CN115176897B - 一种可增强黄牛机体代谢和生长的天然饲用酶及其应用 - Google Patents
一种可增强黄牛机体代谢和生长的天然饲用酶及其应用 Download PDFInfo
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Abstract
本发明属于饲用酶技术领域,具体地说,涉及一种可增强机体代谢能力的饲用酶制剂及其应用,所述的饲用酶包括犏牛瘤胃厌氧真菌和甲烷菌共培养物—命名为:Neocallimastix frontalis和Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle‑YakTZ1降解秸秆所产的发酵液,能有效提高干黄玉米秸秆作为粗饲料的适口性;可降解干黄玉米秸秆中难以被消化利用的木质纤维素;饲用酶添加入玉米秸秆粗饲料后提高了玉米秸秆粗饲料的营养价值,使黄牛平均日增重量和平均日采食量均有所提高;玉米秸秆的表观消化率也显著提高;使黄牛的机体代谢旺盛,粗饲料的吸收利用率提高;对肝脏功能无负担,肝脏的蛋白质合成能力及组织蛋白质的沉淀作用增强。
Description
技术领域
本发明属于饲用酶技术领域,具体涉及一种可增强机体代谢能力的饲用酶及其应用。
背景技术
玉米秸秆含有纤维素、半纤维素和蛋白质等饲草动物可以消化利用的营养成分,全株玉米青贮饲料已成为全世界范围内用于反刍动物日粮的主要饲料[19]。但因其季节性收获的特点,加之农耕条件或习惯等原因,玉米秸秆通常在萎蔫或者干黄后才收获,导致水分和糖分大量流失。干黄玉米秸秆的质地粗糙,适口性差,不适宜直接饲喂反刍动物,且难以满足青贮原料条件。但在偏远地区仍有很多农户将揉碎的干黄玉米秸秆作为黄牛日粮饲喂,导致黄牛营养供给不够引起的生长发育迟缓与当地繁重的耕作作业不匹配,还会间接影响其繁殖性能和产肉产奶性能,导致经济效益发展缓慢。刘兴琳等人在将复方微生态制剂和复方酶制剂添加在荷斯坦犊牛日粮中,发现菌酶协同作用可以降低犊牛的料重比,从而提高犊牛生长性能。何家俊等人研究发现饲粮中添加丁酸梭菌能提高犊牛的生长性能,降低腹泻率,提高血清GLU和lgG含量,增强机体抗氧化能力和免疫功能,改善犊牛健康。
我国牦牛占世界总牦牛数的90%以上,约2000万头,主要分布在我国青海、西藏、四川、甘肃、新疆、云南以及青藏高原及周边国家。黄牛是我国本地品种,具有耐粗饲、适应性强等优点。犏牛是牦牛的衍生牛种,是牦牛种群不断创新结出的果实。犏牛是牦牛与黄牛或改良牛种间远缘杂交的F1代杂种,公黄牛与母牦牛杂交,其杂种称黄犏牛,公牦牛与母黄牛杂交,其杂种称牦犏牛。无论黄犏牛、牦犏牛雄性均不育,其生长发育、产肉、役力等生产性能上均明显优于牦牛亲代,在高海拔地区适应生态环境的能力高于黄牛亲代,对提高牧区人民生活水平、繁荣牧区经济,保持三江源区生态平衡具有十分重要意义。调查发现截止至2012年青海省犏牛占牛总数的3.5%,不少于12万头,且犏牛生长发育快、抗病力强,一般不易得病,适于高山放牧,且犏牛肉、役兼用,产奶性能好,奶质量高,表现出明显的杂种优势。同样饲养条件下,与黄牛和牦牛相比,在体重、体长、体高等各个方面,犏牛均表现出突出的杂种优势。同一地区生长的黄牛、犏牛和牦牛的饲料转化率、肉品质、乳品质不同,造成这种差异的原因除了遗传差异外,我们也推测与瘤胃微生物之间关系紧密。牦牛和犏牛瘤胃液真菌区系遗传距离更为接近,与黄牛瘤胃液真菌差异较大。犏牛适应高寒生态条件,耐粗饲、耐严寒、耐低氧的恶劣自然环境。犏牛瘤胃内栖息着独特的,复杂多样,大量的微生物群落协同高效降解野牧草为犏牛提供生存能量和营养物质。研究表明反刍动物瘤胃内的厌氧真菌能产生一系列高活性降解植物细胞壁的木质纤维素降解酶,包括纤维素酶、半纤维素酶(主要是木聚糖酶)、漆酶和酯酶等,在瘤胃木质纤维素的降解中发挥了重要作用。厌氧真菌可以利用和降解的底物包括可溶性糖,以及纤维素、木聚糖、葡聚糖等复杂的结构性多糖和贮存性多糖等。体外降解实验表明甲烷菌可以利用厌氧真菌的代谢产物,与厌氧真菌形成稳定的共培养物,这个过程中促进了厌氧真菌和甲烷菌的生长,同时显著提高了厌氧真菌对木质纤维素底物的降解和利用,同时产生更高活性的木质纤维素降解酶,以及大量甲烷和乙酸作为主要发酵终产物。
犏牛瘤胃厌氧真菌Neocallimastix sp.属和甲烷菌的自然共培养物不仅能够分泌较全的天然降解木质纤维素的酶系,包括木聚糖酶、纤维素酶、漆酶等,而且能够降解晶体状和复杂结构的纤维素。本发明主要以干黄玉米秸秆为原料,利用从犏牛瘤胃中分离培养的厌氧真菌—新美鞭菌和甲烷菌—甲烷短杆菌的自然共培养物对复杂结构木质纤维素的降解优势,以市售商品木聚糖酶做对照,对比研究犏牛瘤胃厌氧真菌Neocallimastixsp.属和甲烷菌的自然共培养物对黄牛的生长性能、粗饲料表观消化率、血液生化指标,为干黄玉米粗秸秆的饲料化利用提供新思路。
