CN116887689A - 通过使用丝氨酸蛋白酶改善动物饲料中糖酶对碳水化合物的可消化性的方法 - Google Patents
通过使用丝氨酸蛋白酶改善动物饲料中糖酶对碳水化合物的可消化性的方法 Download PDFInfo
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Landscapes
- Fodder In General (AREA)
Abstract
本发明提供一种通过添加一种或多种蛋白酶(优选丝氨酸蛋白酶,例如拟诺卡氏菌属丝氨酸蛋白酶或S8丝氨酸蛋白酶)来改善动物饲料(例如大豆粕、玉米、小麦、小麦麦麸、燕麦、黑麦、大麦和高粱)和/或粗饲料(例如干草和牧草植物)或其混合物中糖酶对碳水化合物的可消化性的方法。本发明还提供了一种适用于上述方法的饲料组合物。
Description
技术领域
本发明涉及一种用于提升动物饲料中的糖酶活性和/或改善动物体内的碳水化合物的可消化性的方法。
背景技术
碳水化合物是由碳、氢和氧构成的提供能量的饲料组分。它们应占动物日粮的约75%。它们提供的能量为肌肉运动提供动力。碳水化合物还产生帮助动物保暖的体热。它们有助于由身体使用蛋白质和脂肪。碳水化合物不存储在体内。它们必须每天在动物的日粮中提供。未使用的碳水化合物会转换成脂肪进行存储。
碳水化合物可分为两类:存储碳水化合物和结构碳水化合物。存储碳水化合物包括淀粉和单糖,例如果糖和蔗糖。这些碳水化合物以及种子胚胎部分中的脂质是从谷物种子长出的新植株的主要能量来源。
另一方面,结构碳水化合物,包括众所周知的非淀粉多糖,负责细胞的形式和结构,并且主要位于外细胞膜中。结构碳水化合物,通常称为“纤维”,由于缺乏合适的内源酶而很难被单胃动物消化。因此,大多数结构碳水化合物在后肠中发酵,在所述后肠中它们可释放有限的可用能量水平。
糖酶是一种特殊的商用酶制备物,其攻击碳水化合物,从而释放能量,否则动物会损失这些能量。它们主要通过打开完整植物细胞的细胞壁结构来工作,由此不仅释放能量(淀粉),而且还释放其他营养物质,例如蛋白质、矿物质和脂质。此外,植物细胞壁级分会增加肠道粘度,所述增加的肠道粘度会导致营养吸收减少、病原体(例如大肠杆菌(Escherichia coli))增殖加速,以及其他问题,例如粘性粪便和脏蛋。如今,糖酶变得在动物饲料中越来越受欢迎。
令人惊讶的是,发现蛋白酶可提升糖酶的活性以水解动物饲料中的碳水化合物,由此潜在地提高动物体内碳水化合物的可消化性。
发明内容
因此,本发明提供了一种用于在动物饲料中提升糖酶的活性或改善糖酶对碳水化合物的水解的方法;以及一种用于通过使用一种或多种蛋白水解酶(即,蛋白酶)来改善动物体内碳水化合物的可消化性的方法。
本发明还提供了一种饲料组合物及其用途,所述饲料组合物包含一种或多种蛋白水解酶(即蛋白酶)和用于改善动物饲料中碳水化合物的水解和/或用于改善动物体内碳水化合物的可消化性的糖酶。
具体实施方式
在本发明中,术语“动物(animal/animals)”是指除人类之外的任何动物。动物的示例是单胃动物,包括但不限于小猪或生猪(包括但不限于仔猪、生长猪和母猪);家禽,例如火鸡、鸭、鹌鹑、珍珠鸡、鹅、鸽子(包括乳鸽)和鸡(包括但不限于肉用仔鸡(在本文中称为肉鸡)、雏鸡、下蛋母鸡(在本文中称为蛋鸡));宠物,例如猫和狗;马(包括但不限于热血马、冷血马和温血马)、甲壳类动物(包括但不限于基围虾和对虾)和鱼(包括但不限于青甘鱼、巨滑舌鱼、鲃鱼、鲈鱼、扁鲹、鲮脂鲤科(bocachico)、鳊鱼、大头鱼、大盖巨脂鲤、鲤鱼、鲶鱼、卡特拉魮、虱目鱼、红点鲑、丽鱼科鱼、军曹鱼、鳕鱼、莓鲈、金头鲷、鼓鱼、鳗鱼、虾虎鱼、金鱼、丝足鱼、石斑鱼、花身副丽鱼、大比目鱼、爪哇鱼、野鲮属(labeo)、濑鱼、泥鳅、鲭鱼、遮目鱼、银鲈、泥鱼、鲻鱼、paco、绿腹丽鱼、银汉鱼、河鲈鱼、梭子鱼、鲳鲹、斜齿鳊、鲑鱼、长丝异鳃鲶、大眼鰤鲈、海鲈鱼、海鲷、闪光鱼、鲈塘鳢、黑鱼、鲷鱼、锯盖鱼、鳎、蓝子鱼、鲟鱼、翻车鱼、香鱼、丁鲷、皇冠鱼、罗非鱼、鳟鱼、金枪鱼、大菱鲆、白鳟鱼、玻璃梭鲈和白鲑鱼)。
在本发明中,术语“动物饲料”、“动物日粮”或“饲料”是指适合于或旨在被动物摄入并且能够在除水外不消耗任何附加物质的情况下维持生命和/或促进动物生产的任何化合物、制备物或混合物。用于单胃动物的动物饲料通常包含浓缩物以及维生素、矿物质、酶、直接饲喂的微生物、氨基酸和/或其他饲料成分(例如在预混物中),而用于反刍动物的动物饲料通常包含草料(包括粗饲料和青贮料)并且还可以包含浓缩物以及维生素、矿物质、酶、直接饲喂的微生物、氨基酸和/或其他饲料成分(例如在预混物中)。
在本发明中,术语“浓缩物”是指具有高蛋白质和能量浓度的饲料,诸如鱼粉、糖蜜、低聚糖、高粱、种子和谷物(整粒玉米、燕麦、黑麦、大麦、小麦,或通过压碎、碾磨等由例如玉米、燕麦、黑麦、大麦、小麦制成)、油料压榨饼(例如,来自棉籽、红花、向日葵、大豆(诸如豆粕)、油菜籽/低芥酸菜籽、花生)、棕榈仁饼、酵母衍生材料和蒸馏酒粕(诸如湿蒸馏酒粕(wet distillers grains,WDS))和具有可溶物的干蒸馏酒粕(dried distillersgrains with solubles,DDGS)。
在本发明中,如本文所定义的术语“草料”还包括粗饲料。草料是来自饲用植物、禾草和其他饲用植物、海藻、发芽谷物和豆类或它们的任何组合的新鲜植物材料,例如干草和青贮料。草料植物的示例为苜蓿(紫花苜蓿)、百脉根(birdsfoot trefoil)、芸苔属(例如羽衣甘蓝、油菜(卡诺拉)、芜菁甘蓝(瑞典芜菁)、芜菁(turnip))、三叶草(例如杂种车轴草、红车轴草、地三叶草、白三叶草)、禾草(例如狗牙根、雀麦草、假燕麦草、羊茅草(fescue)、欧石楠草(heath grass)、草地早熟禾、鸭茅草(orchard grass)、黑麦草、提摩西草(Timothy-grass))、玉米(玉蜀黍)、小米、大麦、燕麦、黑麦、高粱、大豆和小麦以及蔬菜(例如甜菜)。草料进一步包括来自粮食生产的作物残茬(例如玉米秸秆;来自小麦、大麦、燕麦、黑麦和其他谷物的秸秆);来自蔬菜的残茬,如甜菜缨(beet tops);来自油籽生产的残茬,如来自大豆、油菜籽和其他豆类的茎和叶;以及来自供动物或人食用的谷物的提炼或来自燃料生产或其他工业的级分。
