CN107950754A - 宠物饲料添加剂及其制备方法 - Google Patents
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Abstract
本发明涉及宠物饲料添加剂及其制备方法，所述制备方法包括：在活性乳酸菌冷冻干燥粉中混合γ‑氨基丁酸和L‑谷氨酸的步骤；在上述步骤中获得的混合物中进一步添加低聚果糖，以提高活性乳酸菌的恢复率的步骤。该饲料添加剂是在活性乳酸菌中以相同量的重量比(w/w)来混合并添加γ‑氨基丁酸和氨基酸而制得，具有能够提供一种改善肠部及脑部健康的功能性饲料添加剂的显著效果。
Description
技术领域
本发明涉及一种改善肠部及脑部健康的功能性的宠物饲料添加剂及其制备方法。
背景技术
乳酸菌(lactic acid bacteria)作为被广泛用作益生菌(probiotic)的代表性细菌，是一种在人或动物的肠道和发酵食品等中容易发现的安全微生物。乳酸菌附着并寄生在肠道内上皮细胞上，改善肠内菌群的性质，使肠内菌群稳定，并通过抑制有害细菌的停留以减少腐败产物的生成，而且还具有预防疾病、激活免疫、抗癌、降低胆固醇等作用，从而大大有助于宿主动物。乳酸菌对各种腐败性微生物及病原性微生物具有生长抑制作用，其源于乳酸菌的几种代谢特性，也就是乳酸菌能够代谢出的代谢产物如有机酸(organicacid)、过氧化氢(hydrogen peroxide)、罗氏菌素(reuterin)、双乙酰(diacetyl)、乙醛(acetaldehyde)及细菌素(bacteriocin)等抗菌活性因子。
被简称为GABA的γ-氨基丁酸(γ-aminobutyric acid)作为一种非蛋白质氨基酸，大量存在于人的脑部、蔬菜、水果、大米或薏米等谷物中。在包括人类在内的高等动物、无脊椎动物及昆虫等中，γ-氨基丁酸起到神经递质的作用，而且还已知其为在植物体内起到防御机制的物质。此外，随着GABA在抑制人体的精神压力、强化注意力、增强血压方面的效果被人们所知后，市面上出现了包括健康功能食品在内的各种食品和动植物用添加剂等，其应用范围也正在逐渐扩大。
此外，已知GABA对动物具有以下效果。其能够通过缓解压力来抑制疾病的产生，通过促进生长激素的分泌来提高生产效率，还提高牛肉等畜产物的质量。
宠物作为陪伴人类生活并在心理上给予人类稳定感和亲密感的存在，据2015年统计资料显示，饲养宠物的家庭的比例占21.8％，共计约有457万户的家庭在饲养宠物，而且呈现逐年增长的趋势。饲养最为普遍的宠物为狗和猫分别占19.1％和5.2％，并且有关宠物的市场规模也在持续增长。因此，未来不仅是一般饲料的市场，健康功能性饲料组合物的市场也将急剧扩大。
作为必需氨基酸(essential amino acid)，蛋白质在体内分解成氨基酸之后被吸收利用。因此，蛋白质的营养价值由其中所含的氨基酸的种类和含量所决定，氨基酸分为在动物体内由其它氨基酸制得的氨基酸和需要从食物中摄取而非在体内合成的氨基酸。
所谓必需氨基酸是指，不在体内进行合成或者即使合成其含量也非常少而不足以维持生理机能的、必须通过食物进行供给的氨基酸。
作为与本发明相关的现有文献，已知有韩国授权专利第10-1187512号，其涉及一种含有γ-氨基丁酸(GABA)的饲料添加剂，具体涉及一种通过与谷物粉末或黄土粉末赋形剂进行酶反应，从而使L-谷氨酸转化成γ-氨基丁酸的饲料添加剂的制备方法。韩国公开专利第10-2011-0009638号公开了一种含有乳杆菌属乳酸菌复合菌株作为有效成分的饲料添加剂，日本公开专利第10-2012-0036787号公开了一种含有L-谷氨酸、L-谷氨酸钠及/或L-色氨酸的非反刍动物饲料组合物。但是，到目前为止还没有公开过一种在活性乳酸菌中以同量比(相同量的重量比)(w/w/w)混合GABA和必需氨基酸来制备的改善肠部及脑部功能的功能性宠物饲料添加剂。
