包含外源性蛋白酶的基于藻類的動物飼料組合物、動物飼料添加劑、及其用圖
【專利說明】包含外源性蛋白酶的基于藻類的動物飼料組合物、動物飼料 添加劑、及其用途
[0001]本申請要求2013年7月11日提交的美國臨時專利申請61/845,243的優(yōu)先權(quán)，其全 部內(nèi)容在此通過引用并入。 發(fā)明領(lǐng)域
[0002]本發(fā)明涉及基于藻類的動物飼料組合物、動物飼料添加劑、及其用途
【背景技術(shù)】
[0003] 玉米和大豆的種植面積分別占國家耕地的29%和26% (Cromwell，"Soybean Meal_The"Gold Standard，"The Farmer's Pride，KPPA News，Vol.ll，No.20，llHl〇H， 1999)。在美國生產(chǎn)的約60 %的玉米和47 %的大豆被用于飼料業(yè)，主要用來飼養(yǎng)雞和豬 (Olson，"Below Cost Feed Crops：An Indirect Subsidy for Industrial Animal Factories，" Issues 202:222(2011))。盡管它們代表了兩大產(chǎn)肉品種，但其對玉米和大豆 的大量消耗直接與人類的食物供應(yīng)產(chǎn)生競爭。因此，需要替代的飼料成分來維持畜牧業(yè)和 人類食物保障 已經(jīng)在用于雞（Grau et al·，"Sewage-Grown Algae as a Feedstuff for Chicks，"Poult.Sci.36:1046-1051(1957);Mokady et al·，"Algae Grown on Wastewater as a Source of Protein for Young Chickens and Rats，'，Nutr · Rep · Int · 9: 383-390 (1979)；Austic et al·，"Potential and Limitation of a New Defatted Diatom Microalgal Biomass in Replacing Soybean Meal and Corn in Diets for Broiler Chickens，"J.Agric.Food Chem.61(30):7341_7348(2013))和豬（Hintz et al·，"Sewage-Grown Algae as a Protein Supplement for Swine，'，Anim.Prod.9:135-140(1967);Yap et al·，"Feasibility of Feeding Spirulina-Maxima，Arthrospira-Platensis or Chlorel la Sp to Pigs Weaned to a Dry Diet at 4 to 8Day s of Age，'， Nutr.Rep.Int.25：543-552(1982)；Isaacs et al·，"A Partial Replacement of Soybean Meal by whole or Defatted Algal Meal in Diet for Weanling Pigs Does not Affect their Plasma Biochemical Indicators，"J.of Animal Sci.89：723-723(2011)) 的飲食中測試了各種海洋微藻。伴隨著最近在使用微藻作為原料用于第三代生物燃料生產(chǎn) 方面的興趣（Chisti，"Biodiesel from Microalgae，"Biotechnol · Adv · 25 : 294-306 (2007))，已經(jīng)確定了將脫脂娃藻微藻十字脆桿藻亞屬（Staurospira sp.)加入到肉仔雞 (Austic et al·，"Potential and Limitation of a New Defatted Diatom Microalgal Biomass in Replacing Soybean Meal and Corn in Diets for Broiler Chickens，'， J.Agric.Food Chem.61(30):7341_7348(2013))和斷奶仔豬（Isaacs et al·，"A Partial Replacement of Soybean Meal by whole or Defatted Algal Meal in Diet for Weanling Pigs Does not Affect their Plasma Biochemical Indicators，'，J · of Animal Sci .89:723-723(2011))的飲食中的功效。
[0004]在連續(xù)三個雞的實驗中，證明了在肉仔雞飲食中加入7.5%的脫脂硅藻微藻來取 代玉米和豆柏并不破壞其在整個生產(chǎn)周期（從第1天到第42天）中的生長表現(xiàn)或生化狀態(tài) (Austic et al·，"Potential and Limitation of a New Defatted Diatom Microalgal Biomass in Replacing Soybean Meal and Corn in Diets for Broiler Chickens，'， J.Agric.Food Chem.61(30):7341-7348(2013))。然而，加入10%的脫脂硅藻微藻來取代玉 米和豆桕或者加入7.5%的脫脂硅藻微藻來單獨取代豆桕導(dǎo)致了對雞生長表現(xiàn)的負面影 響。由于脫脂硅藻微藻含有比豆桕更低水平的粗蛋白（19.1對48.5%)，所以實際的問題是 肉仔雞能否耐受加入更多的其他類型的具有更優(yōu)的蛋白質(zhì)、氨基酸和其他組分譜的脫脂微 藻生物質(zhì)。雖然在飲食中補充高達10%的脫脂硅藻微藻對雞的一些血漿生化指標(biāo)幾乎沒有 效應(yīng)，但是對禽類氮代謝的血楽尿酸、終產(chǎn)物和敏感生物標(biāo)記物的效應(yīng)(Donsbough et al.