采用从放牧犏牛瘤胃液中分离出的自然共生的厌氧真菌和甲烷菌共培养物以玉米秸秆为底物所产的具有高活性木质纤维素降解酶活性的发酵液添加入黄牛的玉米秸秆粗饲料后再饲喂黄牛,是一个国内外首创的、新颖的、有效的手段。本发明意外地从犏牛瘤胃中分离出了一种厌氧真菌Neocallimastix frontalis和甲烷菌Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1,并使用此共培养物以玉米秸秆为底物发酵4d的发酵液添加入黄牛的玉米秸秆粗饲料后再饲喂黄牛,此发酵液作为一种天然饲用酶,不但能够提高玉米秸秆粗饲料的中性洗涤纤维素的瘤胃降解率,显著改善黄牛的采食量,使黄牛的平均日增重量有所提高,还能够使黄牛机体的蛋白质代谢旺盛,蛋白质的吸收利用率提高,肝脏的蛋白质合成能力及组织蛋白质的沉淀作用增强,从而促进了黄牛的生长。
应当说明的是,新美鞭菌的拉丁名为Neocallimastix frontalis,新美鞭菌是属于厌氧真菌的一种;在本发明中,新美鞭菌、厌氧真菌和Neocallimastix frontalis均代表的是同一株菌。甲烷短杆菌的拉丁名为Methanobrevibacter olleyae,甲烷短杆菌是属于甲烷菌的一种,在本发明中,甲烷短杆菌、甲烷菌和Methanobrevibacter olleyae均代表的是同一株菌。
发明内容
针对上述技术问题,本发明的第一目的是提供一种可增强机体代谢能力的饲用酶,所述的饲用酶为犏牛瘤胃新美鞭菌和甲烷短杆菌自然共培养物,命名为Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养所产高活性木质纤维素降解酶发酵液,所述的Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1于2021年11月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23971,保藏地址为北京是朝阳区北辰西路1号院3号中国科学院微生物研究所,电话为010-64807355。
优选的,所述的饲用酶中木聚糖酶活性为10800mU/mL,羧甲基纤维素酶活性为610mU/mL,漆酶活性是75.0mU/mL。
优选的,所述的Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养的时间为4d。
本发明的第二目的是提供Neocallimastix frontalis和Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养所产高活性木质纤维素降解酶发酵液作为饲用酶的应用。
本发明的第三目的是提供Neocallimastix frontalis和Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养所产高活性木质纤维素降解酶发酵液在提高粗饲料利用率中的应用。
本发明的第四目的是提供Neocallimastix frontalis和Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养所产高活性木质纤维素降解酶发酵液在增强动物代谢能力中的应用。
本发明的第五目的是提供Neocallimastix frontalis和Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养所产高活性木质纤维素降解酶发酵液在促进动物生长中的应用。
本发明的有益效果是:本发明提供了一种可增强机体代谢能力的饲用酶,所述的饲用酶包括犏牛瘤胃Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物厌氧培养4d所产的具有高活性木质纤维素降解酶的发酵液,所述的饲用酶能有效提高干黄玉米秸秆作为粗饲料的适口性,且可降解干黄玉米秸秆中难以被消化利用的木质纤维素,能够提高玉米秸秆粗饲料的中性洗涤纤维的瘤胃降解率,提高了粗饲料的营养价值,使黄牛平均日增重量和平均日采食量均有所提高,并且使黄牛机体蛋白质代谢旺盛,蛋白质的吸收利用率提高,肝脏的蛋白质合成能力及组织蛋白质的沉淀作用增强,从而促进了黄牛的生长。
具体实施方式
下面结合具体实施例对本发明的保护范围进行进一步的限定,应当说明的是,本发明的保护范围并不受实施例的限制。
