CN104206706A - 乳酸杆菌改善玩赏动物食物中赖氨酸的吸收的用途 - Google Patents
乳酸杆菌改善玩赏动物食物中赖氨酸的吸收的用途 Download PDFInfo
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- CN104206706A CN104206706A CN201410340998.XA CN201410340998A CN104206706A CN 104206706 A CN104206706 A CN 104206706A CN 201410340998 A CN201410340998 A CN 201410340998A CN 104206706 A CN104206706 A CN 104206706A
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Classifications
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- A—HUMAN NECESSITIES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
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Abstract
本发明涉及非致病性、刺激赖氨酸吸收的乳酸杆菌菌株的选择和用途；优选路氏乳酸杆菌菌株，例如路氏乳酸杆菌菌株ATCCPTA-6127。本发明还包括使用这种菌株例如用于改善玩赏动物的皮毛和爪的质量的产品和方法。
Description
发明领域
本发明涉及非致病性、刺激赖氨酸吸收的乳酸杆菌菌株的选择和用途，和使用这种菌株用于改善玩赏动物的皮毛和爪的质量的产品和方法。
背景技术
1908年，俄国生物学家EIi Metchnikoff确信某些保加利亚和俄国公民长寿在于消费大量发酵乳产品(1)。这些食物中的关键生物随后被鉴定为嗜酸乳杆菌(lactobacillus acidophilus)-一种乳酸生产细菌(2)。该乳酸生产细菌由此因它们生产乳酸的能力而被命名。然而，生产乳酸仅仅是由这个细菌集合得到的诸多益处之一。
基于Metchnikoff和其他人的工作，科学家们发展了益生微生物的想法，直接将活的乳酸生产细菌和酵母饲喂动物以改善它们的健康和特性。所观察到的益处可能由以下原因引起的：1)竞争消化道中的附着部位，2)竞争必需的营养物质，3)产生抗微生物的物质，4)增加有益细菌的生长和5)刺激免疫***(3)。
一些致病菌通过破坏小肠内壁而降低动物吸收营养物质的能力(4)。研究表明乳酸生产细菌附着于小肠并产生防止致病生物结合肠壁的物质(5)。此外，有益细菌的附着可以增加小肠的吸收表面积并提高酶活性，使动物吸收更多营养物质(8，6)。
促进健康和致病的细菌的生长都需要某些营养物质。乳酸生产细菌可以利用维生素、氨基酸或也另外支持有害细菌的生长的其他营养物质(7)。
相当多的研究集中于直接饲喂微生物培养物产生抑制致病生物的物质的能力。乳酸、乙酸和甲酸降低肠内pH以产生不适合有害生物的环境(4)。乳酸生产细菌还分泌过氧化氢，产生不利于需氧微生物的条件(8)。
已经鉴定了两组抗微生物物质，低分子量的抗微生物物质，例如路氏乳酸杆菌产生的无蛋白菌质(reuterin)；和细菌素。细菌素是微生物产生的物质，抑制通常遗传上相关的细菌的生长(4)。细菌素是多肽且蛋白酶破坏他们的抑制特性，而无蛋白菌质是一种广谱抗微生物物质，不是多肽且它的抗微生物活性不受蛋白酶影响。
研究已证明了乳酸生产细菌抑制大肠杆菌(E.coli)、鼠伤寒沙门氏杆菌(Salmonella typhimurium)、金黄色葡萄球菌(Staphylococcusaureus)和产气荚膜梭菌(Clostridiumperfingens)的能力。减少引起腹泻的生物在新生和幼年动物中尤为重要。