在本发明中,术语“粗饲料”是指具有高纤维水平的干燥植物材料,例如纤维、麸皮、来自种子和谷粒的外皮以及作物残茬(例如秸秆、干椰子肉、糠秕、甜菜渣)。
在本发明中,术语“动物饲料添加剂”是指添加到动物饲料中的成分或成分组合,所述成分或成分组合通常以微量使用并且需要仔细处理和混合。此类成分包括但不限于维生素、氨基酸、矿物质、酶、摄生素、着色剂、促生长添加剂和芳香族化合物/调味剂、多不饱和脂肪酸(PUFA);产活性氧的物质、抗氧化剂、抗微生物肽、抗真菌多肽和真菌毒素管理化合物等。
在本发明中,术语“水解”是指待水解的碳水化合物在酸性或碱性条件下和/或在酶的存在下被分解成可溶性糖分子。例如,通过用浓盐酸处理可以将多糖水解成单糖或二糖或寡糖,并且通过用浓盐酸或淀粉酶处理可以将淀粉分解成麦芽糖。当与其他饲料组合物相比,更多的碳水化合物,例如1%、2%、3%、4%、5%或更多的碳水化合物在本发明的饲料组合物存在下被分解成可溶性糖分子时,碳水化合物的水解得到改善。
在第一方面中,本发明提供了一种用于提升动物饲料中糖酶的活性的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶(即,蛋白酶)。
继第一方面,本发明还提供了一种用于改善动物饲料中碳水化合物的水解的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶(即,蛋白酶)和糖酶。
继第一方面,本发明进一步提供了一种用于改善动物体内碳水化合物的可消化性的方法,所述方法包括向所述动物施用在动物饲料中的一种或多种蛋白水解酶(即,蛋白酶)和糖酶。
在本发明中,蛋白水解酶或蛋白酶分解蛋白质中的肽键,从而将所述蛋白质分解成氨基酸链的片段或肽。
蛋白酶基于其催化机制分类为以下组:丝氨酸蛋白酶(S)、半胱氨酸蛋白酶(C)、天冬氨酸蛋白酶(A)、金属蛋白酶(M)和未知或尚未分类的蛋白酶(U)(参见Handbook ofProteolytic Enzymes,A.J.Barrett,N.D.Rawlings,J.F.Woessner(编辑),AcademicPress(1998))。根据本发明的蛋白酶是丝氨酸蛋白酶,优选地酸稳定性丝氨酸蛋白酶,更优选地S8蛋白酶。
对于根据本发明使用的蛋白酶的来源没有限制。因此,术语蛋白酶不仅包括天然或野生型蛋白酶,而且还包括表现出蛋白酶活性的任何突变体、变体、片段等,以及合成蛋白酶,例如改组蛋白酶和共有蛋白酶。此类经遗传工程改造的蛋白酶可以按照本领域通常已知的方式制备,例如通过定点诱变、通过PCR(使用含有所需突变的PCR片段作为PCR反应中的引物之一)或通过随机诱变。共有蛋白的制备描述于例如EP 0 897 985中。
优选地,根据本发明的蛋白酶是微生物蛋白酶,术语微生物的指示所述蛋白酶来源于或起源于微生物,或者是来源于微生物的类似物、片段、变体、突变体或合成蛋白酶。它可以在原始野生型微生物菌株中,在另一种微生物菌株中或在植物中产生或表达;即所述术语涵盖野生型、天然存在的蛋白酶的表达,以及在任何宿主中重组的、经遗传工程改造的或合成的蛋白酶的表达。
微生物的示例是细菌,例如放线菌门(Actinobacteria phy.nov.),例如纲I的细菌;放线菌,例如亚纲V的放线菌;放线菌亚纲,例如目I的放线菌亚纲;放线菌目,例如亚目XII的放线菌目;链孢囊菌亚目(Streptosporangineae),例如科II的链孢囊菌亚目;拟诺卡氏菌科(Nocardiopsaceae),例如属I的拟诺卡氏菌科;拟诺卡氏菌属(Nocardiopsis),例如拟诺卡氏菌属种属NRRL 18262和白色拟诺卡氏菌(Nocardiopsis alba);例如,表现出蛋白酶活性的芽孢杆菌属(Bacillus)物种或其突变体或变体。这种分类法基于Berge's Manualof Systematic Bacteriology,第2版,2000,Springer(preprint:Road Map to Bergey's)。
微生物的另外示例是真菌,例如酵母或丝状真菌。
根据本发明的优选蛋白酶是从以下属获得的酸稳定性丝氨酸蛋白酶或可从以下属获得:拟诺卡氏菌属,例如来源于达松维尔拟诺卡氏菌(Nocardiopsis dassonvillei)亚种达松维尔DSM 43235(A1918L1)、葱绿拟诺卡氏菌(Nocardiopsis prasina)DSM 15649(NN018335L1)、葱绿拟诺卡氏菌(先前称为白色拟诺卡氏菌)DSM 14010(NN18140L1)、拟诺卡氏菌属种属DSM 16424(NN018704L2)、嗜碱盐拟诺卡氏菌(Nocardiopsis alkaliphila)DSM 44657(NN019340L2)和卢森坦拟诺卡氏菌(Nocardiopsis lucentensis)DSM 44048(NN019002L2);或以下属:芽孢杆菌属,例如霍内克芽胞杆菌(Bacillus horneckiae)、芽孢杆菌属种属TY145、芽孢杆菌属种属13380、病研所芽孢杆菌(Bacillus idriensis)、芽孢杆菌属种属62451和海洋沉积物芽胞杆菌(Bacillus oceanisediminis);以及同源蛋白酶。
对于根据本发明使用的酸稳定性丝氨酸蛋白酶的来源没有限制。因此,术语蛋白酶不仅包括天然或野生型蛋白酶,而且还包括其表现出蛋白酶活性的任何突变体、变体、片段等,以及合成蛋白酶,例如改组蛋白酶和共有蛋白酶。此类经遗传工程改造的蛋白酶可以按照本领域通常已知的方式制备,例如通过定点诱变、通过PCR(使用含有所需突变的PCR片段作为PCR反应中的引物之一)或通过随机诱变。共有蛋白的制备描述于例如EP 0 897 985中。
根据本发明使用的酸稳定性蛋白酶的示例是
a)来源于拟诺卡氏菌属种属NRRL 18262的蛋白酶和白色拟诺卡氏菌的蛋白酶;
b)与(i)的蛋白酶中的任何蛋白酶具有至少60%、65%、70%、75%、80%、85%、90%或至少95%氨基酸同一性的蛋白酶;
