CN110157771A - 一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法 - Google Patents
一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法 Download PDFInfo
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Abstract
本发明属于生物饲料活性度评价方法技术领域，提供了一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法。该评价方法包括：通过检测发酵豆粕对动物肠道菌群正向或者负相调节作用以及动物小肠绒毛的特征来评价发酵豆粕活性度。该评价方法通过动物小肠绒毛健康程度，以及动物肠道菌群丰度和均匀度与粪便中菌群的关系来评价猪群的健康程度和发酵豆粕活性度，更加准确和全面。
Description
技术领域
本发明属于生物饲料活性度评价方法技术领域，具体地说，涉及一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法。
背景技术
近年来，我国养猪业得到长期发展，规模化、集约化程度越来越高，然而在现有条件下，即使猪群个体间有相同的遗传背景及饲养环境，其生长性能及猪群健康程度仍有明显差异，这给规模化养殖生产在圈舍利用、疾病防治等环节造成了一定的损失，增加饲养成本，有研究表明，猪群的健康程度与肠道菌群有密切关联。
在长期进化过程中，微生物与动物体及环境之间形成了相互依赖、相互制约的微生态***，在肠道微生态***下，肠道中的有益菌群与有害菌群达成动态平衡，肠道中的有益菌分泌有益成分，维持肠道正常的功能与结构，当有害菌占据主导地位，动物机体便会发病，影响营养成分吸收及动物生长；因此研究猪群肠道菌群及肠道健康程度对养殖具有重要意义。
专利文献“CN106591458A-生物饲料活性度的评价方法”涉及一种生物饲料活性度的评价方法。具体而言，涉及以肠道菌群为靶标的一种生物饲料活性度的评价方法，其利用无菌动物通过16S rDNA测序来检测动物肠道菌群正向或负向波动，以评价生物饲料的活性度。该方法可以用于各种生物饲料活性度的评估，通过体内外实验检测测试物对肠道菌群的作用，以菌群的波动变化反映样品的优劣性。然而上述专利文献仍然存在评价不够全面等缺点。
发明内容
针对现有技术中上述的不足，本发明的目的在于提供一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，该评价方法通过动物小肠绒毛健康程度，以及动物肠道菌群丰度和均匀度与粪便中菌群的关系来评价猪群的健康程度和发酵豆粕活性度，更加准确和全面。
为了达到上述目的，本发明采用的解决方案是：
一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，包括：通过检测发酵豆粕对动物肠道菌群正向或者负相调节作用以及动物小肠绒毛的特征来评价发酵豆粕活性度。
进一步地，在本发明较佳的实施例中，动物小肠绒毛的特征包括：动物小肠绒毛的长度，以及动物小肠绒毛的腺窝深度。
进一步地，在本发明较佳的实施例中，动物小肠绒毛的长度越长以及动物小肠绒毛的腺窝深度越深，发酵豆粕活性度越高。
进一步地，在本发明较佳的实施例中，动物小肠绒毛的特征的测评步骤包括：(1)猪源菌群小鼠模型的建立：以4周龄无菌小鼠作为试验小鼠饲养于无菌隔离室中，室温21±2℃，湿度40％～70％，按照12h明暗转换，试验小鼠所用垫料及食用食物均经过无菌处理；采集猪场中健康猪群的新鲜粪便制成粪便悬液，取0.3mL的粪便悬液灌胃每只试验小鼠，3d后进行第二次灌胃，灌胃量为0.3mL，一周后采用强制排便的方式，采集试验小鼠的粪便，若粪便中含有菌群即可认为试验小鼠成为猪源菌群小鼠；(2)动物试验：向猪源菌群小鼠喂食基础日粮或添加有发酵豆粕的基础日粮，自由采食饮水，四周后收集猪源菌群小鼠的新鲜粪便，并将猪源菌群小鼠杀死，采取猪源菌群小鼠的小肠段，至于无菌环境中，观察猪源菌群小鼠小肠绒毛的长度和腺窝深度。