然而对伴生动物中的益生菌研究很少。最近的一项研究(9)调查了嗜酸乳杆菌DSM 13241在犬中的应用。根据它的生长特征、对病原体的抗微生物活性和在肠模型中的存活率选择这个菌株。饲喂健康狗导致粪便中可回收乳酸杆菌数量显著增加，梭菌属数量伴随减少。研究人员做出结论饲喂益生菌剂引起肠微生物学和全身效应方面的积极改变，提示免疫***刺激，如在人类消耗乳酸杆菌种属后在他们中观察到的(10)。
像狗、猫、貂(Mustela vison)和蓝狐(Alopex lagopus)属于食肉目哺乳动物。食肉动物适合于相对浓缩和很易消化的饮食，其特点是胃和相对短且简单的肠道。貂缺少盲肠且消化道短，结肠中细菌活性非常有限。狗和狐具有一个小容量的盲肠和一个非囊状的结肠，但是一些细菌发酵在盲肠和结肠进行(11)。
蛋白对于身体组织的生长、修复和维持是至关重要的。它形成酶和抗体的基础。对于动物和鸟类，很多蛋白被用作制造皮毛、羽毛和爪的能量，主要是α-角蛋白。α-角蛋白由长的α螺旋多肽组成，这些多肽彼此旋绕形成三股螺旋。通常对于毛皮动物，含硫氨基酸通常被认为是最有限制作用的氨基酸(12)。制造毛皮、羽毛和爪的蛋白只能来自饮食。饮食必须供给充足的氨基酸以满足必需氨基酸的需要，足够过量的氨基酸供给制造非必需氨基酸的氮。饮食中额外的蛋白不能储存并将转变成脂肪或被肾脏***出去。
赖氨酸是必需氨基酸，因此不能通过转氨基作用合成。它有助于通过产生肉毒碱使脂肪变得对身体有用，从身体去除毒素，制造皮毛、羽毛和骨头。高水平增加对精氨酸的需要。赖氨酸在完整蛋白中高度不稳定。它与葡萄糖反应，并且在温暖和潮湿环境下反应加快。对幼年猪和家禽的研究发现赖氨酸是对生长最有限制作用的氨基酸(例如Boisen et al.，2000；(13，14)。
甲硫氨酸和半胱氨酸是营养氨基酸，并且是基本氨基酸中最具活性的氨基酸。它们用于制造这种重要的分子，如肉毒碱(用于运输脂肪酸)、肌酸、烟酸、聚胺和嘌呤(用于产生尿酸)。它们还用于制造皮毛和羽毛。半胱氨酸可以从甲硫氨酸合成，因此它被分类为非必需氨基酸。然而，半胱氨酸及其氧化产物胱氨酸可以满足总含硫氨基酸的大约百分之五十的需要，这样可以降低对甲硫氨酸的需要。甲硫氨酸不能从半胱氨酸合成，因此它是必需氨基酸。在缺乏胱氨酸的情况下，甲硫氨酸可以满足对含硫氨基酸的全部需要。一些研究已经发现甲硫氨酸是对貂的毛发生长和皮毛质量最具限制作用的氨基酸(15，16，17，18，19，20，21)。
在大多数家庭宠物中，健康的皮和毛通常表明动物身体健康。因为皮和毛问题在家庭宠物中是普遍的，已经进行很多研究提供修复皮和毛状况变质的饮食，由此提供基本水平的健康皮和毛，如下文所述。
研究表明补充锌及亚油酸的全面且均衡的商业狗粮可以使狗的皮和毛状况显著和实质性提高。与接受标准饮食的对照组相比时，狗在皮毛光泽度和皮毛大小方面表现出显著的改善。专利EP0987961B1中也描述了这个方法。
营养可能影响皮肤屏障功能。国际专利申请WO0207531A1公开了含抗微生物的脂肪酸的饮食脂类在制备用于改善或维持皮肤健康和/或皮毛质量的食品组合物中的用途。
在Kerminen-Hakkio的研究(21)中，貂中蛋白质量粗劣使毛皮长度平均降低4cm。在优质、高水平蛋白组观察到最好的总体皮毛质量。作者做出结论增加饮食中蛋白水平不能完全弥补必需氨基酸供给的缺乏。
有些情况下，仍应添加非必需氨基酸以确保最佳的可利用量。我们发现可利用的甲硫氨酸应该与猫和狗的食物中也存在的赖氨酸保持平衡，以改善皮毛和爪的生长和质量。一个更好地利用来自食物的赖氨酸的值得称赞的方法不是仅仅饲喂更多，而是寻找改善它吸收的方法。
众所周知含乳酸杆菌(LAB)的饲料能促进生长，这是因为其通常能改善营养物质的吸收。在今天的现代动物育种中，很多国家已经使用含益生微生物的饲料添加剂以催肥动物。动物消耗LAB时，它们移植于消化道并开始产生消化淀粉(淀粉酶)、蛋白(蛋白酶)、脂肪(脂肪酶)的酶。这些酶有助于将饲料中复合有机化合物分解为低分子化合物。这些动物可以更好利用这些低分子化合物。它们的食欲改善，饲料转化率更好和生产性能改善。LAB在动物饲养中的应用还有助于免疫防御病原体。LAB将碳水化合物分解为乳酸，乳酸降低消化道内容物的pH，形成极其不利于病原体微生物的环境。