，"Uric Acid，Urea，and Ammonia Concentrations in Serum and Uric Acid Concentration in Excreta as Indicators of Amino Acid Utilization in Diets for Broilers，"Poult.Sci.89:287_294(2010);Hernandez et al.，"Effect of Low-Protein Diets and Single Sex on Production Performance ? Plasma Metabolites， D i ge s t ib i I i ty，andNi trogen Excretion in I -to 48_Day-〇ld Broilers，'， Poult.Sci .91:683-692(2012))仍然不確定（Austic et al·，"Potential and Limitation of a New Defatted Diatom Microalgal Biomass in Replacing Soybean Meal and Corn in Diets for Broiler ChickensZiJ-Agric-Food Chem.61(30)：7341-7348(2013)； Leng et al·，"Effect of Dietary Defatted Diatom Biomass on Egg Production and Quality of Laying Hens，" J .Anim · Sci · Biotechnol .5:3(2014))。還沒有確定補充性的脫 脂硅藻微藻對血漿氨基酸譜的效應(yīng)。此外，雖然通常在肉仔雞的飲食中摻入非淀粉多糖 (NSP)降解酶來改善營養(yǎng)價值，但是以往的研究僅測試了在含7.5%脫脂硅藻微藻飼料中的 補充性的可商購蛋白酶的效應(yīng)（Lum et al·，"Effects of Various Replacements of Corn and Soy by Defatted Microalgal Meal on Growth Performance and Biochemical Status of Weanling Pigs，"J.Anim.Sci.90:701-701(2012);Simbaya et al.?uThe Effects of Protease and Carbohydrase Supplementation on the Nutritive Value of Canola Meal for Poultry：In Vitro and In Vivo Studies，'， Anim.Feed Sci.Technol.61：2I9-234(1996)；Ghazi et al.，"The Potential for the Improvement of the Nutritive Value of Soya-Bean Meal by Different Proteases in Broiler Chicks and Broiler CockerelsBr.Poult.Sci.43：70-77(2002)；07Shea et al.，"The Effect ofProtease and Xylanase Enzymes on Growth Performance， Nutrient Digestibility?and Manure Odour in Grower-Finisher Pigs，'，Anim.Feed Sci.Technol. 189:88-97(2014))。補充NSP酶可能在含微藻的飲食中特別有用，這歸因于其 生物質(zhì)中存在復(fù)雜的碳水化合物（Brown et al. /'Nutritional Properties of Microalgae for Mariculture ,Aquaculture 151:315-331(1997);Chen et al·， uMicroalgae-Based Carbohydrates for Biofuel Production，'，Biochem.Eng.J.78:1_10 (2013))，并且其他微藻生物質(zhì)可以呈現(xiàn)出與脫脂硅藻微藻不同的對外源性蛋白酶的易感 性（Austic et al·，"Potential and Limitation of a New Defatted Diatom Microalgal Biomass in Replacing Soybean Meal and Corn in Diets for Broiler Chickens，"J.Agric.Food Chem.61(30):7341-7348(2013))〇
[0005] 盡管將微藻作為新的或者替代的蛋白補充劑進行了探討，以前的研究 (Venkataraman et al·，"Replacement Value of Blue-Green Alga(Spirulina platensis)for Fishmeal and a Vitamin-Mineral Premix for Broiler Chicks，'， Br.Poult.Sci.35：373-381(1994)；Becker?uMicro-Algae as a Source of ProteinZ， Biotechnol.Adv. 25:207-210(2007))并沒有考查飼喂藻類對機體新陳代謝的影響或者潛 在的調(diào)節(jié)機制。具體地，哺乳動物雷帕霉素靶蛋白（mTOR)通路是細胞生長的關(guān)鍵調(diào)節(jié)物，其 整合來自營養(yǎng)物、能量狀態(tài)和生長因子的信號來調(diào)節(jié)新陳代謝（Sarbassov et al.， "Growing Roles for the mTOR Pathway，"Curr.0pin.Cell Biol.l7:596_603(2005))。飼 喂方式（Boussaid-Om et al ·，"Regulators of Protein Metabolism are Affected by Cyclical Nutritional Treatments with Diets Varying in Protein and Energy Content，" J ·Nutr · Biochem. 