c)与SEQ ID NO:1和/或SEQ ID NO:2中任一者具有至少60%、65%、70%、75%、80%、85%、90%或至少95%同一性的蛋白酶。
为了计算同一性百分比,可以使用本领域已知的任何计算机程序。此类计算机程序的示例是Clustal V算法(Higgins,D.G.,and Sharp,P.M.(1989),Gene(Amsterdam),73,237-244);以及在GCG版本8程序包中提供的GAP程序(Program Manual for the WisconsinPackage,版本8,Genetics Computer Group,575Science Drive,Madison,Wisconsin,USA53711)(Needleman,S.B.和Wunsch,C.D.,(1970),Journal of Molecular Biology,48,443-453)。
根据本发明使用的蛋白酶可在原始野生型微生物菌株中,在另一种微生物菌株中或在植物中产生或表达;即所述术语涵盖野生型、天然存在的蛋白酶的表达,以及在任何宿主中重组的、经遗传工程改造的或合成的蛋白酶的表达。
在本发明上下文中,术语酸稳定性意指纯蛋白酶,在对应于A280=1.0的稀释度下,并在37℃下在以下缓冲液中孵育2小时后:
·100mM琥珀酸、100mM HEPES、100mM CHES,
·100mM CABS、1mM CaCl2、150mM KCl、0.01% TritonX-100,pH 3.5,
的蛋白酶活性是参考活性的至少40%,如使用本文实施例1中所述的测定测量的(底物:Suc-AAPF-pNA,pH 9.0,25℃)。
在上述酸稳定性定义的具体实施方式中,蛋白酶活性是参考活性的至少45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或至少97%。
术语参考活性是指相同蛋白酶以纯形式在对应于A280=1.0的稀释度下在以下缓冲液中于5℃孵育2小时后的蛋白酶活性:100mM琥珀酸、100mM HEPES、100mM CHES、100mMCABS、1mM CaCl2、150mM KCl、0.01% Triton X-100,pH 9.0,其中所述活性如上所述测定。
换句话说,测定酸稳定性的方法包括以下步骤:
a)将待测蛋白酶样品(纯形式,A280=1.0)分为两等份(I和II);
b)将等分试样I于37℃和pH 3.5下孵育2小时;
c)测量等分试样I的残余活性(pH 9.0和25℃);
d)将等分试样II在5℃和pH 9.0下孵育2小时;
e)测量等分试样II的残余活性(pH 9.0和25℃);
f)计算等分试样I的残余活性相对于等分试样II的残余活性的百分比。
或者,在酸稳定性的上述定义中,步骤b)缓冲液的pH值可为1.0、1.5、2.0、2.5、3.0、3.1、3.2、3.3或3.4。
在涉及上述替代步骤b)缓冲液pH值的上述酸稳定性定义的其他替代实施方式中,与参考相比的残余蛋白酶活性为至少5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、或至少97%。
在替代实施方式中,6.0、6.5、7.0、7.5、8.0或8.5的pH值可应用于步骤d)缓冲液。
在上述酸稳定性定义中,术语A280=1.0意指相对于缓冲液空白,在1cm光程长度比色皿中在280nm处产生1.0的吸收的所述纯蛋白酶的浓度(稀释度)。
并且在上述酸稳定性定义中,术语纯蛋白酶是指A280/A260比率高于或等于1.70的样品。
在另一具体实施方式中,用于根据本发明使用的蛋白酶除了是酸稳定的之外,还是热稳定的。
术语热稳定意指以下中的一者或多者:最佳温度为至少50℃、52℃、54℃、56℃、58℃、60℃、62℃、64℃、66℃、68℃,或至少70℃。
根据本发明的另一种优选的蛋白酶是由具有S8蛋白酶活性的多肽定义的蛋白酶,其中所述多肽选自由以下组成的列表:
a)与SEQ ID NO:3至SEQ ID NO:6中的任一者具有至少70%的序列同一性的多肽;
b)SEQ ID NO:3至SEQ ID NO:6中的任一者的变体,其中所述变体具有蛋白酶活性并且包含在1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个位置中的一个或多个取代、和/或一个或多个缺失、和/或一个或多个插入或它们的任意组合;
c)多肽,所述多肽包含(a')或(b')的多肽以及N末端和/或C末端His标签和/或HQ标签;
d)多肽,所述多肽包含(a')或(b')的多肽以及至多10个氨基酸(例如1、2、3、4、5、6、7、8、9或10个氨基酸)的N末端和/或C末端延伸;以及
e)(a')或(b')的多肽的片段,所述多肽的片段具有蛋白酶活性并具有成熟多肽的长度的至少90%。
由上述定义所涵盖的市售蛋白酶是ProAct(DSM NutritionalProducts AG,Switzerland)、/>ProAct360(DSM Nutritional Products Ltd.,Switzerland)、AxtraPro(枯草杆菌蛋白酶,Dupont,USA)、Poultrygrow(不同蛋白酶的混合物,Jefo,USA)、Cibenza DP100(Novus,USA)。
在优选实施方式中,蛋白酶的预期剂量为0.01-200mg蛋白酶/kg最终饲料。
根据本发明,蛋白酶可以以1,000单位/kg动物饲料至1,000,000单位/kg动物饲料的剂量提供,例如以以下量(剂量范围)中的一者提供:1,000单位/kg动物饲料、2,000单位/kg动物饲料、4,000单位/kg动物饲料、6,000单位/kg动物饲料、8,000单位/kg动物饲料、10,000单位/kg动物饲料、15,000单位/kg动物饲料、20,000单位/kg动物饲料、30,000单位/kg动物饲料、50,000单位/kg动物饲料、80,000单位/kg动物饲料、100,000单位/kg动物饲料、150,000单位/kg动物饲料、200,000单位/kg动物饲料、250,000单位/kg动物饲料、300,000单位/kg动物饲料、500,000单位/kg动物饲料、600,000单位/kg动物饲料、800,000单位/kg动物饲料、1,000,000单位/kg动物饲料。一个蛋白酶单位(PROT)是指在pH 9.0和37℃下每分钟从1mM底物(例如N-琥珀酰-Ala-Ala-Pro-Phe-pNA)释放1μmol对硝基苯胺(pNA)的酶的量。