我们惊讶地发现食物或胃肠道中存在的不同LAB菌株可以不同地辅助氨基酸的吸收，尤其是赖氨酸。而且这种差异可以测定，从而使得选择会最大量改善赖氨酸吸收的特定乳酸杆菌菌株是可能的。
已经证明Caco-2细胞***是对营养物质[肽和氨基酸(23，24，25，26)]和药物产品(27，24)的肠上皮渗透性研究都合适的模型***。Twaites 1996年进行的研究描述了肠内Caco-2细胞运输二碱氨基酸赖氨酸的能力(28)。
然而对乳酸杆菌促进或增加玩赏动物中来自饮食的氨基酸吸收很少研究。
使用限定的四种不同细菌(荚膜甲基球菌(Methylococcuscapsulatus)、食酸产碱菌(Alcaligenes acidovorans)、短芽孢杆菌(Bacillus brevis)和坚强芽孢杆菌(Bacillus firmus))的混合物，和天然气作为碳源和能源，经由连续细菌发酵产生的细菌蛋白膳食(BPM)是一种新的高蛋白饲料成分。使用蓝狐作为模型动物进行研究，扩展对BPM作为狗粮成分的认识。动物通常显示出很好的生长，但是没有数据显示皮毛质量改善(7)。
专利WO 01/17365A1描述了改善和保养宠物皮毛***的方法，其中营养剂可以是前生物或任何益生微生物。该申请人提及益生菌类的十七种不同细菌，和将近三十种益生微生物的具体实例。凝结芽孢杆菌(Bacillus coagulans)Lacris-S菌株(SANKYO LIFETECH CO.，LTD，Hongo，Bunkyo-ku，Tokyo，Japan)是唯一描述的特定菌株，没有提及优选菌株的选择，也没有任何基于LAB菌株刺激赖氨酸吸收的能力。
尽管为了动物皮毛和羽毛更好而使用益生菌是本领域已知的，但是以前没有认识到不同的益生菌株改善动物，例如狗或猫的皮毛和爪的质量的能力差异，和这种能力与益生菌株对动物肠道吸收赖氨酸的影响有关。
因此本发明的一个目的是通过分析它们直接促进或改善动物吸收赖氨酸的能力来选择这种更合适的菌株，和将该选择出的菌株用于动物饲料或添加剂中，例如以改善狗或猫的皮毛和爪质量的应用。路氏乳酸杆菌的一个新菌株ATCC PTA-6127显示出刺激皮毛动物中的赖氨酸吸收和这个特性引起皮毛和爪质量改善。
由下列公开内容和所附权利要求，其他目的和优点将更加明显。
发明内容
本发明涉及非致病性、刺激赖氨酸吸收的乳酸杆菌菌株的选择和用途，和使用这种菌株用于治疗和预防玩赏动物的皮毛和爪的质量的产品和方法。
附图说明
图1条线图显示了7.0×107cfu/ml不同的LAB菌株对Caco-2肠细胞吸收赖氨酸的影响。所用菌株是路氏乳酸杆菌ATCCPTA-6127、路氏乳酸杆菌1068、路氏乳酸杆菌SD2112和凝结芽孢杆菌Lacris-S菌株。
具体实施方式
本发明涉及非致病性、刺激赖氨酸吸收的乳酸杆菌菌株的选择和用途，和使用这种菌株用于治疗和预防玩赏动物的皮毛和爪的质量的产品和方法。
本发明的一个目的是通过分析它们直接促进或改善动物吸收赖氨酸的能力来选择最好的合适菌株，和将该选择出的菌株用于动物饲料或添加剂中，例如以改善狗或猫的皮毛和爪质量的应用。路氏乳酸杆菌的一个新菌株(ATCC PTA-6127)显示出刺激皮毛动物中的赖氨酸吸收和这个特性引起皮毛和爪质量改善。这个菌株根据布达佩斯条约于2004年7月22日保藏在美国典型培养物保藏中心(Manassas，VA)。
本发明的产品可以是饲料或片剂或胶囊或其他制剂等剂型，和制备本领域已知的下述产品的标准方法，包括所选择的LAB培养物。
尽管这里陈述了某些代表性的实施方案，但是本领域技术人员会很容易地认识到可以进行改动而不背离本发明的精神或范围。
实施例1.细胞培养物的制备
Caco-2细胞(ATCC号：HTB-37，Manassas，VA，USA)如(Thwaiteset al.，1993a；1993b)所述进行培养。细胞单层制备如下：高密度(4.4-5.0°-105细胞/cm2)接种在12或24.5mm直径组织培养插片上[Transwell聚碳酸酯滤膜(Costar)]。细胞单层维持在37℃，含5％CO2的湿润空气下。显微术估测细胞融合并测定跨上皮电阻。
接种后18-25天和饲喂后18-24小时进行放射性同位素标记的赖氨酸流通量测定。
实施例2.选择刺激肠吸收赖氨酸的路氏乳酸杆菌菌株的方法