发明内容
因此，本发明的目的在于，提供一种改善肠道及脑部健康的功能性宠物饲料添加剂，并对该宠物添加剂进行评价。
本发明的以上目的是通过下述方式实现的。包括：在乳酸菌活菌冷冻干燥粉中混合GABA和必需氨基酸的步骤；在上述步骤中获得的混合物中添加低聚果糖(fructooligosaccharide)，以提高活性乳酸菌的恢复率(recovery rate)，从而制得改善肠道及脑部健康的功能性动物饲料添加剂的步骤，然后通过将上述饲料添加剂用作测试材料来实施动物的免疫、压力及胆固醇含量实验从而进行评价。
本发明的另一目的在于，提供一种饲料添加剂的制备方法，所述制备方法还包括以同量比(w/w)来混合pH敏感型(sensitive)羟丙基甲基纤维素邻苯二甲酸酯(hydroxypropyl methyl cellulose phthalate，HPMCP)的步骤。
本发明的宠物饲料添加剂对改善动物的肠道及脑部功能健康具有显著的效果。
附图说明
图1为示出根据本发明制备的饲料添加剂粉末剂型的图。
图2为示出根据本发明制备的饲料添加剂的最终制品的图。
图3为示出施用根据本发明制备的饲料添加剂的先天性免疫细胞活性的图。
具体实施方式
本发明提供一种作为狗和猫等宠物饲料添加剂的、以相同量的重量比(w/w/w)来含有活性乳酸菌(probiotic)、GABA及必需氨基酸作为有效成分的改善宠物的肠部及脑部健康的功能性饲料添加剂组合物。
根据本发明，经过多次实验，结果，优选活性乳酸菌剂、GABA和必需氨基酸的混合比为相同量的重量比(w/w)。
此外，根据本发明，所述活性乳酸菌优选为选自片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)、双歧杆菌属(Bifidobacterium)、明串珠菌属(Leuconostoc)及链球菌属(Streptococcus)中的任一种以上的菌株，最优选的是选自耐酸性菌株的片球菌属的乳酸菌。
根据本发明制备的饲料添加剂，以组合物比例计，每日剂量最优选为总饲料剂量的2重量％，如果小于该用量时，效果会显著降低而不优选。
此外，根据本发明制备的饲料添加剂可以采用本领域通常的方法来制备成任何剂型，例如，可以制备成粉末、颗粒或片剂等多种剂型，并可装入玻璃瓶容器或塑料材质等多种容器中后产品化(参见图1至图2)。但是，从乳酸菌的肠内存活率和稳定性方面考虑，最优选的剂型为片剂。
将通过以下具体实施例、实验例及剂型例来更加详细地说明本发明，但是，下述实施例、实验例及剂型例只是为了例示本发明，并非意在将本发明的权利范围限定于此。
根据本发明，优选以普通饲料1日摄取量的2重量％的量来施用粉末或颗粒剂添加剂。但是，为了提高活菌剂的肠内存活率以及增加稳定性，在制备所述粉末剂或颗粒剂添加剂时，优选以活菌剂的约10倍的添加量来添加耐酸性载体(pH敏感型载体(sensitivecarrier))来进行制备。即相当于以与所述饲料添加剂相同的重量比(w/w)来混合耐酸性载体，从而制备片剂。
这种片剂(tablet)剂型在宠物的肠道内提高乳酸菌的存活效率，在热带地区能够使储存稳定性得以增加。载体有HPMCP(羟丙甲纤维素邻苯二甲酸酯)和HPMCAS(醋酸羟丙基甲基纤维素琥珀酸酯)、HPMCMC(羟丙基甲基羧甲基纤维素)等，但最优选为pH敏感型HPMCP。
下面，对本发明饲料添加剂成分的优选混合比及制备例进行说明。
实施例1本发明饲料添加剂的制备
作为宠物的肠部及脑部健康功能性饲料添加剂组合物的最优选混合比，采用下述表1所示的重量单位来进行混合配制。如下所述，本发明的实验采用了粉末剂型，在各个实验结果中，在功效方面与颗粒剂型没有很大的差异，而片剂(tablet)与粉末或颗粒剂相比，功效增强了约10～20％，所有的实验采用粉末剂型实施。此外，去除了低聚果糖的对照组1和去除了必需氨基酸的对照组2以及去除了GABA的对照组3各自的实验结果表明，饲料添加剂的适合性(suitability)降低(表10)。