在本发明中,糖酶可以是任何可以添加到用于饲喂动物的动物饲料中的糖酶。糖酶的示例包括但不限于半纤维素酶、果胶酶、葡聚糖酶、淀粉酶(例如α-淀粉酶、β-淀粉酶和γ-淀粉酶)、木聚糖酶、半乳糖苷酶、麦芽糖酶,以及它们的混合物。
优选的木聚糖酶是1,4-β-木聚糖酶,例如由里氏木霉(Trichoderma reesei)产生的内切-1,4-β-木聚糖酶。糖酶混合物的示例是不同木聚糖酶的混合物或包含至少两种选自由以下组成的组的酶的糖酶共混物:木聚糖酶、葡聚糖酶、半纤维素酶、果胶酶、淀粉酶、半乳糖苷酶、麦芽糖酶。仅通过一种非经遗传修饰的真菌(如例如多能篮状菌(Talaromycesversatilis))即可生产含有多于10种活性酶的组合的混合物。
由上述定义涵盖并且可以被根据本发明的蛋白酶提升的市售糖酶是VP(葡聚糖酶,DSM Nutritional Products AG,Switzerland)、/>WX(木聚糖酶,DSM Nutritional Products Ltd.,Switzerland)、/>HiStarch(来自地衣芽孢杆菌(Bacillus licheniformis)的淀粉酶,DSM NutritionalProducts AG,Switzerland)、Rovabio(糖酶共混物,Adisseo,France)、/>(木聚糖酶,AB Enzymes,Germany)、CIBENZA CSM(木聚糖酶、β-葡聚糖酶和半乳糖苷酶的混合物,Novus International,USA)和AllzymeSSF(糖酶共混物,Alltech USA)。
根据本发明,糖酶以10单位/kg动物饲料至5,000单位/kg动物饲料的剂量提供,例如以以下量中的一者提供:10单位/kg动物饲料、20单位/kg动物饲料、40单位/kg动物饲料、50单位/kg动物饲料、60单位/kg动物饲料、80单位/kg动物饲料、100单位/kg动物饲料、200单位/kg动物饲料、500单位/kg动物饲料、800单位/kg动物饲料、1,000单位/kg动物饲料、2,000单位/kg动物饲料、3,000单位/kg动物饲料、4,000单位/kg动物饲料和5,000单位/kg动物饲料。
在本发明中,糖酶的活性可以通过糖酶对碳水化合物的水解来表示,其可以通过本领域已知的方法容易地测量,例如如本申请的实施例中所示通过测试由所述糖酶处理的碳水化合物的水解产物。当与没有蛋白酶的相同条件相比,在蛋白酶存在下更多的碳水化合物(例如1%、2%、3%、4%、5%或更多)分解成可溶性糖分子时,糖酶的活性被提升。
在本发明中,基于从用糖酶水解获得的水解产物的量,糖酶活性可被提升或者碳水化合物的水解可被改善1%、2%、3%、4%、5%、10%、15%、20%或更多。
如本领域中已知,碳水化合物是所有动物的基本能量来源并且必须在动物日粮中每天提供。适合于根据本发明的动物饲料的碳水化合物可以是单糖,例如半乳糖、葡萄糖、果糖、核糖、阿拉伯糖和木糖;二糖,例如麦芽糖、蔗糖和乳糖;寡糖,例如阿拉伯糖基木聚糖;和/或多糖,例如淀粉,包括直链淀粉、支链淀粉和改性淀粉、非淀粉多糖(NSP),包括戊聚糖、糖原、纤维、纤维素、半纤维素、几丁质、果胶和/或亲水胶体。
在本发明中,待由糖酶水解的碳水化合物可以是寡糖和/或多糖。优选地,碳水化合物是阿拉伯糖基木聚糖、淀粉或NSP,例如纤维、纤维素、半纤维素和果胶。
在本发明中,碳水化合物可以是浓缩物的形式,例如大豆粕、玉米、小麦、小麦麦麸、燕麦、黑麦、大麦和高粱;和/或粗饲料,例如干草和牧草植物,或它们的混合物。优选地,动物饲料中的碳水化合物为大豆粕、玉米、小麦、小麦麦麸、燕麦、黑麦或大麦或它们的混合物的形式。
优选地,根据本发明的动物饲料是基于大豆粕、玉米和/或小麦的动物日粮。
如本领域任何技术人员所预期的,根据本发明的动物饲料还可包含微量成分。
所述微量成分包括但不限于芳香族化合物;抗微生物肽;多不饱和脂肪酸(PUFA);产活性氧的物质;至少一种酶,和脂溶性和水溶性维生素,以及矿物质。
抗微生物肽(AMP)的示例是CAP18、明串珠菌素A(leucocin A)、protegrin-1、thanatin、防御素、乳铁蛋白、乳铁素和ovispirin,例如novispirin(Robert Lehrer,2000)、菌丝霉素和他汀类。
多不饱和脂肪酸的示例是C18、C20和C22多不饱和脂肪酸,例如花生四烯酸、二十二碳六烯酸,二十碳五烯酸和γ-亚油酸。
产活性氧的物质的示例是例如过硼酸盐、过硫酸盐或过碳酸盐的化学物质;以及例如氧化酶、加氧酶或合成酶的酶。
酶的示例是植酸酶(EC 3.1.3.8或3.1.3.26)、半乳聚糖酶(EC 3.2.1.89)、α-半乳糖苷酶(EC 3.2.1.22)、磷脂酶A1(EC 3.1.1.32)、磷脂酶A2(EC 3.1.1.4)、溶血磷脂酶(EC3.1.1.5)、磷脂酶C(EC 3.1.4.3)和/或磷脂酶D(EC 3.1.4.4)。
脂溶性维生素的示例包括但不限于维生素A、维生素D3和维生素K,例如维生素K3。
水溶性维生素的示例包括但不限于维生素B12、生物素和胆碱、维生素B1、维生素B2、维生素B6、烟酸、叶酸和泛酸盐,例如D-泛酸钙。
矿物质的示例包括但不限于钙、磷、钠、钾、镁、氯、碘、铁、锰、铜、钼、钴和锌。饲料中常用的矿物质补充剂是:石灰石、骨粉、牡蛎壳、氯化钠、磷酸二钙、硫酸锰、碘化钾和过磷酸盐。矿物质的来源包括肉类下脚料、鱼粉、乳产品、磨碎的石灰石(钙)、磨碎的牡蛎壳(钙)、磷酸二钙(钙、磷)、脱氟磷酸岩(磷、钙)、蒸骨粉(磷、钙)、盐(钠、氯、碘)、硫酸锰(锰)、氧化锰(锰)、碳酸锌(锌)、氧化锌(锌)。
