含有7.5ml经过过滤的瘤胃流体)(10％v/v瘤胃流体)一起孵育。
在孵育2、4、6、8、10、12、16和24小时时，频繁记录气体体积或压力，在6、8、10和24小时后取样。
分析
在RPT系统的浆液瓶中记录气体压力，使用实验测定的校正曲线将其转化为体积。每次测量后，从浆液瓶中释放气体。通过将孵育内容转移到预先称重的聚酯袋(Ankom 51μm孔径)中来测量体外的真实消化性。将所述的袋热密封，在中性洗涤剂溶液中烹煮1小时，在105℃干燥过夜，称量，用于测定消化性。使用装有GP 10％SP10001％H3PO4、ChromosobW AW不锈钢柱(Suppelco Inc.Bellafonte，PA)，通过气相色谱来测定SCFA。向0.9ml孵育上清液中，加入0.1ml含有内部标准(1％甲基丁酸)的甲酸。蛋白质在4℃沉淀过夜。样品被离心(30,000g，10分钟，4℃)，收集上清液至合适的GC管中，用于分析。
按照改进苯酚氯仿方案来抽提RAN。将600μl pH 5.1的苯酚、270μlpH 5.1的缓冲液、30μl SDS(20％w/v)和1.0g锆珠粒加入到样品(300μl)中。通过在珠粒研磨器(bead-beater，50Hz)中对样品进行2x2分钟的研磨使细胞被裂解，2x2分钟之间是10分钟在60℃的孵育。样品在冰上冷却10分钟，加入300μl氯仿。在剧烈摇晃以及室温下再放置10分钟后，离心样品(10,000g，5分钟，4℃)，分离水相和有机相。定量移走水相，将其转移至含有300μl乙酸铵(7.5M)和900μl异丙醇的管中。在-20℃对样品进行过夜孵育，通过离心(16,000g，10分钟，4℃)沉淀出核酸。弃去上清液，在80％乙醇中洗涤一次样品。将核酸上样至琼脂糖凝胶，用溴化溴乙非啶染色后，针对校正曲线(25-300ng/μl)进行光密度测量定量。
针对对发酵一般影响的读数方法
用读取式压力技术[RPT]进行一系列的十三次连续发酵，每周一次，以确定包括进这些测试材料对基础草料(玉米青贮饲料)的发酵和降解特征的潜在影响。所有材料都按照三份重复在两种内含水平(100和400mgg-1DM底物)被检验，除了β-香叶烯例外(10和40mg g-1内含水平)。玉米青贮饲料被预先干燥，磨碎，经过2mm的筛。将总共1.0g共混的底物(青贮饲料加底物)放置到每个发酵瓶中。然后加入经过缓冲的培养基(90ml)，密封所述的瓶，在室温下贮藏过夜。在用制备好的瘤胃流体接种之前，将瓶中内容物升温至39℃。在喂饲之前(07.00小时)，从两只泌乳奶牛手工获得瘤胃流体(手挤出的内容物)，所述奶牛被提供有随意的玉米/草青贮饲料∶浓缩物(60∶40)比例。将流体通过两层细棉布过滤，保持于39℃、CO2下，直到使用。将10ml制备好的流体加入到每只瓶中，在研究持续期在39℃对其进行孵育。每轮包括六份阴性对照(仅有缓冲过的培养基加瘤胃流体)，以对于直接的瘤胃流体影响，校正气体值和降解剩余物。此外，所有轮次(每轮六瓶)中都包括不含补充剂的玉米青贮饲料(1.0g瓶-1)。研究结果相对阳性对照值表示。
在孵育后2、4、6、8、10、12、14、16、18、21和24小时获得顶部空间的气压读数。最后一次测量后，按照测试材料/内含水平，从每瓶中取大约3.0ml发酵培养基盛装作为样品，冷冻(-20℃)贮藏，直到使用Varian 3600GC针对VFA组合物进行后续分析。通过在减压下将瓶中内容物通过60mlGooch坩埚(孔径1,100至160μm)过滤来回收发酵剩余物。然后对剩余物进行干燥(100℃，24小时)、称重、灰化(500℃，过夜)以及重新称重。测得的干物质(DM)和有机物质(OM)的量被用于评估iDMD和iOMD(分别是体外DM和OM降解)。用以前导出的二次函数转化压力为体积，来产生发酵气体释放的程度(ml气体g-1孵育的OM)和速率(ml h-1)。饲料发酵效率(FE)被评估为iDMD(g kg-1)/在孵育后24小时积累的气体(ml)释放。用学生T-检验以及P＞0.05的显著性，来评估处理平均值和阳性对照(仅含玉米青贮饲料)之间的统计学差异。
针对对发酵一般影响的León方法
用由75％紫花苜蓿干草和25％大麦组成的膳食喂饲的瘤胃插管绵羊(自由获取微生物-矿物质补充剂和水)作为瘤胃流体的供体。就在早晨喂饲之前收集瘤胃流体。
用于分批培养物的底物由50％紫花苜蓿干草、40％草料干草和10％大麦谷粒组成，其在具有1mm筛的锤式粉碎机中被磨碎。使用的底物的量为500mg，测试植物的内含比例为10％(ca.50mg)。将二者都称重到浆液瓶中，其中，50ml经过缓冲的瘤胃流体(含有10ml经过滤的瘤胃流体和40ml培养基，如Goering & Van Soest，1970所述)被厌氧分散。瓶被密封，在39℃孵育。24小时孵育之后，记录以下测量：