表1 本发明饲料添加剂的混合比
| 成分 | 混合比(％) |
| 混合乳酸菌(益生菌(probiotics)，100亿CFU/g) | 5～15 |
| 低聚果糖(益生元(prebiotics)) | 3～7 |
| GABA(γ-氨基丁酸) | 10～20 |
| 混合必需氨基酸-PET | 10～20 |
| 乳酸菌发酵提取粉末 | 10～20 |
| 酵母提取粉末 | 5～15 |
| 无水葡萄糖 | 3～7 |
| 糊精 | 10～20 |
| 其它成分(复合维生素、二氧化硅、硬脂酸镁等) | 余量 |
| 合计 | 100.0 |
实验例1血液内免疫指标IgG(免疫球蛋白G)分析
为了确认本发明的所述实施例1的饲料添加剂对小鼠血液内免疫指标的影响，在普通饲料中添加普通饲料总重量的2重量％的上述实施例1中获得的本发明的饲料添加剂来实施动物实验。为了测定血液内的免疫指标，在试验开始前和试验结束时(5周后)，以每个处理组为4只，从同一个体的尾静脉中取血后，在4℃下以2,000xg离心分离30分钟后，将获得的血清用于血液内免疫指标(白细胞(WBC)，红细胞(RBC)，淋巴细胞(Lymphocyte)，IgG，血清总蛋白(total protein)，白蛋白(albumin))分析。
实验结果如下述表2所示，对各实验动物的血液内的IgG测定的结果，相比于对照组而言，实验组中的血液内IgG的量显著增加。
表2
| IgG(mg/mL) | 对照组 | 实验组 |
| 0天 | 331.5 | 340.3 |
| 14天 | 320.8 | 402.5 |
| 35天 | 342.5 | 501.1 |
| 差异 | 11 | 160.8 |
实验例2根据本发明的先天性免疫细胞活性
为了确认本发明是否具有先天性免疫细胞内的活性增强效果，对免疫细胞活性的指标的变化进行了调查。COX-2(环氧化酶2：Cyclooxygenase-2)和IFNβ(干扰素β)为一种在先天性免疫细胞的巨噬细胞中，当TLR4(Toll样受体4)被细菌或病毒活化时会增加的具代表性的免疫蛋白质，NF-kB和IRF3是被TLR4活化的具有代表性的转录因子(transcriptionfactor)。
为此，将小鼠巨噬细胞(macrophage)以1×105的浓度进行培养，注入分别具有COX-2-启动子、IFNβ-启动子、NF-kB结合位点(binding site)及IRF3结合位点的荧光素酶报告质粒(luciferase reporter plasmid)后，使用100μg/mL及500μg/mL的浓度的上述实施例1获得的饲料添加剂处理8小时。获取细胞提取物后，使用发光酶基因分析试剂盒(luciferase reporter assay system TM，普洛麦格有限公司(Promega Co.)，麦迪逊(Madison)，威斯康辛州(WI))分别测定了COX-2和IFNβ在基因水平上的表达和NF-kB、IRF3的活化。
实验结果如图3所示，可知本发明的饲料添加剂分别在100μg/mL及500μg/mL的浓度下，COX-2、IFNβ的表达及NF-kB、IRF3的活化得到显著性增加，通过该结果能够确认，本发明的饲料添加剂能够增强与先天性免疫相关的转录因子的活性以及增强免疫蛋白质的表达，从而能够增强先天性免疫细胞的活化。
实验例3大肠杆菌感染诱导动物模型的免疫增强效果
为了查明上述实施例1中获得的本发明的饲料添加剂的免疫增强效果，将大肠杆菌向小鼠腹腔内进行给药，通过对大肠杆菌细菌感染所引起的死亡的预防效果，来确认了对病原性细菌的防御效果。