还如本领域技术人员所预期的,根据本发明的动物饲料可进一步包含任何数量的动物饲料典型组分,例如蛋白质、如上定义的碳水化合物、脂肪和附加添加剂。
可包含在饲料中的合适类型的蛋白质的示例包括但不限于肉类下脚料(赖氨酸)、鱼粉(赖氨酸、甲硫氨酸)、家禽副产品粉(色氨酸、赖氨酸)、血粉、肝脏和腺粉、羽毛粉(水解的)、动物残渣、乳产品、棉籽粉、花生粉、大豆粕、芝麻粉、葵花籽粉。
大多数饲料成分(玉米、大麦、红花、蜀黍、小麦、米糠等)含有大约2-5%的脂肪和亚油酸。脂肪的来源包括动物油脂(牛肉)、荤油、玉米油和其他植物油。
附加添加剂包括但不限于如上所定义的矿物质;抗氧化剂,如BHT(丁羟甲苯)、山都魁(santoquin)、乙氧喹(ethoxyquin)、丁基化羟基茴香醚和二苯基对苯二胺;丸粒粘合剂,例如钠质膨润土(粘土)、木浆工业的液体或固体副产品、糖蜜和瓜尔豆粕;着色剂,例如叶黄素、合成类胡萝卜素和角黄素;益生菌,例如乳杆菌属(lactobacillus)和链球菌属(streptococcus)的菌株;和/或抗生素,例如青霉素、链霉素、四环素和金霉素。
在一个实施方式中,本发明提供了一种用于提升动物饲料中糖酶的活性的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶(即,蛋白酶),其中:
a)所述蛋白酶是酸稳定性丝氨酸蛋白酶,更优选地S8蛋白酶,所述蛋白酶的剂量为10,000单位/kg饲料至30,000单位/kg饲料;
b)所述糖酶为半纤维素酶、果胶酶、淀粉酶、木聚糖酶,或它们的混合物;
c)所述动物饲料是基于大豆粕、玉米和/或小麦的动物日粮;并且
d)任选地,所述糖酶的活性增加了5%或更多。
在另一实施方式中,本发明提供了一种用于改善动物饲料中碳水化合物的水解的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶(即,蛋白酶)和糖酶,其中:
a)所述蛋白酶是酸稳定性丝氨酸蛋白酶,更优选地S8蛋白酶,所述蛋白酶的剂量为10,000单位/kg饲料至30,000单位/kg饲料;
b)所述糖酶为半纤维素酶、果胶酶、淀粉酶、木聚糖酶,或它们的混合物;
c)所述动物饲料是基于大豆粕、玉米和/或小麦的动物日粮;并且
d)任选地,所述糖酶的活性增加了5%或更多。
在第二方面中,本发明提供了一种饲料组合物,所述饲料组合物包含一种或多种如下所定义的蛋白酶,以及糖酶或糖酶混合物。
此类组合物的蛋白酶是:
A:酸稳定性蛋白酶选自由以下组成的组:
a)来源于拟诺卡氏菌属种属NRRL 18262的蛋白酶和白色拟诺卡氏菌的蛋白酶;
b)与(i)的蛋白酶中的任何蛋白酶具有至少60%、65%、70%、75%、80%、85%、90%或至少95%氨基酸同一性的蛋白酶;
c)与SEQ ID NO:1和/或SEQ ID NO:2中任一者具有至少60%、65%、70%、75%、80%、85%、90%或至少95%同一性的蛋白酶。
或者
B:由具有S8蛋白酶活性的多肽定义的蛋白酶,其中所述多肽选自由以下项组成的列表:
a)(a')与SEQ ID NO:3至SEQ ID NO:4中的任一者具有至少70%的序列同一性的多肽;
b)(b')SEQ ID NO:3至SEQ ID NO:6中的任一者的变体,其中所述变体具有蛋白酶活性并且包含在1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个位置中的一个或多个取代、和/或一个或多个缺失、和/或一个或多个插入或它们的任意组合;
c)(c')多肽,所述多肽包含(a')或(b')的多肽以及N末端和/或C末端His标签和/或HQ标签;
d)(d')多肽,所述多肽包含(a')或(b')的多肽以及至多10个氨基酸(例如1、2、3、4、5、6、7、8、9或10个氨基酸)的N末端和/或C末端延伸;以及
e)(e')(a')或(b')的多肽的片段,所述多肽的片段具有蛋白酶活性并具有成熟多肽的长度的至少90%。
此类组合物的糖酶可以是可添加到用于饲喂动物的动物饲料中的任何糖酶。糖酶的示例包括但不限于半纤维素酶、果胶酶、葡聚糖酶、淀粉酶(例如α-淀粉酶、β-淀粉酶和γ-淀粉酶)、木聚糖酶、半乳糖苷酶、麦芽糖酶。
优选的木聚糖酶是1,4-β-木聚糖酶,例如由里氏木霉(Trichoderma reesei)产生的内切-1,4-β-木聚糖酶。糖酶混合物的示例是不同木聚糖酶的混合物或包含至少两种选自由以下组成的组的酶的混合物:木聚糖酶、葡聚糖酶、半纤维素酶、果胶酶、淀粉酶、半乳糖苷酶、麦芽糖酶。仅通过一种非经遗传修饰的真菌(如例如多能篮状菌(Talaromycesversatilis))即可生产含有多于10种活性酶的组合的混合物。
由上述定义涵盖的市售糖酶是VP(葡聚糖酶,DSM NutritionalProducts AG,Switzerland)、/>WX(木聚糖酶,DSM Nutritional ProductsLtd.,Switzerland)、/>HiStarch(来自地衣芽孢杆菌(Bacilluslicheniformis)的淀粉酶,DSM Nutritional Products AG,Switzerland)、Rovabio(糖酶共混物,Adisseo,France)、/>(木聚糖酶,AB Enzymes,Germany)、CIBENZA CSM(木聚糖酶、β-葡聚糖酶和半乳糖苷酶的混合物,Novus International,USA)、AllzymeSSF(糖酶共混物,Alltech USA)。
所述饲料组合物和/或所述组合物所含有的组分(例如糖酶和蛋白酶)可以配制为液体制剂或固体制剂。因此,根据本发明的饲料组合物还可包含一种或多种配制试剂。
所述配制剂可以选自由以下组成的组:多元醇,例如甘油、山梨糖醇、乙二醇、二甘醇、三甘醇、1,2-丙二醇、1,3-丙二醇、二丙二醇和聚乙二醇(PEG);盐,例如有机或无机锌盐、钠盐、钾盐、钙盐或镁盐(例如,硫酸镁、乙酸钙、苯甲酸钙、碳酸钙、氯化钙、柠檬酸钙、山梨酸钙、硫酸钙、乙酸钾、苯甲酸钾、碳酸钾、氯化钾、柠檬酸钾、山梨酸钾、硫酸钾、乙酸钠、苯甲酸钠、碳酸钠、氯化钠、柠檬酸钠、硫酸钠、乙酸锌、苯甲酸锌、碳酸锌、氯化锌、柠檬酸锌、山梨酸锌和硫酸锌);以及淀粉或糖或糖衍生物,例如蔗糖、糊精、葡萄糖、乳糖和山梨糖醇;有机小分子、面粉、纤维素和矿物质以及粘土矿物(也称为水合页铝硅酸盐,例如高岭石或高岭土)。
