CN114504056A - 提高机体抗氧化和免疫功能的氨基酸包及其在断奶仔猪无抗低蛋白质饲粮中应用 - Google Patents
提高机体抗氧化和免疫功能的氨基酸包及其在断奶仔猪无抗低蛋白质饲粮中应用 Download PDFInfo
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Abstract
本发明属于饲料技术领域，具体涉及一种提高机体抗氧化和免疫功能的氨基酸包及其在断奶仔猪无抗低蛋白质饲粮中应用。该提高机体抗氧化和免疫功能的氨基酸包组成为：L－赖氨酸盐酸盐、DL－蛋氨酸、L－苏氨酸、L－色氨酸、L－缬氨酸、L‑异亮氨酸、L‑谷氨酸。该复合氨基酸可将断奶仔猪饲粮蛋白质水平降低至15.93%，并能改善肠道健康、提高机体抗氧化应激、增强免疫机能，能更加有效解决断奶仔猪禁抗问题的一种基础技术，同时对促进猪肉安全和消费者健康等方面也具有重要意义，并将产生巨大的生态、经济和社会效益。
Description
技术领域
本发明属于饲料技术领域，具体涉及一种提高机体抗氧化和免疫功能的氨基酸包及其在断奶仔猪无抗低蛋白质饲粮中应用。
背景技术
规模化养猪生产中断奶引发了仔猪心理、营养、环境等系列剧烈反应，仔猪出现严重的氧化应激，导致仔猪肠道损伤、免疫力下降、肠道菌群失调、腹泻、生长缓慢等问题。生产中采用抗生素来解决此类问题，但随着我国于2020年7月在饲料中禁止使用抗生素，对断奶仔猪生长产生较大影响。
通常生产中使用的断奶仔猪饲粮蛋白质水平较高，未被消化的蛋白质在肠道内被微生物发酵，增加病源微生物繁殖，同时产生更多的氨氮等有害物质，损伤肠道上皮组织。研究证明，降低饲粮粗蛋白质水平2-4个百分点，补充相应的合成氨基酸（赖氨酸、蛋氨酸、苏氨酸和色氨酸），不影响猪的生长性能，且有利于改善猪肠道健康、降低氨氮排放量。
配制低蛋白质饲粮关键技术是合理的氨基酸平衡模式的构建，建立在常规饲粮蛋白质水平基础上的氨基酸平衡模式参数不能简单应用于低蛋白质饲粮中，国内有关断奶仔猪氨基酸精准需要和氨基酸间平衡技术尚不够完善，综合国内相关文献，过多降低饲粮蛋白质水平会影响猪的生长性能，如当饲粮蛋白质水平降低超过4个百分点，即使补充合成氨基酸，对断奶仔猪生长性能也会造成一定的影响，分析原因可能与断奶仔猪饲粮氨基酸间平衡比例、某些必需氨基酸缺乏或某些非必需氨基酸不足等因素有关。
因此，本项目提出的一种依据特定断奶仔猪重要氨基酸平衡模式结合应用功能性氨基酸（谷氨酸）配制的氨基酸包，及其在无抗低蛋白质饲粮中应用，使饲粮蛋白质水平从21.16%降低至15.93%，达到提高断奶仔猪抗氧化和免疫功能、优化肠道菌群结构、促进肠道健康、降低仔猪腹泻率和减少氨氮排放的一种技术，是更加有效解决断奶仔猪禁抗问题的一种基础技术，同时对促进猪肉安全和消费者健康等方面也具有重要的意义，将产生巨大的生态、经济和社会效益。
发明内容
本发明目的在于针对现有技术的不足，提供一种依据特定断奶仔猪重要氨基酸平衡模式结合应用功能性氨基酸（谷氨酸）配制的氨基酸包，及其在无抗低蛋白质饲粮中应用，使饲粮蛋白质水平从21.16%降低至15.93%，达到提高断奶仔猪抗氧化和免疫功能、优化肠道菌群结构、促进肠道健康、降低仔猪腹泻率和减少氨氮排放的一种技术，是更加有效解决断奶仔猪禁抗问题的一种基础技术，同时对促进猪肉安全和消费者健康等方面也具有重要的意义，将产生巨大的生态、经济和社会效益。
为实现上述目的，本发明采用如下技术方案：
一种提高机体抗氧化和免疫功能的氨基酸包，其原料组成按质量分数计为：L－赖氨酸盐酸盐16.37%、DL－蛋氨酸8.55%、L－苏氨酸8.18%、L－色氨酸2.85%、L－缬氨酸5.69%、L-异亮氨酸4.98%、L-谷氨酸53.38%。
利用上述氨基酸包制备的断奶仔猪无抗低蛋白质饲粮，其原料组成按质量分数计为：玉米47.00%，豆粕6.20%，膨化大豆12.00%，鱼粉7.00%，乳清粉16.00%，蔗糖2.00%，柠檬酸2.00%，豆油2.56%，石粉0.23%，磷酸氢钙0.82%，食盐0.30%，氯化胆碱0.08%，氨基酸包2.81%，预混料1.00%。