-总气体产量(ml)，使用压力变换器来进行，在经过校正的注射器中收集气体。
孵育培养基中的pH。
-孵育培养基中的挥发性脂肪酸浓度，通过GC来测量(还在接种时间测量VFA，以计算24小时后的VFA产量)。
-DM孵育剩余物，这是通过将瓶中内容物在熔结坩埚中过滤以及干燥后称量剩余物来进行的。此后，测定剩余物的中性洗涤纤维(NDF)含量，以计算未降解的NDF的量。由此来评估DM和NDF的消失。
对每种植物孵育三份重复样品，使用空白(无底物无植物)和对照(500mg底物，没有任何植物，以及550mg底物，没有任何植物)。
瘤胃消化物的甲烷形成
对通过León常规方法进行的孵育进行甲烷形成分析。获取产生的气体的代表性样品，用于随后的分析。使用特殊的气密性玻璃注射器来进行取样，所述注射器配有阀门，允许样品被封闭和贮存。将针头通过塞子隔膜插入后，直接从顶部空间收集样品，在注射器中取样，关上阀门。在测量总的气体产量之后收集该样品，假设释放的气体和留在顶部空间中的气体的气体组成一样。
产生的气体中甲烷浓度是通过气相色谱来测定的。将气体样品(300至500μl)从气密性注射器直接注射到相应端口。用于分析的GC参数设定如下：
仪器：Shimadzu GC-14B，提供有火焰离子化检测器(FID)
柱：2.3米长x2.1mm内部直径的不锈钢柱，装有60/80目的Carboxen1000固定相
载气：氦，流速(100kPa)，恒定流量模式
温度：
检测器：FID，温度200℃，合成空气流速(50kPa)，H2流速(50kPa)
注射器：温度200℃
柱-炉：170℃(等温)
用于校正的标准是纯甲烷(99.9％)。通过绘制注射的甲烷标准量相对峰面积的线性回归来建立标准曲线，获得应答因子。
酸中毒检验
使用如下模型，其中，在48小时的发酵期间，相对于对照的的孵育培养基pH的降低和产生的乳酸的浓度，提供了对于测试底物抵消可能的酸中毒影响的能力的指示。在该研究中，磨碎的小麦谷粒(2mm)被用作为基础底物，测试材料以100mg g-1DM(或10mg g-1DM β-香叶烯)被包括进来。如以前一样，向每只瓶中加入1.0g总的底物。为增加乳酸生产，制备的缓冲溶液被稀释百分之五十。这用于确保：孵育培养基的缓冲能力被超出，得到的pH降低促进Lactobacilli和其它产乳酸细菌的生长。获得瘤胃流体，按照前文所述对其进行制备，例外之处在于，向所用的两只奶牛提供高水平粗糙磨碎小麦的泌乳早期配给(early lactation ration)。通过向每只瓶中直接插入pH电极，于接种后1小时(起始pH)以及再过23小时和47小时，对流体pH进行测量。最后一次测量之后，从每只瓶中立即取大约3.0ml流体，用作为样品，冷冻贮存于-20℃，直到针对L[+]乳酸进行酶分析。
结果作为相对对照的绝对差异展示。对酸中毒的正面影响被鉴定为：到24小时的pH降低速率减慢，更高的终点pH，以及更低的乳酸浓度。
结果
下表2a、2b和2c显示了从对发酵活性的测量获得的结果。
在原生动物检验、方法1的蛋白质水解检验和甲烷形成中获得的结果也被示于同样的表中。
图1至图7显示了：体外实验中，选择的植物/植物提取物在不同内含物比例下对原生动物溶菌活性的影响。瘤胃流体中原生动物溶菌活性的持续性要高于缓冲液或水中的，尤其是用G.asclepiadea进行的实验中(表1)。K.arvensis的影响示于图8中。
表1.瘤胃流体或Coleman’s缓冲液预孵育对于样品抑制瘤胃纤毛原生动物溶菌活性的能力的影响
表2a.选择的植物材料、植物提取物或天然等同组分对于发酵的影响。没有植物材料的情况下值为100。
表2b.选择的植物材料、植物提取物或天然等同组分对于发酵的影响。没有植物材料的情况下值为100。
表2c.选择的植物材料、植物提取物或天然等同组分对于发酵的影响。没有植物材料的情况下值为100。
获得的结果明显表明，本发明的测试组分对于瘤胃发酵具有正面影响，这种影响将支持本发明的目的。
引用文献
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Claims (3)
1.熊果(Arctostaphylos uva-ursi)的植物材料及其提取物用于生产用于影响瘤胃发酵以及增加反刍动物对能量和氮源的可利用性的添加剂的用途。
2.如权利要求1所述的用途，其特征在于，所述添加剂用于降低所述瘤胃消化的蛋白水解活性。
3.如权利要求1或2所述的用途，其特征在于，施予的组分的总量为每天每kg反刍动物体重0.02mg至20g。
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| KR20140019307A (ko) | 2010-12-09 | 2014-02-14 | 와이앤드비 마더스 초이스 엘티디. | 사포닌을 포함하는 제형 및 이의 용도 |