Caco-2细胞单层用500ml改良的Krebs缓冲液(其组分(全部为mmol/l)，NaCl 140，KCl 5.4，CaCl22.8，MgSO41.2，NaH2PO40.3，KH2PO40.3，HEPES 10，葡萄糖10(用Tris碱调pH至7.4，37℃))洗涤4次，或适当时，用不含Na+的Krebs缓冲液(如上，除了用胆碱Cl替换NaCl和NaH2PO4，并放置于含2ml预热(37℃)的不含Na+的Krebs缓冲液的6孔板(Life Technologies)。等分的新鲜Krebs缓冲液或不含Na+的Krebs缓冲液置于容器中。使用追踪浓度(0.2microCi/ml)的放射性同位素标记的赖氨酸((3H)Amersham)。然后上皮层与各种待测试的LAB孵育；路氏乳酸杆菌ATCC PTA-6127、路氏乳酸杆菌1068、路氏乳酸杆菌SD2112和凝结芽孢杆菌Lacris-S菌株以7.0x107cfu/ml的浓度在37℃孵育6小时。每60分钟从基础溶液中取200微升样品测定跨上皮运输。用闪烁计数法测定放射性同位素标记。结果参见图1。
实施例3.含所选择菌株的饲料产品的制备
在这个实施例中，为了将菌株添加到商品中，通常基于良好的生长特性和实施例2中在先提及的选择中的有利结果来选择路氏乳酸杆菌"Shiny"(ATCC PTA-6127)。使用工业上培养乳杆菌的标准方法来培养路氏乳酸杆菌Shiny菌株并将其冻干。
饲料混合物由玉米、玉米麸质、鸡和鱼粉、盐、维生素和矿物质组成。将饲料混合物装入预调节器并润湿。离开预调节器的润湿饲料接着被装入挤压机-蒸煮器并胶质化。离开挤压机的胶质化基质通过模具冲压并压出。离开模具压头的压出物切成适于饲喂给狗的块，在大约140℃干燥大约20分钟并冷却形成球丸。球丸的水活度大约为0.6。
球丸用包含动物脂肪的包被基质喷雾。在动物脂肪凝结之前，通过干燥喷雾以107 CFU/克产品的水平应用益生菌路氏乳酸杆菌“Shiny”(ATCC PTA-6127)，以便其附着于或部分渗入脂肪层中。37℃保存8周的结果表明微生物显示出极好的稳定性且在正常条件下保存一年后可能仍是稳定的。
实施例4.狗试验
使用45只比格犬进行试验。试验开始之前，给狗饲喂与实施例3中饮食相当的标准干燥饮食一周。在试验开始之前，立即通过实施例5描述的评价小组评价参与的狗的皮毛状况。将狗分为三组，每组15只狗。一组狗饲喂覆盖有路氏乳酸杆菌“Shiny”(ATCC PTA-6127)的干燥球丸，另一组狗饲喂覆盖有路氏乳酸杆菌菌株1068的干燥球丸和第三组继续饲喂标准饮食，由此提供对照饮食。所有的组自由摄取食物和水。12周后，再次评价每只狗的皮毛状况。饲喂覆盖有路氏乳酸杆菌“Shiny”的球丸的狗比饲喂覆盖有路氏乳酸杆菌1068菌株的球丸的狗和对照组的狗具有明显更光亮的外观并且没有显示出明显的皮屑。
实施例5.爪和皮毛的生长特性
技术熟练的趾甲美容师对实施例4中的狗爪进行打分，两位有经验的专门研究比格犬品种的狗形象裁判对皮毛进行打分。试验之前，根据皮毛和爪的质量将狗平均分布在各组之中。12周后，评价趾甲被切割生长时对抗裂开的质量并以五个水平的等级进行打分，1是低劣和5是极好。评价皮毛的总体质量、皮屑、皮毛长度和光泽度，等级范围从1(最差)至5(最好)。
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Claims (9)
1.一种路氏乳杆菌菌株在改善伴生动物的皮毛和爪质量的应用，所述应用包括选择非致病性、刺激赖氨酸吸收的路氏乳杆菌菌株，和给动物提供含有所选择菌株的生物学上纯培养物的产品。
2.根据权利要求1所述的应用，其中，所述路氏乳杆菌菌株为路氏乳酸杆菌1068、路氏乳酸杆菌SD2112。
3.一种用于改善玩赏动物的皮毛和爪质量的产品，所述产品包括：
a)一种根据权利要求1或2所选择的路氏乳酸杆菌菌株的生物学上纯培养物；和b)一种饲料混合物，其中所述饲料混合物覆盖有包被基质，随后被所选择菌株的生物学上纯培养物的喷雾干燥覆盖。
4.根据权利要求3所述的产品，其中，所述饲料混合物被制成球丸。