23 :1467_1473(2012))、氨基酸豐度和可用度（Kimball et al·，"Control of Protein Synthesis by Amino Acid Availability，'，Curr.Opinion Cl in.Nutr.Metab.Care 5：63-67(2002)；KimbalI et al·，"Signaling Pathways and Molecular Mechanisms Through which Branched-Chain Amino Acids Mediate Translational Control of Protein Synthesis，"J.Nutr.l36:227S_231S(2006))、蛋白 質(zhì)豐度（Boussaid-Om et al ·，"Regulators ofProtein Metabolism are Affected by Cyclical Nutritional Treatments with Diets Varying in Protein and Energy Content，" J· Nutr · Biochem. 23:1467-1473(2012))和蛋白質(zhì)來源（Luo et al ·，"Effects of Different Dietary Protein Sources on Expression of Genes Related to Protein Metabolism in Growing Rats，"Br.J.Nutr.l04:1421(2010))能夠影響此通路D mTOR的下游祀點包括介導(dǎo)翻譯起始步驟的真核翻譯起始因子4E結(jié)合蛋白I (4E-BP1)和真核 翻譯起始因子4E(eIF4E)以及被認為調(diào)節(jié)蛋白質(zhì)翻譯的核糖體蛋白S(S6)和核糖體蛋白S6 激酶（S6Kl)(Wang et al·，"The mTOR Pathway in the Control ofProtein Synthesis，" Physiol. 21:362-369(2006))。由于肉仔雞在生長期間經(jīng)歷高強度的蛋白質(zhì)合成，所以確定 在其飲食中加入微藻生物質(zhì)對涉及mTOR通路的信號蛋白的功能性表達的效應(yīng)不僅有新意， 而且相關(guān)。
[0006] 正如肉仔雞一樣，斷奶仔豬以較快的速度生長并伴有高強度的蛋白質(zhì)代謝 (Robison，"Growth Patterns in Swine，" J.Anim.Sci .42:1024-1035(1976))。同樣，以前 的石開究（Lum et al·，"Effects of Various Replacements of Corn and Soy by Defatted Microalgal Meal on Growth Performance and Biochemical Status of Weanling Pigs/'J.Anim.Sci. 90:701-701 (2012))顯示，斷奶仔豬在以含 7.5%脫脂硅藻微 藻的飲食取代玉米和豆桕情況下生長良好，但在用15%的脫脂硅藻微藻替代玉米和豆桕的 條件下沒有此效果。隨后，出現(xiàn)了同樣的問題:斷奶仔豬能否耐受加入的更高水平的含有更 高蛋白的脫脂綠色微藻生物質(zhì)。然而，對于豬和雞的研究有不同的考慮。首先，在豬的研究 中，加入的微藻生物質(zhì)對營養(yǎng)狀況的血漿生化量度，包括抗酒石酸酸性磷酸酶（"TRAP"）、堿 性磷酸酶（"AKP"）和丙氨酸轉(zhuǎn)氨酶（"ALT"）（Isaacs et al.，"A Partial Replacement of Soybean Meal by whole or Defatted Algal Meal in Diet for Weanling Pigs Does not Affect their Plasma Biochemical Indicators，"J·of Animal Sci.89：723-723 (2011))的效應(yīng)沒有肉仔雞研究中的效應(yīng)那么確定。通常使用血漿尿素氮（"PUN"）和氨基酸 譜來估計飼料蛋白質(zhì)對于豬的營養(yǎng)價值(Kohn et al.，"Using Blood Urea Nitrogen to Predict Nitrogen Excretion and Efficiency of Nitrogen Utilization in Cattle， Sheep，Goats，Horses，Pigs，and Rats，" J.Anim. Sci · 83:879-889(2005))。雖然對于豬能方 便地將氧化鉻作為指示劑加入飲食中來估計總氨基酸全腸道表觀消化率而對于雞這是不 可能的（Stein et al ·，"Comparative Protein and Amino Acid Digestibilities in Growing Pigs and Sows，"J.Anim.Sci.77:1169_1179(1999))，但是，從前者進行組織取樣 來研究飲食治療的代謝性調(diào)節(jié)比從后者中取樣更困難并且更昂貴（Suryawan et al.， "Developmental Regulation of the Activation of Signaling Components Leading to Translation Initiation in Skeletal Muscle of Neonatal Pigs，'， Am.J.Physiol.Endocrinol.Metab.291：E849-59(2006)；Escobar et al.?uAmino Acid Availability and Age Affect the Leucine Stimulation of Protein Synthesis and eIF4F Formation in Muscle/iAm-J-Physiol.Endocrinol.Metab.293：E1615-21(2007))〇 此外，在斷奶仔豬的飼料中經(jīng)常只補充外源性蛋白酶，而不補充NSP酶（Guggenbuhl et al·，"Effects ofDietary Supplementation with a Protease on the Apparent Ileal Digestibility of the Weaned Piglet/iJ.Anim.Sci.90(Suppl 4)：152-154(2012))〇