进一步地，所述的预混料按每1000g由五水硫酸铜3.0g、一水硫酸亚铁23.0g、一水硫酸锌20.0g、一水硫酸锰4.8g、1％碘酸钙2.5g、1％亚硒酸钠1.3 g、维生素A 2.5g、维生素D3 2.4g、维生素E 9.0g、维生素K3 0.2g、维生素B1 0.4g、维生素B2 0.6g、维生素B6 0.4g、1%维生素B12 0.2g、烟酸1.5g、泛酸1.0g、叶酸0.10g、2%d-生物素2.0g、米糠粕925.10g组成。
本发明的有益效果：
（1）本发明的特点在于构建特定的饲粮氨基酸平衡模式，结合应用功能性氨基酸（谷氨酸），谷氨酸（Glutamate，Glu）是一种功能性氨基酸，在细胞代谢和生理调节方面具有多种重要作用，是小肠粘膜生长和更新的能量物质，能缓解氧化应激和毒素引起的肠道损伤，是维持肠道健康的关键因素；当仔猪饲粮添加谷氨酸后，饲粮中超过 90% 谷氨酸会在门静脉回流组织中被代谢供能，从而减少门静脉回流组织对其他必需氨基酸的消耗，使得进入肝脏的氨基酸数量更为平衡，而且谷氨酸是几乎所有氨基酸代谢的中枢，当机体其他氨基酸不足时，谷氨酸可以通过转氨基作用予以补充，因此，当饲粮蛋白质水平较大幅度降低时，谷氨酸可以弥补由此造成的必需氨基酸或非必需氨基酸的不足。由此可见，本发明采用特定的饲粮氨基酸平衡模式添加多种合成氨基酸，结合应用功能性氨基酸（谷氨酸），更加提高了饲粮氨基酸的平衡性，并在机体抗氧化、免疫功能和维护肠道健康方面具有显著优势。
（2）本发明的提高机体抗氧化和免疫功能的氨基酸包可将断奶仔猪饲粮蛋白质水平降低至15.93%，并能改善肠道健康、提高机体抗氧化应激、增强免疫机能，能更加有效解决断奶仔猪禁抗问题的一种基础技术。
具体实施方式
本发明用下列实施例来进一步说明本发明，但本发明的保护范围并不限于下列实施例。
实施例1
一种提高机体抗氧化和免疫功能的氨基酸包，其原料组成按质量分数计为：L－赖氨酸盐酸盐16.37%、DL－蛋氨酸8.55%、L－苏氨酸8.18%、L－色氨酸2.85%、L－缬氨酸5.69%、L-异亮氨酸4.98%、L-谷氨酸53.38%。
利用上述氨基酸包制备的断奶仔猪无抗低蛋白质饲粮，其原料组成按质量分数计为：玉米47.00%，豆粕6.20%，膨化大豆12.00%，鱼粉7.00%，乳清粉16.00%，蔗糖2.00%，柠檬酸2.00%，豆油2.56%，石粉0.23%，磷酸氢钙0.82%，食盐0.30%，氯化胆碱0.08%，氨基酸包2.81%，预混料1.00%。
进一步地，所述的预混料按每1000g由五水硫酸铜3.0g、一水硫酸亚铁23.0g、一水硫酸锌20.0g、一水硫酸锰4.8g、1％碘酸钙2.5g、1％亚硒酸钠1.3 g、维生素A 2.5g、维生素D3 2.4g、维生素E 9.0g、维生素K3 0.2g、维生素B1 0.4g、维生素B2 0.6g、维生素B6 0.4g、1%维生素B12 0.2g、烟酸1.5g、泛酸1.0g、叶酸0.10g、2%d-生物素2.0g、米糠粕925.10g组成。
一、试验设计与试验饲粮：
采用单因子随机试验设计。选用96头遗传背景相似、健康状况良好的“杜长大”三元杂交断奶仔猪为试验对象（平均断奶日龄为26±3 d，平均初始体重为6.72±0.36 kg），对照组饲粮参照NRC（2012）5－10 kg仔猪营养需要标准配制，饲粮蛋白水平为21.16%，对照组添加饲用抗生素（硫酸抗敌素），试验组在保持饲粮其它营养水平与对照组相同的情况下，将饲粮蛋白质水平从21.16%（对照组）降低到15.93%，试验组饲粮中添加按特定氨基酸平衡模式结合功能性氨基酸（谷氨酸）配制的氨基酸包。对照组、试验组各设6个重复，每个重复8头仔猪（公母各半），预试期3天，试验期16天，饲粮组成及营养水平见表1。
表1饲粮组成与营养水平