进一步地，在本发明较佳的实施例中，正向调节作用的表现为在动物肠道中有益菌群丰度及均匀度提升，负向调节作用的表现为在动物肠道中致病菌群丰度及均匀度提升。
进一步地，在本发明较佳的实施例中，有益菌群包括双歧杆菌、乳杆菌、芽孢杆菌、梭菌等；致病菌群包括沙门氏菌、大肠杆菌。
进一步地，在本发明较佳的实施例中，丰度及均匀度的检测包括：采用微生物平板计数法或16S rDNA高通量测序法对动物肠道中的微生物数量进行检测。
进一步地，在本发明较佳的实施例中，16S rDNA高通量测序法包括：1)基因组DNA提取；2)高可变区域引物设计和PCR扩增；3)测序文库建立；4)上机测序；5)结果分析。
进一步地，在本发明较佳的实施例中，有益菌群所占比例越大，发酵豆粕活性度越高；有益菌群所占比例就越小，发酵豆粕活性度越低。
本发明的有益效果是：
本发明提供的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，通过动物小肠绒毛健康程度，以及动物肠道菌群丰度和均匀度与粪便中菌群的关系来评价猪群的健康程度和发酵豆粕活性度，更加准确和全面。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例
(1)猪源菌群小鼠模型的建立：
以4周龄无菌小鼠作为试验小鼠饲养于无菌隔离室中，室温21±2℃，湿度40％～70％，按照12h明暗转换，试验小鼠所用垫料及食用食物均经过无菌处理；采集猪场中健康猪群的新鲜粪便制成粪便悬液，取0.3mL的粪便悬液灌胃每只试验小鼠，3d后进行第二次灌胃，灌胃量为0.3mL，一周后采用强制排便的方式，采集试验小鼠的粪便，若粪便中含有菌群即可认为试验小鼠成为猪源菌群小鼠。
(2)动物试验：将400只猪源菌群小鼠随机分为4组，100只/组,对照组A和试验组B-D：其中，其中对照组A只饲喂基础日粮；试验组B为发酵豆粕添加量低剂量组(发酵豆粕的质量分数为5％)，试验组C为发酵豆粕添加量中剂量组(发酵豆粕的质量分数为10％)，试验组D为发酵豆粕添加量高剂量组(发酵豆粕的质量分数为15％)；试验周期为4周，自由采食饮水，试验过程中遵循试验动物福利原则。
试验结束后，收集对照组A和试验组B-D的猪源菌群小鼠的新鲜粪便，其后将猪源菌群小鼠杀死，迅速采取猪源菌群小鼠的小肠段，至于无菌环境中，观察猪源菌群小鼠小肠绒毛密度和腺窝深度，结果见表1所示；同时定点采集和猪源菌群小鼠的小肠绒毛粘液层，搜集培养，收集菌体提取菌体DNA，通过高通量测序的方法比对菌体16S rDNA，然后对同源性、多样本间差异性进行分析，做出菌的***发育树，通过分析不同样品的操作分类单位(OTU)反应样本件差异，分析菌群的丰度和均匀度反应发酵豆粕活性度对肠道菌群健康程度的影响，结果见表2所示。
试验结果：
表1试验小鼠的小肠绒毛健康程度
表2试验相关微生物菌群丰度及均匀度
| 细菌 | 对照组A | 试验组B | 试验组C | 试验组D |
| 双歧杆菌 | 5.25±0.25 | 6.05±0.25 | 7.23±0.32 | 6.85±0.23 |
| 乳杆菌 | 5.2±0.12 | 5.5±0.15 | 6±0.21 | 6.23±0.15 |
| 肠球菌 | 5.98±0.13 | 6.2±0.14 | 7.1±0.21 | 7±0.15 |
| 梭菌 | 4.85±0.02 | 5.23±0.12 | 6.25±0.23 | 6±0.12 |
| 大肠杆菌 | 5.85±0.23 | 5.2±0.14 | 5±0.2 | 4.7±0.12 |
| 沙门氏菌 | 4.56±0.28 | 4.2±0.15 | 3.5±0.14 | 3.7±0.31 |
| 葡萄球菌 | 4.39±0.12 | 4.1±0.18 | 3.5±0.7 | 3.71±0.17 |
注：菌数量级为108。