5.根据权利要求3所述的产品，其中，所述路氏乳杆菌菌株为路氏乳酸杆菌1068、路氏乳酸杆菌SD2112。
6.根据权利要求3所述的产品，其中，所述饲料混合物包含玉米、玉米麸质、鸡和鱼粉、盐、维生素和矿物质。
7.根据权利要求3所述的产品，其中，所述包被基质包含动物脂肪。
8.根据权利要求7所述的产品，其中，在动物脂肪凝结之前，通过喷雾干燥以107CFU/克产品的水平应用生物学上纯培养物，以便其附着于或部分渗入脂肪层中。
9.根据权利要求3所述的产品，其中，所述产品的剂型选自由球丸、片剂和胶囊组成的组。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108366582A (zh) * | 2015-11-09 | 2018-08-03 | 杜邦营养生物科学有限公司 | 饲料添加剂组合物 |
| CN114437993A (zh) * | 2022-03-10 | 2022-05-06 | 中国农业科学院农产品加工研究所 | 罗伊氏乳杆菌及其在制备预防猫腹泻制剂中的应用 |
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| US9210945B2 (en) * | 2009-07-31 | 2015-12-15 | The Iams Company | Animal food having low water activity |
| US10104903B2 (en) | 2009-07-31 | 2018-10-23 | Mars, Incorporated | Animal food and its appearance |
| US9173423B2 (en) * | 2009-07-31 | 2015-11-03 | The Iams Company | Animal food kibble with electrostatically adhered dusting |
| CN102711512A (zh) * | 2010-01-22 | 2012-10-03 | 爱默思公司 | 用于制备涂覆的粗磨食物形式的宠物食品的方法 |
| CN102367433B (zh) * | 2011-11-08 | 2013-05-01 | 四川农业大学 | 一种无胃温水性鱼肠细胞悬浮培养方法 |
| KR20150083225A (ko) * | 2014-01-09 | 2015-07-17 | 씨제이제일제당 (주) | 영양성분이 강화된 축우용 배합사료 및 이의 제조방법 |
| EP3258792A1 (en) | 2015-02-16 | 2017-12-27 | Mars, Incorporated | Interlocking kibble |
| CA2982836A1 (en) | 2015-04-28 | 2016-11-03 | Mars, Incorporated | Process of preparing a sterilized wet pet food product |
| JP2020502039A (ja) * | 2016-12-15 | 2020-01-23 | ソシエテ・デ・プロデュイ・ネスレ・エス・アー | 小型イヌ科動物用の組成物及び方法 |
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| JP2010519900A (ja) | 2010-06-10 |
| CA2679095A1 (en) | 2008-09-12 |
| US20110086126A1 (en) | 2011-04-14 |
| US8496924B2 (en) | 2013-07-30 |
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| AU2008221660B2 (en) | 2010-11-18 |
| EP2117341A4 (en) | 2011-05-11 |
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