注：1）氨基酸包含有L－赖氨酸盐酸盐、DL－蛋氨酸、L－苏氨酸、L－色氨酸、L－缬氨酸、L-异亮氨酸、L-谷氨酸。2）1.00%预混料每1000g由五水硫酸铜3.0g、一水硫酸亚铁23.0g、一水硫酸锌20.0g、一水硫酸锰4.8g、1%碘酸钙2.5g、1％亚硒酸钠1.3 g、维生素A2.5g、维生素D3 2.4g、维生素E 9.0g、维生素K3 0.2g、维生素B1 0.4g、维生素B2 0.6g、维生素B6 0.4g、1%维生素B12 0.2g、烟酸1.5g、泛酸1.0g、叶酸0.10g、2%d-生物素2.0g、米糠粕（载体）925.10g组成。3）粗蛋白质为实测值，其它营养指标为计算值。
二、测定指标与测定方法：
1、生长性能
于试验开始第1天及最后1天，以重复为单位，仔猪在禁喂16小时（只给饮水）条件下进行逐头称重，计算仔猪的平均日增重(average daily weight gain，ADG)。以重复为单位记录每天投料量及剩料量，统计每个重复的耗料量，计算仔猪的平均日采食量(avergaedaily feed intake，ADFI)及料重比(the feed to gain ratio，F/G)。
2、腹泻率
试验期间每天8:00、10:00、14:00和16:00专人负责观察仔猪排泄情况，记录仔猪腹泻的头次和天数，以栏为单位统计仔猪腹泻率。
腹泻率（%）＝（仔猪腹泻天数×腹泻仔猪头数）/（试验仔猪总头数×正试期天数）×100。
3、样品采集
饲料样品：在加工试验饲料时，以处理组为单位从每个包装袋中各取150 g饲料，混匀后按照四分法收集饲料样品，冷藏保存待测。每种试验料分别取2个样品，表1中饲粮粗蛋白质含量为2个样品测定值的平均值。
试验期结束称重后，每个重复选取接近平均体重的2头仔猪(1头公猪和1头母猪)，前腔静脉采集血液约10 mL，置于含肝素钠(肝素抗凝，20 iu•mL-1) 的离心管中，血液样品室温静置1 h，经过3500g离心15 min，制备血浆并于-20 ℃保存备测。
在试验的第11至13天连续3天于早晨饲喂前采用直肠收集法采集无污染粪样少量于5 mL冻存管中，放入液氮罐中迅速冷冻，之后转移至-80 ℃冰箱保存待测。
4、血液生化指标
血浆乳酸脱氢酶（LDH）、碱性磷酸酶（AKP）、谷丙转氨酶（GPT）和谷草转氨酶（GOT）浓度采用酶联免疫吸附试验法（ELISA）测定，检测仪器为华东电子DG5033A酶标仪（南京华东电子集团医疗装备有限责任公司）。血浆二胺氧化酶（ADO）浓度采用分光光度计法，血浆尿素氮（PUN）浓度采用二乙酰肟法，仪器为UNICO-UV2000分光光度计（尤尼柯（上海）仪器有限公司），试剂盒均购自南京建成生物工程研究所，操作方法按试剂盒说明书进行。
5、免疫指标
血浆免疫球蛋白G（IgG）、免疫球蛋白A（IgA）、免疫球蛋白M（IgM）、白细胞介素-1β（IL-1）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）及肿瘤坏死因子-α（TNF-α）采用酶联免疫吸附试验法（ELISA）测定，仪器为华东电子DG5033A酶标仪（南京华东电子集团医疗装备有限责任公司），试剂盒购自南京建成生物工程研究所，检测方法按试剂盒说明书进行。
6、抗氧化指标
血浆总抗氧化能力（Total antioxidation capability，T-AOC）采用FRAP法测定，血浆超氧化物岐化酶（Superoxide dismutase，SOD）活力采用羟胺法测定，血浆谷胱甘肽过氧化物酶（Glutathione peroxidase，GSH-Px）活力采用硫代二巯基苯甲酸法测定，血浆丙二醛（Malondialdehyde，MDA）含量采用硫代巴比妥法测定。样品测定的试剂盒均购自南京建成生物工程研究所，操作按试剂盒说明书进行。仪器为华东电子DG5033A酶标仪（南京华东电子集团医疗装备有限责任公司）和UNICO-UV2000分光光度计（尤尼柯（上海）仪器有限公司）。
7、激素指标