由表1数据结果可知，通过测定猪源菌群小鼠的小肠绒毛的长度及腺窝深度，试验组B-D的猪源菌群小鼠的小肠绒毛相较于对照组A，其小肠绒毛长度提高，小肠绒毛腺窝深度增加。上述结果表明，饲喂发酵豆粕有利于小肠的发育，且随着发酵豆粕的添加量的增加对小肠的发育越好。
由表2数据结果可知，通过测定16S rDNA，试验组B-D的定猪源菌群小鼠的小肠内的有益菌种含量均高于对照组A，且致病菌群含量有明显降低，维护了肠道健康；结果表明，试验组B-D采用了发酵豆粕进行饲喂，发酵豆粕的活性度高。
综上所述，采用本发明提供的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法；该评价方法通过动物小肠绒毛健康程度，以及动物肠道菌群丰度和均匀度与粪便中菌群的关系来评价猪群的健康程度和发酵豆粕活性度，更加准确和全面。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (9)
1.一种以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于，包括：通过检测发酵豆粕对动物肠道菌群正向或者负向调节作用以及动物小肠绒毛的特征来评价发酵豆粕活性度。
2.根据权利要求1所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述动物小肠绒毛的特征包括：动物小肠绒毛的长度，以及动物小肠绒毛的腺窝深度。
3.根据权利要求2所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述动物小肠绒毛的长度越长以及所述动物小肠绒毛的腺窝深度越深，所述发酵豆粕活性度越高。
4.根据权利要求2所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述动物小肠绒毛的特征的测评步骤包括：
(1)猪源菌群小鼠模型的建立：以4周龄无菌小鼠作为试验小鼠饲养于无菌隔离室中，室温21±2℃，湿度40％～70％，按照12h明暗转换，所述试验小鼠所用垫料及食用食物均经过无菌处理；采集猪场中健康猪群的新鲜粪便制成粪便悬液，取0.3mL的所述粪便悬液灌胃每只所述试验小鼠，3d后进行第二次灌胃，灌胃量为0.3mL，一周后采用强制排便的方式，采集所述试验小鼠的粪便，若粪便中含有菌群即可认为所述试验小鼠成为猪源菌群小鼠；
(2)动物试验：向所述猪源菌群小鼠喂食基础日粮或添加有所述发酵豆粕的基础日粮，自由采食饮水，四周后收集所述猪源菌群小鼠的新鲜粪便，并将所述猪源菌群小鼠杀死，采取所述猪源菌群小鼠的小肠段，至于无菌环境中，观察所述猪源菌群小鼠小肠绒毛的长度和腺窝深度。
5.根据权利要求3所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述正向调节作用的表现为在动物肠道中有益菌群丰度及均匀度提升，所述负向调节作用的表现为在动物肠道中致病菌群丰度及均匀度提升。
6.根据权利要求5所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述有益菌群包括双歧杆菌、乳杆菌、芽孢杆菌、梭菌等；所述致病菌群包括沙门氏菌、大肠杆菌。
7.根据权利要求5所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述丰度及均匀度的检测包括：采用微生物平板计数法或16S rDNA高通量测序法对动物肠道中的微生物数量进行检测。
8.根据权利要求7所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述16S rDNA高通量测序法包括：1)基因组DNA提取；2)高可变区域引物设计和PCR扩增；3)测序文库建立；4)上机测序；5)结果分析。
9.根据权利要求5所述的以猪肠道菌群为靶标的发酵豆粕活性度体内外评价方法，其特征在于：所述有益菌群所占比例越大，所述发酵豆粕活性度越高；所述有益菌群所占比例就越小，所述发酵豆粕活性度越低。
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