为此，用大肠杆菌(E.coli)对30只6周龄的雄性ICR小鼠进行处理。将实验分别分成无处理对照组、接种大肠杆菌阳性对照组、给予饲料添加剂后接种大肠杆菌的实验组，共3组，每组使用5只小鼠。在饲料中添加饲料总重量的2重量％的饲料添加剂来进行实验。将大肠杆菌以培养液0.5mL的量向腹腔内进行注射接种。记录接种后4天期间的死亡数，从而对饲料添加剂的病原性细菌防御率进行调查。
实验结果如下述表3所示，接种大肠杆菌阳性对照组的动物在接种大肠杆菌后，第一天10只小鼠全部死亡，显示出了100％的死亡率，而给予饲料添加剂的实验组在注射大肠杆菌后的第一天死亡5只，第二天死亡1只，第三天死亡1只，在试验结束时还存活有3只小鼠，该结果表明，在病原性大肠杆菌接种防御效果实验中，显示出了30％的存活率，从而确认了具有细菌防御效果。
表3
实验例4明暗移动实验(明暗穿箱实验)
明暗移动实验是以小鼠先天不喜欢亮光且在新的环境下自发地进行探索活动为前提而实施的实验。即，具有因压力引起的不安症状的小鼠，不会活跃地进行自发的探索活动，从而从暗室向明室移动的次数会变少。通过测定小鼠从暗室向明室移动的次数，来确认抗压力效果。
为此，将雄性ICR小鼠用于实验。在亚克力笼(acrylic cage)(45×60×25cm)中饲养，并恒定维持20～24℃的温度，使其适应一周后用于实验。将实验动物分成只给予饲料的阴性对照组，在普通饲料中以普通饲料总重量的2重量％来添加本发明饲料添加剂来给予的实验组，以及给药安定(diazepam)的阳性对照组。
实验结果如下述表4所示，可以确认与阴性对照组相比，给予了本发明饲料添加剂的实验组中，从暗室向明室移动的次数增加。
表4
| 实验动物个数 | 从暗室向明室移动的次数 | |
| 阴性对照组 | 5 | 5.0±4.38 |
| 阳性对照组 | 5 | 12.6±4.89 |
| 实验组 | 5 | 11.5±4.81 |
实验例5激素浓度变化
实验动物及实验组的组成按照与实验例4相同的方式进行处理。使实验动物自由取食4周。4周后，对各个小鼠以5秒为间隔且以每秒施加0.6mA的方式共实施30分钟的电击(electric foot shock)，以诱导压力。对被诱导了压力的小鼠分别进行尸体剖检，从而取出血液及脑组织(大脑皮质、海马体及纹状体)，然后利用酶联免疫吸附试验(ELISA，enzyme-linked immunosorbent assay)对取出的血液及脑组织所分泌的多巴胺、皮质脂酮及去甲肾上腺素进行分析。
实验结果如下述表5所示，可以确认与阴性对照组相比，将本发明饲料添加剂以普通饲料总重量的2重量％来给予的实验组中，多巴胺、去甲肾上腺素及皮质脂酮的分泌量减少。
表5
| 多巴胺浓度(％) | 去甲肾上腺素浓度(％) | 皮质脂酮浓度(％) | |
| 阴性对照组 | 100 | 100 | 100 |
| 阳性对照组 | 43.2 | 37.5 | 16.1 |
| 实验组 | 62.5 | 60.0 | 33.2 |
实验例6血液中胆固醇
为了确认上述实施例1的本发明饲料添加剂的给药对血液中胆固醇所产生的效果，作为实验动物，使用了自发性高血压大鼠(SHR)和作为对照组的正常血压的大鼠(WKY)。实验分成对WKY大鼠给药普通饲料的阴性对照组、对WKY大鼠给药普通饲料中添加本发明饲料添加剂的实验组1、对SHR大鼠给药普通饲料的阳性对照组，以及对SHR给药普通饲料和本发明饲料添加剂的实验组2，在普通饲料中添加并进行给药4周后，测量了血压。本发明的饲料添加剂的加入量为，普通饲料总重量的2重量％。
实验结果如下述表6所示，在WKY大鼠中没有效果，而可以确认在高血压的大鼠SHR大鼠中血压显著性降低。
表6
| 血压(mmHg) | |
| WKY(普通饲料) | 128.38 |
| WKY(普通饲料+本发明饲料添加剂) | 125.19 |