| CN102526223A (zh) * | 2010-12-09 | 2012-07-04 | 天津瑞贝特科技发展有限公司 | 具有保肝利胆功能的中药组合物及其制备方法 |
| CN102106462B (zh) * | 2011-01-20 | 2013-01-16 | 塔里木大学 | 一种减少反刍动物瘤胃甲烷释放的添加剂 |
| EP2653039A1 (en) * | 2012-04-19 | 2013-10-23 | Interquim, S.A. | Feed composition for reducing ruminant methanogenesis |
| CN103404699B (zh) * | 2013-08-12 | 2014-08-20 | 浙江大学 | 一种促进瘤胃发酵的天然产物制剂 |
| CN103518965B (zh) * | 2013-10-11 | 2015-04-01 | 中国农业科学院饲料研究所 | 可降低牛羊甲烷排放量的预混合饲料 |
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- 2005-04-15 CN CN2005800114393A patent/CN101005848B/zh active Active
- 2005-04-15 WO PCT/EP2005/051673 patent/WO2005099729A2/en active Application Filing
- 2005-04-15 ES ES05747206T patent/ES2392539T3/es active Active
- 2005-04-15 KR KR1020067022880A patent/KR20070033968A/ko not_active Application Discontinuation
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100904A (zh) * | 1994-05-27 | 1995-04-05 | 张进 | 熊果滋补液及其制造方法 |
Non-Patent Citations (2)
| Title |
|---|
| Dykes GA等.Enhancement of nisin antibacterial activity by a bearberry (Arctostaphylos uva-ursi) leaf extract.《Food Microbiology》.2003,第20卷(第2期),第211-216页. * |
| Parejo I等.A single extraction step in the quantitative analysis of arbutin inbearberry (Arctostaphylos uva-ursi) leaves by high-performance liquidchromatography.《Phytochemical Analysis》.2001,第12卷(第5期),第336-339页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1765371B1 (en) | 2012-08-08 |
| CN101005848B (zh) | 2011-04-20 |
| EP1765371A2 (en) | 2007-03-28 |
| ES2392539T3 (es) | 2012-12-11 |
| JP2007532609A (ja) | 2007-11-15 |
| US20080008774A1 (en) | 2008-01-10 |
| CN101005848A (zh) | 2007-07-25 |
| AU2005232408B2 (en) | 2010-07-01 |
| CN102132778A (zh) | 2011-07-27 |
| SE0400996D0 (sv) | 2004-04-16 |
| WO2005099729A3 (en) | 2005-12-22 |
| WO2005099729A2 (en) | 2005-10-27 |
| JP4964125B2 (ja) | 2012-06-27 |
| KR20070033968A (ko) | 2007-03-27 |
| AU2005232408A1 (en) | 2005-10-27 |
| NZ550402A (en) | 2009-12-24 |
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