血浆胰岛素样生长因子1（IGF-1）和胆囊收缩素（CCK）采用酶联免疫吸附试验法（ELISA）测定，试剂盒购自南京建成生物工程研究所，检测方法按试剂盒说明书进行，仪器为华东电子DG5033A酶标仪（南京华东电子集团医疗装备有限责任公司）。
8、粪样微生物组成
粪样微生物组成采用实时荧光定量PCR法检测。粪样总DNA采用Omege公司的DNA提取试剂盒（Omege D40-15）操作说明进行提取，用核酸浓度测定仪（ND-1000）检测其浓度，采用0.8%琼脂糖凝胶电泳检测DNA完整性，总细菌DNA分别和不同浓度大肠杆菌、乳酸杆菌、双歧杆菌和总细菌标准质粒进行定量PCR检测，将标准质粒浓度和Ct值进行回归分析，制定标准曲线，计算出目标菌属DNA拷贝数，结果用每克粪样中细菌拷贝数的常用对数表示（lg拷贝数/g）。
9、统计分析
所有数据先用Excel软件进行统计处理，用SPSS26.0统计软件进行方差分析，用Duncan氏法对数据进行多重差异显著性比较，试验结果采用平均值±标准差表示，以P<0.01作为极显著、P<0.05作为显著性、（P>0.05）作为差异不显著判断标准。
三、试验结果与说明：
1、添加氨基酸包的低蛋白质饲粮对断奶仔猪生长性能和腹泻率的影响。
从表2可知，断奶仔猪腹泻率试验组比对照组降低58.95%，差异极显著（P<0.01）。
表2 添加氨基酸包的低蛋白质饲粮对断奶仔猪生长性能和腹泻率的影响
同列数据不同肩标小写字母表示差异显著（P <0.05），不同肩标大写字母表示差异极显著（P <0.01），无字母表示差异不显著（P>0.05），下表同。
2、添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆生化指标的影响。
由表3可知，血浆DAO浓度试验组比对照组降低18.17%，差异显著（P<0.05）；血浆PUN浓度试验组比对照组降低36.92%，差异显著（P<0.05）。血浆DAO 活性是监测肠道屏障功能受损和肠上皮细胞通透性改变的重要指标，当肠道黏膜受损时，DAO 通过肠黏膜进入血液循环导致血浆DAO 活性升高，本研究结果表明，对照组仔猪肠道通透性增加，肠道上皮组织可能受到损伤，而试验组血浆中 DAO 活性显著降低，说明试验组仔猪肠道损伤较轻，肠道屏障功能较完善。血浆PUN值降低说明试验组断奶仔猪氮摄入量显著减少、或试验组饲粮中氨基酸的平衡性比对照组好，猪只氮排放明显减少。
表3 添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆生化指标的影响
3、添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆抗氧化指标的影响。
由表4可知，断奶仔猪血浆T-AOC深度试验组比对照组提高66.67%，差异显著（P<0.05），说明试验组仔猪机体总抗氧化能力明显高于对照组。
表4 添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆抗氧化指标的影响
4、添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆免疫指标的影响。
由表5可知，断奶仔猪血浆IgA浓度试验组比对照组提高42.62%，差异显著（P<0.05）；血浆IgG浓度试验组比对照组提高44.28%，差异显著（P<0.05）；血浆IgM浓度试验组比对照组提高30.43%，差异显著（P<0.05）；血浆IL-10浓度试验组比对照组提高25.96%，差异显著（P<0.05）。试验结果说明试验组仔猪机体免疫功能明显高于对照组。
表5 添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆免疫指标的影响
5、添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆激素指标的影响.