| SHR(普通饲料) | 202.67 |
| SHR(普通饲料+本发明饲料添加剂) | 159.36 |
制备例1容器制品
容器制品按照下述表7所示配制后被制备成粉末或颗粒剂型，然后如图所示被装入容器中产品化。
表7
制备例2条棒(stick)制品
将1日所需的饲料添加剂按照下述表8那样进行配制，从而制备成粉末或颗粒剂型后，包装成小包装的条棒制品，从而使携带便利。
表8
| 原料名 | 加入量(g) |
| 混合乳酸菌(益生菌，1,000亿CFU/g) | 0.2 |
| 低聚果糖或低聚半乳糖(益生元) | 0.1 |
| GABA(γ-氨基丁酸) | 0.3 |
| 混合必需氨基酸-PET | 0.3 |
| 乳酸菌发酵提取粉末 | 0.3 |
| 酵母提取粉末 | 0.2 |
| 无水葡萄糖 | 0.1 |
| 糊精 | 0.3 |
| 其它成分(复合维生素、二氧化硅、硬脂酸镁等) | 余量 |
| 合计 | 2 |
制备例3药片(tablet)制品
将1日所需的饲料添加剂按照下述表9那样进行配制，从而制备成药片制品后，装入图2所示那样的容器中产品化。
表9
| 原料名 | 加入量(g) |
| 混合乳酸菌(益生菌，1,000亿CFU/g) | 3.00 |
| 低聚果糖或低聚半乳糖(益生元) | 1.50 |
| GABA(γ-氨基丁酸) | 4.50 |
| 混合必需氨基酸-PET | 4.50 |
| 乳酸菌发酵提取粉末 | 4.50 |
| 酵母提取粉末 | 3.00 |
| 无水葡萄糖 | 1.50 |
| 糊精 | 3.00 |
| 其它成分(复合维生素、二氧化硅、硬脂酸镁等) | 余量 |
| 合计 | 30 |
| pH敏感型羟丙基甲基纤维素邻苯二甲酸酯55(HPMCP55) | 30 |
| 总计 | 60 |
比较例1本发明饲料添加剂的效果比较
此外，为了实施按照上述实施例1的方法制备的本发明的饲料添加剂(实验组)和去除益生元(prebiotics)而制备的饲料添加剂(对照组1)，去除必需氨基酸而制备的饲料添加剂(对照组2)及去除GABA而制备的饲料添加剂(对照组3)的比较实验，以与实验例1至实验例5相同的方法，在普通饲料中添加总重量的2重量％来进行给药，从而实施了比较实验。
实验结果如下述表10所示，可以确认本发明的饲料添加剂(实验组)相比于对照组1至3具有优异的改善肠部及脑部功能的效果。
表10
工业实用性
如上所述，如实施例1那样以一定比例添加了活性乳酸菌、GABA及必需氨基酸的本发明的饲料添加剂具有改善肠部及脑部健康的功能性显著效果，因此是在宠物健康功能性饲料产业上非常有用的发明。
Claims (6)
1.一种宠物饲料添加剂的制备方法，其特征在于，包括：
在活性乳酸菌冷冻干燥粉中混合γ-氨基丁酸和L-谷氨酸的步骤；在上述步骤中获得的混合物中进一步添加低聚果糖，以提高活性乳酸菌的恢复率的步骤。
2.根据权利要求1所述的宠物饲料添加剂的制备方法，其特征在于，
所述活性乳酸菌为选自片球菌属、乳杆菌属、肠球菌属、双歧杆菌属、明串珠菌属及链球菌属中的一种以上的菌株。
3.根据权利要求1所述的宠物饲料添加剂的制备方法，其特征在于，
所述γ-氨基丁酸通过乳酸发酵、化学合成及从含有γ-氨基丁酸的植物体中提取的方法中的一种方法来获得。
4.一种宠物饲料添加剂，其特征在于，通过权利要求1-3中任一项所述的制备方法来制得，并且是改善肠部及脑部健康的功能性的宠物饲料添加剂。
5.根据权利要求4所述的宠物饲料添加剂，其特征在于，
所述宠物饲料添加剂的剂型选自粉末、颗粒及片剂中的一种。
6.根据权利要求4所述的宠物饲料添加剂，其特征在于，
所述宠物饲料添加剂的包装制品选自容器制品及条棒制品中的一种。
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