根据本发明的饲料组合物还可以包含一种或多种乳化剂。所述乳化剂可以有利地选自由以下项组成的组:聚甘油脂肪酸酯,例如酯化的蓖麻油酸或脂肪酸的丙二醇酯、蔗糖酯或糖甘油酯、聚乙二醇、卵磷脂等。
在根据本发明的饲料组合物中,蛋白酶可以以1,000单位/kg动物饲料至1,000,000单位/kg动物饲料的剂量提供,例如以以下量(剂量范围)中的一者提供:1,000单位/kg动物饲料、2,000单位/kg动物饲料、4,000单位/kg动物饲料、6,000单位/kg动物饲料、8,000单位/kg动物饲料、10,000单位/kg动物饲料、15,000单位/kg动物饲料、20,000单位/kg动物饲料、30,000单位/kg动物饲料、50,000单位/kg动物饲料、80,000单位/kg动物饲料、100,000单位/kg动物饲料、150,000单位/kg动物饲料、200,000单位/kg动物饲料、250,000单位/kg动物饲料、300,000单位/kg动物饲料、500,000单位/kg动物饲料、600,000单位/kg动物饲料、800,000单位/kg动物饲料、1,000,000单位/kg动物饲料。
在根据本发明的饲料组合物中,糖酶可以以10单位/kg动物饲料至5,000单位/kg动物饲料的剂量提供,例如以以下量中的一者提供:10单位/kg动物饲料、20单位/kg动物饲料、40单位/kg动物饲料、50单位/kg动物饲料、60单位/kg动物饲料、80单位/kg动物饲料、100单位/kg动物饲料、200单位/kg动物饲料、500单位/kg动物饲料、800单位/kg动物饲料、1,000单位/kg动物饲料、2,000单位/kg动物饲料、3,000单位/kg动物饲料、4,000单位/kg动物饲料和5,000单位/kg动物饲料。
如本领域任何技术人员所理解的,根据本发明的饲料组合物可以配制为饲料添加剂或动物饲料,并且因此可以进一步包含如上所定义的适合于动物饲料的成分。
本发明的第三方面提供了一种或多种蛋白水解酶(即,蛋白酶)和糖酶在动物饲料中用于改善动物体内碳水化合物的可消化性的用途,其中所述蛋白酶、糖酶、碳水化合物、动物饲料和相关剂量如上所定义。
本发明将通过以下实施例进一步说明。
实施例
实施例1:包含蛋白酶的组合物
通过混合如表1中所示的以下成分和量来制备根据本发明的包含蛋白酶的组合物1至3。
表1
*由DSM Nutritional Products AG,Switzerland销售的酶产品的商品名。
实施例2:包含蛋白酶的动物饲料
通过以如表2所示的以下配方将蛋白酶添加到饲料中来制备根据本发明的包含蛋白酶的动物饲料。
表2
1每千克日粮供应:100mg的丁羟甲苯;0.2mg的生物素;12.8mg的泛酸钙;60μg的胆钙化醇;0.017mg的氰钴胺;5.2mg的叶酸;4mg的甲萘醌;35mg的烟酸;10mg的吡哆醇;3.33mg的反式视黄醇;12mg的核黄素;3.0mg的硫胺素;60mg的dl-α-生育酚乙酸酯;638mg的氯化胆碱;0.3mg的Co;3.0mg的Cu;25mg的Fe;1mg的I;125mg的Mn;0.5mg的Mo;200μg的Se;60mg的Zn(DSM Nutritional Products,Wagga Wagga,NSW,Australia)
实施例3:动物饲料中蛋白酶对SBM的影响
通过体外实验研究用于应用于动物饲料中的蛋白酶对大豆粕(soy-bean meal,SBM)的影响。
在25mL的0.1M乙酸盐缓冲液+5mM CaCl2 pH 6.0中提取80.1mg的VP颗粒。将样品用0.2μm过滤器过滤,并在0.1M乙酸盐缓冲液+5mM CaCl2pH 6.0中稀释四倍。将100μL所获得的酶溶液加入4mL 10% SBM重量/体积中。将11.2μL纯化蛋白酶溶液(浓度21.5mg/mL)用1989μL的0.1M乙酸盐缓冲液+5mM CaCl2 pH 6.0稀释。将100μL所获得的酶溶液加入4mL 10% SBM重量/体积中。
在与酶一起孵育后,通过在4700g和0℃下离心15min去除固体,并将500μL上清液在99℃下进行酸水解(1.6M HCl)1小时。通过高性能阴离子交换色谱结合脉冲电流检测(pulsed amperometric detection,HPAEC-PAD)分析所得样品的中性单糖含量。在40℃的温度下用流率为0.2mL/min的1mM KOH等度洗脱液在CarboPac分析PA-210柱(内径2mm)和CarboPac PA-210保护柱(Thermofisher)上实现分离。
对对照组和VP组实施相同的工序,不同之处在于在对照组中不添加酶,而仅在/>VP组中添加/>VP。
实验一式四份地运行。对六种单糖标准品(岩藻糖、阿拉伯糖、葡萄糖、木糖、甘露糖和半乳糖醛酸)进行分析,并且将以测定平均值的形式给出的结果记录在表3中。
表3
实验表明,VP从SBM分解并溶解出NSP,并且当添加蛋白酶时,从SBM水解和溶解的NSP增加了5%。
实施例4:蛋白酶在动物饲料中对脱淀粉玉米的用途
通过体外实验研究用于应用于动物饲料中的蛋白酶对脱淀粉玉米的影响。
在25mL的0.1M乙酸盐缓冲液+5mM CaCl2 pH 6.0中提取100mg的WX CT颗粒。将样品用0.2μm过滤器过滤。将100μL获得的酶溶液添加到4mL的10%脱淀粉玉米重量/体积中。将11.2μL纯化蛋白酶溶液(浓度21.5mg/mL)用1989μL的0.1M乙酸盐缓冲液+5mM CaCl2 pH 6.0稀释。将100μL所获得的酶溶液加入4mL 10%脱淀粉玉米重量/体积中。
在与酶一起孵育后,通过在4700g和0℃下离心15min去除固体,并将500μL上清液在99℃下进行酸水解(1.6M HCl)1小时。通过高性能阴离子交换色谱结合脉冲电流检测(pulsed amperometric detection,HPAEC-PAD)分析所得样品的中性单糖含量。在40℃的温度下用流率为0.2mL/min的1mM KOH等度洗脱液在CarboPac分析PA-210柱(内径2mm)和CarboPac PA-210保护柱(Thermofisher)上实现分离。