实施例一、一种可促进黄牛生长的天然饲用酶的制备
犏牛瘤胃Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1:将Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1在以玉米秸秆为底物(1%(w/v))厌氧培养72h的发酵液25mL接种在225mL通用厌氧真菌培养基中,以玉米秸秆为底物(1%(w/v))培养4d,将发酵液1000×g离心10min获得上清酶液备用。此上清液在瘤胃条件下—即pH 6.8,39℃时木聚糖酶活性为10800mU/mL,羧甲基纤维素酶活性为610mU/mL,漆酶活性是75.0mU/mL。
实施例二、一种可促进黄牛生长的天然饲用酶的应用
1.材料与方法
1.1试验设计与样品处理
1.1.1试验材料
玉米秸秆:取自甘肃省定西市,收集时间2020年12月,收集时含水量5.25%,属于干黄状态,粉碎至0.5—1cm备用。
商品木聚糖酶:来自白银赛诺生物科技有限公司,粉末状木聚糖酶加蒸馏水至饱和,酶活性为10万u/g,加蒸馏水溶解致饱和,取上清液弃沉淀备用。
1.1.2试验动物的饲喂管理
试验动物为甘肃省定西市通渭县李岘村黄牛养殖场,每天饲喂三次,早上8:00、中午12:00、下午7:00,自由饮水。
日粮组成(风干基础%):玉米秸秆90%,麦麸4%,豆粕3%,尿素1%,小苏打1%,碳酸钙1%。
1.1.3实验设计及样品采集
随机选取试验用9头牛分为3组分别标记,其中空白组3只黄牛(K1—K3)饲喂干黄玉米秸秆,对照组3只黄牛(D1—D2)在饲喂前玉米秸秆中加入木聚糖酶饱和液,实验组3只黄牛(S1—S3)在饲喂前玉米秸秆中加入犏牛瘤胃新美鞭菌和甲烷短杆菌的自然共培养物,试验预试期5d,正式期14d,在正式试验第一天开始先将9头牛进行48h空腹准备。将准备好的不同预处理的三组秸秆对试验牛进行饲喂,早晚各一次,自由饮水。
1.2测定指标与测定方法
1.2.1生长性能测定
正式实验开始和结束时,试验黄牛均空腹禁食12h后,称重记为初重、末重,计算平均日增重。试验期间每日准确记录每组黄牛的给料量和剩料量,计算平均日增重量、料重比和平均日采食量。
平均日增重量(ADG)=(末重-初重)/试验天数
平均日采食量(ADFI)=每头牛试验期内的采食量/试验天数
料重比(F/G)=平均日采食量/平均日增重量
1.2.2养分表观消化率的测定
取一定量样品烘干测定其水分含量和干物质(DM),取烘干后的样品用粉碎机粉碎,然后过40目筛,取过筛后的样品测定干物质(DM)、中性洗涤纤维(NDF)、酸性洗涤纤维(ADF),干物质(DM)测定采用105℃烘干法[111]。木质纤维组分测定:NDF、ADF采用阿尔法F6800型纤维测定仪。
干物质消化率=[(食入的干物质-排出的干物质)/干物质总量)]*100%
某养分的表观消化率=[(某养分的食入含量-粪便中该养分的含量)/某养分的食入含量]*100%[131]
1.2.3血液生化指标的测定
试验最后1d晨饲前,每组黄牛采用含抗凝剂的抗凝管颈静脉采血,分析各组黄牛血液生化学指标[丙氨酸氨基转移(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、甘油酸酯(TG)、葡萄糖(GLU)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLB)]
1.3数据分析
基础数据经Excel 2010初步整理并制作图表,利用SPSS 20.0软件对试验数据进行统计分析,采用单因子ANOVO模型处理,并用Duncan方法对数据进行多重比较分析,以平均值±标准差表示测定结果,P<0.05代表数据存在显著性差异。
2试验结果
2.1不同添加酶对黄牛生长性能测定
由表1可以看出,用Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物(1%(w/v))培养4d的发酵液添加入玉米秸秆粗饲料然后饲喂黄牛后,黄牛平均日增重量和平均日采食量均显著高于对照组(商品木聚糖酶组)和空白组(P>0.05),空白组与对照组的料重比无显著性差异(P<0.05),实验组料重比显著低于对照组和空白组(P>0.05)。
表1 3组不同添加物的黄牛生长性能
2.2不同添加酶对黄牛玉米秸秆粗饲料的表观消化率的影响