由表6可知，断奶仔猪血浆CCK浓度试验组比对照组提高32.17%，差异显著（P<0.05）。血浆CCK浓度升高，能够刺激胆囊收缩和胰酶分泌，有助于饲料消化吸收，促进断奶仔猪生长。
表6 添加氨基酸包的低蛋白质饲粮对断奶仔猪血浆激素指标的影响
6、添加氨基酸包的低蛋白质饲粮对断奶仔猪粪样微生物的影。。
由表7可知，断奶仔猪粪样大肠杆菌数试验组比对照组降低9.54%，差异显著（P<0.05）；粪样乳酸杆菌数试验组比对照组提高7.05%，差异显著（P<0.05）；粪样总细菌数试验组比对照组降低5.43%，差异显著（P<0.05）。试验结果表明，试验组提高有益菌的增殖，抑制有害菌繁殖，优化仔猪肠道菌群结构，减少断奶仔猪腹泻。
表7 添加氨基酸包的低蛋白质饲粮对断奶仔猪粪样微生物的影响
上述试验结果表明，断奶仔猪采食粗蛋白质水平为15.93%、添加本研究的氨基酸包的无抗低蛋白质饲粮，与常规饲粮（根据NRC标准配制）相比，显著降低断奶仔猪腹泻率，提高断奶仔猪抗氧化和免疫水平，优化肠道菌群结构，减轻肠道屏障功能损伤，减少氨氮排放量，使仔猪安全渡过“断奶关”，有效提高养猪生产水平、改善养猪生态环境。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (3)
1.一种提高机体抗氧化和免疫功能的氨基酸包，其特征在于：其原料组成按质量分数计为：L－赖氨酸盐酸盐16.37%、DL－蛋氨酸8.55%、L－苏氨酸8.18%、L－色氨酸2.85%、L－缬氨酸5.69%、L-异亮氨酸4.98%、L-谷氨酸53.38%。
2.一种利用权利要求1所述的提高机体抗氧化和免疫功能的氨基酸包制备的断奶仔猪无抗低蛋白质饲粮，其特征在于：其原料组成按质量分数计为：玉米47.00%，豆粕6.20%，膨化大豆12.00%，鱼粉7.00%，乳清粉16.00%，蔗糖2.00%，柠檬酸2.00%，豆油2.56%，石粉0.23%，磷酸氢钙0.82%，食盐0.30%，氯化胆碱0.08%，氨基酸包2.81%，预混料1.00%。
3.根据权利要求2所述的断奶仔猪无抗低蛋白质饲粮，其特征在于：所述的预混料按每1000g由五水硫酸铜3.0g、一水硫酸亚铁23.0g、一水硫酸锌20.0g、一水硫酸锰4.8g、1％碘酸钙2.5g、1％亚硒酸钠1.3 g、维生素A 2.5g、维生素D3 2.4g、维生素E 9.0g、维生素K30.2g、维生素B1 0.4g、维生素B2 0.6g、维生素B6 0.4g、1%维生素B12 0.2g、烟酸1.5g、泛酸1.0g、叶酸0.10g、2%d-生物素2.0g、米糠粕925.10g组成。
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| CN108419938A (zh) * | 2018-06-21 | 2018-08-21 | 中国农业大学 | 一种断奶仔猪低蛋白无抗生素的日粮及其应用 |
| CN110973382A (zh) * | 2019-10-31 | 2020-04-10 | 广东省农业科学院动物科学研究所 | 一种无抗生素环保型低蛋白仔猪保育料及其制备方法 |
| CN112369518A (zh) * | 2020-11-09 | 2021-02-19 | 广东省农业科学院动物科学研究所 | 一种仔猪饲料及其应用 |
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| CN108419938A (zh) * | 2018-06-21 | 2018-08-21 | 中国农业大学 | 一种断奶仔猪低蛋白无抗生素的日粮及其应用 |
| CN110973382A (zh) * | 2019-10-31 | 2020-04-10 | 广东省农业科学院动物科学研究所 | 一种无抗生素环保型低蛋白仔猪保育料及其制备方法 |
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