对对照组和WX组实施相同的工序,不同之处在于在对照组中不添加酶,而仅在/>WX组中添加/>WX。
实验一式四份地运行。对阿拉伯糖和木糖进行分析,并且将以测定平均值的形式给出的结果记录在表4中。
表4
实验表明,WX可从小麦分解并溶解出阿拉伯糖基木聚糖,并且在添加蛋白酶时,溶解的阿拉伯糖基木聚糖多了23%。
实施例5:蛋白酶在动物饲料中对玉米的用途
通过体外实验研究用于应用于动物饲料中的蛋白酶对玉米的影响。
在25mL的0.1M乙酸盐缓冲液+5mM CaCl2 pH 6.0中提取160.2mg的HiStarch颗粒。将样品用0.2μm过滤器过滤,并在0.1M乙酸盐缓冲液+5mMCaCl2 pH 6.0中稀释八倍。将100μL所获得的酶溶液加入4mL 10%玉米重量/体积中。将11.2μL纯化蛋白酶溶液(浓度21.5mg/mL)用1989μL的0.1M乙酸盐缓冲液+5mM CaCl2 pH6.0稀释。将100μL所获得的酶溶液加入4mL10%玉米重量/体积中。
实验一式四份地运行。在与酶一起孵育后,通过在4700g和0℃下离心15min去除固体,并将500μL上清液在99℃下进行酸水解(1.6M HCl)1小时。通过高性能阴离子交换色谱结合脉冲电流检测(pulsed amperometric detection,HPAEC-PAD)分析所得样品的中性单糖含量。在40℃的温度下用流率为0.2mL/min的1mM KOH等度洗脱液在CarboPac分析PA-210柱(内径2mm)和CarboPac PA-210保护柱(Thermofisher)上实现分离。
对对照组和HiStarch组实施相同的工序,不同之处在于在对照组中不添加酶,而仅在/>HiStarch组中添加/>HiStarch。
实验一式四份地运行。对葡萄糖进行分析,并且将以测定平均值的形式给出的结果记录在表5中。
表5
实验表明,HiStarch水解玉米淀粉,并且当添加蛋白酶时,水解的玉米淀粉多了21%。/>
序列表
<110> 帝斯曼知识产权资产管理有限公司(DSM IP Assets BV)和诺维信专利公司(Novozymes A/S)
<120> 通过使用丝氨酸蛋白酶改善动物饲料中糖酶对碳水化合物的可消化性的方法
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<170> PatentIn第3.5版
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Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val
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Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly
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<213> 人工序列(Artificial Sequence)
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<223> 蛋白工程化变体
<220>
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<222> (1)..(311)
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Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly
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Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val
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<221> MISC_FEATURE
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<221> MISC_特征
<222> (6)..(6)
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<221> MISC_特征
<222> (7)..(7)
<223> 7位中的氨基酸是任何氨基酸。
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<222> (8)..(8)
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Claims (14)
1.一种用于提升动物饲料中糖酶的活性的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶,即,蛋白酶,其中所述蛋白酶是丝氨酸蛋白酶,优选地酸稳定性丝氨酸蛋白酶或S8蛋白酶,并且其中所述糖酶选自由以下组成的组:半纤维素酶、果胶酶、葡聚糖酶、淀粉酶、木聚糖酶、麦芽糖酶以及它们的混合物。
2.根据权利要求1所述的方法,其中所述酸稳定性蛋白酶选自由以下组成的组:
a.来源于拟诺卡氏菌属种属NRRL 18262的蛋白酶,和白色拟诺卡氏菌的蛋白酶;
b.与(i)的蛋白酶中的任何蛋白酶具有至少60%、65%、70%、75%、80%、85%、90%或至少95%氨基酸同一性的蛋白酶;
c.与SEQ ID NO:1和/或SEQ ID NO:2中任一者具有至少60%、65%、70%、75%、80%、85%、90%或至少95%同一性的蛋白酶。
3.根据权利要求1所述的方法,其中所述蛋白酶由选自由以下项组成的列表的具有S8蛋白酶活性的多肽定义:
a.与SEQ ID NO:3至SEQ ID NO:6中的任一者具有至少70%的序列同一性的多肽;
b.SEQ ID NO:3至SEQ ID NO:6中的任一者的变体,其中所述变体具有蛋白酶活性并且包含在1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个位置中的一个或多个取代、和/或一个或多个缺失、和/或一个或多个插入或它们的任意组合;
c.多肽,所述多肽包含(a')或(b')的多肽以及N末端和/或C末端His标签和/或HQ标签;
d.多肽,所述多肽包含(a')或(b')的多肽以及至多10个氨基酸(例如1、2、3、4、5、6、7、8、9或10个氨基酸)的N末端和/或C末端延伸;以及
e.(a')或(b')的多肽的片段,所述多肽的片段具有蛋白酶活性并具有成熟多肽的长度的至少90%。
4.根据权利要求1-3中任一项所述的方法,其中所述蛋白酶以1,000单位/kg动物饲料至1,000,000单位/kg动物饲料的剂量提供,例如以以下量(剂量范围)中的一者提供:1,000单位/kg动物饲料、2,000单位/kg动物饲料、4,000单位/kg动物饲料、6,000单位/kg动物饲料、8,000单位/kg动物饲料、10,000单位/kg动物饲料、15,000单位/kg动物饲料、20,000单位/kg动物饲料、30,000单位/kg动物饲料、50,000单位/kg动物饲料、80,000单位/kg动物饲料、100,000单位/kg动物饲料、150,000单位/kg动物饲料、200,000单位/kg动物饲料、250,000单位/kg动物饲料、300,000单位/kg动物饲料、500,000单位/kg动物饲料、600,000单位/kg动物饲料、800,000单位/kg动物饲料、1,000,000单位/kg动物饲料。
5.根据权利要求1-3中任一项所述的方法,其中所述糖酶以10单位/kg动物饲料至5,000单位/kg动物饲料的剂量提供,例如以以下量中的一者提供:10单位/kg动物饲料、20单位/kg动物饲料、40单位/kg动物饲料、50单位/kg动物饲料、60单位/kg动物饲料、80单位/kg动物饲料、100单位/kg动物饲料、200单位/kg动物饲料、500单位/kg动物饲料、800单位/kg动物饲料、1,000单位/kg动物饲料、2,000单位/kg动物饲料、3,000单位/kg动物饲料、4,000单位/kg动物饲料和5,000单位/kg动物饲料。
6.根据权利要求1-3中任一项所述的方法,其中所述动物饲料含有选自由以下组成的组的碳水化合物:阿拉伯糖基木聚糖、淀粉和/或非淀粉多糖(NSP),例如纤维、纤维素、半纤维素和果胶,或它们的混合物。
7.根据权利要求6所述的方法,其中所述碳水化合物是浓缩物的形式,例如大豆粕、玉米、小麦、小麦麦麸、燕麦、黑麦、大麦和高粱;和/或粗饲料,例如干草和牧草植物,或它们的混合物。
8.根据权利要求1-3中任一项所述的方法,其中所述动物饲料是基于大豆粕、玉米和/或小麦的动物日粮。
9.一种用于提升动物饲料中糖酶的活性的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶,即,蛋白酶,其中:
a)所述蛋白酶是酸稳定性丝氨酸蛋白酶,更优选地S8蛋白酶,所述蛋白酶的剂量为10,000单位/kg饲料至30,000单位/kg饲料;
b)所述糖酶为半纤维素酶、果胶酶、淀粉酶、木聚糖酶,或它们的混合物;
c)所述动物饲料是基于大豆粕、玉米和/或小麦的动物日粮;并且
d)任选地,所述糖酶的活性增加了5%或更多。
10.一种用于改善动物饲料中碳水化合物的水解的方法,所述方法包括向所述动物饲料中添加一种或多种蛋白水解酶,即,蛋白酶,和糖酶。
11.一种用于改善动物体内碳水化合物的可消化性的方法,所述方法包括向所述动物施用在动物饲料中的一种或多种蛋白水解酶,即,蛋白酶,和糖酶。
12.根据权利要求10或11所述的方法,其中所述蛋白酶是丝氨酸蛋白酶,优选地酸稳定性丝氨酸蛋白酶,更优选地S8蛋白酶。
13.根据权利要求10或11所述的方法,其中所述糖酶选自由以下组成的组:半纤维素酶、果胶酶、葡聚糖酶、淀粉酶、木聚糖酶、麦芽糖酶,以及它们的混合物。
14.一种饲料组合物,所述饲料组合物包含一种或多种蛋白酶和一种或多种糖酶,其中所述蛋白酶是丝氨酸蛋白酶或S8蛋白酶并且其中所述糖酶选自由以下组成的组:半纤维素酶、果胶酶、葡聚糖酶、淀粉酶、木聚糖酶、麦芽糖酶以及它们的混合物,并且其中
a.所述酸稳定性蛋白酶选自由以下组成的组:
i.来源于拟诺卡氏菌属种属NRRL 18262的蛋白酶,和白色拟诺卡氏菌的蛋白酶;
ii.与(i)的蛋白酶中的任何蛋白酶具有至少60%、65%、70%、75%、80%、85%、90%或至少95%氨基酸同一性的蛋白酶;
iii.与SEQ ID NO:1和/或SEQ ID NO:2中任一者具有至少60%、65%、70%、75%、80%、85%、90%或至少95%同一性的蛋白酶;或者
b.所述蛋白酶由选自由以下项组成的列表的具有S8蛋白酶活性的多肽定义:
i.与SEQ ID NO:3至SEQ ID NO:6中的任一者具有至少70%的序列同一性的多肽;
ii.SEQ ID NO:3至SEQ ID NO:6中的任一者的变体,其中所述变体具有蛋白酶活性并且包含在1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个位置中的一个或多个取代、和/或一个或多个缺失、和/或一个或多个插入或它们的任意组合;
iii.多肽,所述多肽包含(a')或(b')的多肽以及N末端和/或C末端His标签和/或HQ标签;
iv.多肽,所述多肽包含(a')或(b')的多肽以及至多10个氨基酸(例如1、2、3、4、5、6、7、8、9或10个氨基酸)的N末端和/或C末端延伸;以及
v.(a')或(b')的多肽的片段,所述多肽的片段具有蛋白酶活性并具有成熟多肽的长度的至少90%。
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