由表2可以看出,添加商品木聚糖酶的干黄玉米秸秆其干物质表观消化率与空白组差异不显著(P<0.05),而添加瘤胃Neocallimastix frontalis和Methanobrevibacterolleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1的干物质表观消化率显著高于对照组与空白组(P>0.05)。实验组和对照组的中性洗涤纤维、酸性洗涤纤维素的表观消化率相较于空白组差异显著(P>0.05),其中实验组的NDFD、ADFD表观消化率显著高于空白组和对照组(P>0.05),且实验组NDFD、ADFD表观消化率最高(P>0.05)。
表2 3组不同添加物的营养物质表观消化率
2.3不同添加酶对黄牛血液生化指标的影响
由表3可以看出,实验组的ALT、AST和ALP均显著低于对照组和空白组(P>0.05),对照组与空白组的TG差异不显著,实验组TG添加木聚糖酶的干黄玉米秸秆其干物质表观消化率与空白组差异不显著(P>0.05),实验组GLU显著高于对照组(P>0.05),对照组与空白组、实验组的TP、ALB和GLB含量差异不显著(P>0.05)。
表3 3组不同预添加物的血液生化指标
3讨论
3.1不同添加酶对黄牛生长性能的影响
动物生长性能反映了动物与的生长状况对某一因素的变化,是机体代谢的综合性指标。本实验中用犏牛瘤胃Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1的发酵液添加到玉米秸秆粗饲料中再饲喂黄牛后,使黄牛饲喂平均日增长量和平均日采食量增加,料重比降低,说明犏牛瘤胃Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1的发酵液能够有效提高干黄玉米秸秆作为粗饲料的适口性,且Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1的发酵液可降解干黄玉米秸秆中难以被消化利用的木质纤维素,添加后提高了玉米秸秆粗饲料的营养价值,使黄牛平均日增重量和平均日采食量均有所提高。
3.2不同添加酶对黄牛秸秆粗饲料消化率的影响
养分表观消化率是体现反刍动物肠道整体机能强弱的重要指标,反刍动物对饲料的消化能力越强,反映相似饲料的养分表观消化率就越高。同种饲料经过不同的处理后干物质表观消化率也存在差异,玉米秸秆经过瘤胃新美鞭菌和甲烷短杆菌的自然共培养物所产的酶液的作用后纤维素、半纤维素和木质素得到有效降解,更容易被瘤胃微生物消化利用,使得实验组的玉米秸秆干物质在瘤胃中的有效降解明显提高。本试验测得干物质表观消化率:实验组>对照组>空白组,对照组由于酶系单一,只能针对性降解半纤维素,而纤维素与木质素未被降解,使得干物质降解率(DMD)低于实验组。中性洗涤纤维素降解率(NDFD)和酸性洗涤纤维素降解率(ADFD)的量一般对纤维素、半纤维素等指标有着代表性意义。在其他条件保持一定,提高中性洗涤纤维的瘤胃降解率可以显著改善反刍动物的采食量,提高其产肉量。这与本试验的研究结果相同,添加商品木聚糖酶和犏牛瘤胃Neocallimastixfrontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1组的NDFD、ADFD和CPD消化率显著高于空白组。
3.3不同添加酶对黄牛血液生化指标的影响
血液生化指标反映机体组织器官的功能和生理状态、营养物质的代谢及免疫水平等,可作为预测犊牛健康的关键指数。AST和ALT在机体蛋白合成和代谢中发挥转氨基酸的作用,其活性是准确反映心肌细胞和肝脏功能的重要指标。ALP与骨骼的生长密切相关,也可以反映机体蛋白质和脂肪代谢的效率。本试验中AST、ALT和ALP均有提高,这表明使用Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1组的发酵液添加入玉米秸秆粗饲料有利于改善黄牛肝脏的功能。GLU是机体代谢直接的能量来源,能反映机体能量水平的高低、糖生成与消耗的动态平衡。TG时机体体脂的重要组成部分,其含量可作为脂肪肝诊断的依据。实验组血清GLU含量显著高于空白组和对照组,这表明实验组能在一定范围内增强机体代谢能力。TP包括ALB和GLB,是反应机体蛋白质合成代谢的重要指标,TP含量的提高可促进机体蛋白质的沉积和免疫力的加强等,ALB用于修补组织和提高能量,还可以维持血液渗透压,而GLB与机体的抗体水平密切相关。本实验中三个不同处理组对血清TP、ALB和GLB含量无显著影响,表明实验组能够使黄牛机体蛋白质代谢旺盛,蛋白质的吸收利用率提高,肝脏的蛋白质合成能力及组织蛋白质的沉淀作用增强。
迄今为止,没有将犏牛瘤胃厌氧真菌和甲烷菌的自然共培养物的发酵液作为日粮添加剂饲喂黄牛的研究,本发明专利具有显著的创新性,取得了良好应用效果.