[0007] 在早前的為期8周的研究中（Leng et al ·，"Defatted Algae Biomass may Replace One-Third of Soybean Meal in Diets for Laying Hens，'，J · Anim. Sci · 90 (Suppl. 3): 701 (2012))，發(fā)現(xiàn)蛋雞耐受7.5%而非15%的脫脂硅藻（十字脆桿藻亞屬 Staurosira sp)微藻。已發(fā)現(xiàn)：同更低的加入率以及對照組相比，在15 %的加入率時鳥中主 要的氮排泄產(chǎn)物血漿尿酸更低。此外，15%的藻類加入率恢復(fù)了在7.5%藻類加入率時降低 的白蛋白重量。
[0008] 蛋白質(zhì)的質(zhì)量和組成會影響一些轉(zhuǎn)運系統(tǒng)，包括陽離子氨基酸轉(zhuǎn)運體（"CAT"）家 族、寡肽轉(zhuǎn)運體1( "PEPT"）和NA2+非依賴性支鏈和氨基酸轉(zhuǎn)運體（"LAT"）以及腸酶例如氨肽 酉每（"APN"）（Gilbert et al·，"Developmental Regulation of Nutrient Transporter and Enzyme mRNA Abundance in the Small Intestine of Broilers，'，Poult·Sci·86: 1739-1753(2007);Gilbert et al·，"Dietary Protein Quality and Feed Restriction Influence Abundance Nutrient Transporter mRNA in the Small Intestine of Broiler Chicks，"J.Nutr.l38:262_271(2008);Gilbert et al·，"Dietary Protein Composition Influences Abundance of Peptide and Amino Acid Transporter Messenger Ribonucleic Acid in the Small Intestine of 2Lines of Broiler Chicks，"Poult.Sci.89:1663-1676(2010);Speier et al·，"Gene Expression of Nutrient Transporters and Digestive Enzymes in the Yolk Sac Membrane and Small Intestine of the Developing Embryonic Chick,Poult·Sci·91:1941-1949 (2012)hS6核糖體蛋白（S6)及其上游調(diào)節(jié)物(包括P70S6激酶1(P70)和哺乳動物雷帕霉素 靶蛋白（mTOR))，連同真核起始因子4E(elF4E)的磷酸化，在引發(fā)蛋白質(zhì)合成中發(fā)揮重要作 用并且響應(yīng)飲食蛋白的變化。
[0009] 從本研究和其他類似的實驗中出現(xiàn)了許多問題(Blum et al .，"Food Value of Spiruline Algae for Growth of the Broiler-Type Chicken，'，Annales de la Nutrition et de I'Alimentation29:651-674( 1975);Lipstein et al.，"The Nutritional Value of Algae for Poultry. Dried Chlorella in Layer Diets，'' Br.Poultry Sci.21：23-27(1980)；Ginzberg et al·，"Chickens Fed with Biomass of the Red Microalga Porphyridium sp.Have Reduced Blood Cholesterol Level and Modified Fatty Acid Composition in Egg Yolk/'J.Appl.Phycol.12:325-330(2000)； Al-Harthi et al.，"The Effects of Preparing Methods and Enzyme Supplementation on the Utilization of Brown Marine Algae(Sarassum dentifebium)Meal in the Diet of Laying Hens，"ItalianJ.Anim.Sci·10:195-203(201I);Halle et al·，"Effect of Microalgae Chlorel la Vulgaris on Laying Hen Performance，''Archiva Zootechnica 12:5-13(2009);El_Deek et al·，"The Use of Brown Algae Meal in Finisher Broiler Diets/'Egypt.Poult.Sci.31：767-781(2011))〇 [0010]本發(fā)明的目的是克服本領(lǐng)域中基于藻類的動物飼料的相關(guān)缺陷。
[0011] 發(fā)明概述
[0012] 本發(fā)明一方面涉及動物飼料組合物，其包括總計為所述組合物48-70 % w/w的量的 一種或多種谷物;總計為所述組合物15-30 %w/w的量的非藻類蛋白源;總計為所述組合物 3-15%w/w的量的藻類;總計為所述組合物0.01-0.1 %w/w的量的外源性蛋白