4.小结
综上所述,本发明专利提供了一种可增强机体代谢能力的饲用酶,所述的天然饲用酶包括犏牛瘤胃Neocallimastix frontalis和Methanobrevibacter olleyae的自然共培养物(N.frontalis+M.olleyae)Cattle-YakTZ1以玉米秸秆为底物(1%(w/v))培养4d的高活力发酵液。所述的天然饲用酶添加入黄牛的玉米秸秆粗饲料中然后饲喂黄牛能够有效提高干黄玉米秸秆作为粗饲料的适口性,降解干黄玉米秸秆中难以被消化利用的木质纤维素;能够提高玉米秸秆粗饲料的中性洗涤纤维的瘤胃降解率,提高了粗饲料的营养价值,使黄牛的平均日增重量和平均日采食量均有所提高;并使黄牛机体蛋白质代谢旺盛,蛋白质的吸收利用率提高,肝脏的蛋白质合成能力及组织蛋白质的沉淀作用增强,从而促进了黄牛的生长,因此它可以作为一种天然的饲用酶添加剂推广应用,应用前景极其广阔。
Claims (6)
1.一种增强黄牛机体代谢能力的饲用酶,其特征在于,所述的饲用酶是犏牛瘤胃新美鞭菌Neocallimastix frontalis和甲烷短杆菌Methanobrevibacter olleyae的自然共培养物Cattle-Yak TZ1在以玉米秸秆为底物厌氧培养所产生的木质纤维素降解酶发酵液,所述犏牛瘤胃新美鞭菌Neocallimastix frontalis和甲烷短杆菌Methanobrevibacterolleyae的自然共培养物Cattle-YakTZ1于2021年11月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23971;所述的饲用酶在反刍动物瘤胃温度39℃的条件下,木聚糖酶活性为10800mU/mL,羧甲基纤维素酶活性为610mU/mL,漆酶活性是75.0mU/mL。
2.如权利要求1所述的饲用酶,其特征在于,所述的是犏牛瘤胃新美鞭菌Neocallimastix frontalis和甲烷短杆菌Methanobrevibacter olleyae的自然共培养物Cattle-YakTZ1以玉米秸秆为底物厌氧培养的时间为4d。
3.如权利要求1所述的饲用酶在提高粗饲料干黄玉米秸秆利用率中的应用。
4.如权利要求1所述的饲用酶在增强黄牛代谢能力中的应用。
5.如权利要求1所述的饲用酶在促进黄牛生长中的应用。
6.犏牛瘤胃新美鞭菌Neocallimastix frontalis和甲烷短杆菌Methanobrevibacterolleyae的自然共培养物Cattle-YakTZ1在以玉米秸秆为底物厌氧培养所产生的木质纤维素降解酶发酵液作为饲用酶的应用,其特征在于,所述的犏牛瘤胃新美鞭菌Neocallimastix frontalis和甲烷短杆菌Methanobrevibacter olleyae的自然共培养物Cattle-YakTZ1于2021年11月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23971;所述的饲用酶在反刍动物瘤胃温度39℃的条件下,木聚糖酶活性为10800mU/mL,羧甲基纤维素酶活性为610mU/mL,漆酶活性是75.0mU/mL。
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