CN114831317B - 一种具有免疫调节作用的功能性组合物及其制备方法和应用 - Google Patents
一种具有免疫调节作用的功能性组合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种溶解性高的且具有增强免疫力的功能性组合物，其特征在于：原料按重量配比如下：乳清蛋白50‑100份；酪蛋白磷酸肽5‑15份；促溶成分2.5‑7.5份。本发明功能性食品有良好的溶解性，易于机体吸收。同时，本发明功能性食品有良好的免疫调节作用；增强免疫抑制小鼠脾脏和小肠谷胱甘肽和超氧化物歧化酶的含量和活性，增强机体抗氧化能力；改善免疫抑制小鼠脾脏和小肠的组织病理损伤。本发明保健食品具有较高的免疫增强活性，可应用于制备增强免疫力的药物或营养保健品的开发中，对于维持机体健康、预防疾病的发生具有重要的意义，同时也为乳清蛋白在食品和医药领域的健康指导和开发利用奠定理论基础。
Description
技术领域
本发明属于食品技术领域，涉及一种具有免疫调节作用的功能性组合物及其制备方法和应用。
背景技术
人体免疫力来于自身的免疫系统。免疫系统由免疫器官、免疫细胞及免疫因子组成，具有识别和排除抗原性异物、与机体其他系统相互协调，共同维持机体内环境稳定和生理平衡的功能。当机体受损时，免疫系统可以发挥功能，使人体免于病毒、细菌、污染物质及疾病的攻击，并且能够清除体内损伤变性、衰老的细胞和体内代谢废物。免疫系统是维持机体内环境稳定的关键，对预防疾病的发生具有重要的意义。免疫功能受到许多因素的不利影响，包括营养不良、心理和氧化应激、外源性病原体和抗原等。在人体免疫功能不足的情况下，人体难以抵抗细菌、病毒、真菌等的病原微生物侵袭感染，例如上呼吸道感染，尿路感染、败血症、脑膜炎、水痘、麻疹、肺结核等。
干扰素、卡介苗多糖核酸、胸腺肽、左旋咪唑等是医学上常用的免疫调节剂，用于调节免疫反应。然而，大多数免疫调节剂由于其昂贵的价格和不可避免的副作用，例如肝功能受损、过敏等，不适合长期治疗。因此，营养和功能性食物摄入的营养干预因其在免疫调节中的积极作用而受到广泛关注。
机体抵抗力、新陈代谢、机体组织修复这些维持人体健康的过程，都需要蛋白质的参与。乳清蛋白由β-乳球蛋白、α-乳白蛋白、免疫球蛋白等活性成分组成，具有吸收率高、氨基酸组成合理等诸多优势，被认为是人体所需的优质蛋白质来源之一。利用乳清蛋白来提高机体免疫力是一个很好的选择。然而，蛋白质是由氨基酸以“脱水缩合”的方式组成的多肽链经过盘曲折叠形成的具有一定空间结构的物质，这导致了蛋白质在水系中只能形成胶体，不能形成溶液。解决蛋白粉的溶解性能不佳、溶解过程易结块的问题，更易提高蛋白粉的口感和吸收，溶解性好的蛋白质的其他功能特性也会增强。
本发明研究了通过复配的方法得到乳清蛋白功能性组合物，获得的乳清蛋白功能性组合物溶解性高、具有增强机体免疫力的功效。
通过检索，尚未发现与本发明专利申请相关的专利公开文献。
发明内容
为了解决现有免疫调节药物的副作用高的问题，本发明提供了具有免疫调节作用的功能性组合物及其制备方法和应用，该组合物安全性高、效果明显的具有增强免疫力。同时，该组合物解决了目前市面上蛋白粉溶解性能不佳的问题。
本发明所采用的技术方案是：
一种溶解性高的且具有增强免疫力的功能性组合物，原料按重量配比如下：乳清蛋白50-100份；酪蛋白磷酸肽5-15份；促溶成分2.5-7.5份。
进一步地，所述的促溶成分，原料按重量配比如下：低聚果糖1-3份；大豆卵磷脂0.5-2.5份；异麦芽酮糖0.5-1.5份；维生素C0.2-1份；维生素E0.01-0.1份。
进一步地，原料按重量配比如下：乳清蛋白85份；酪蛋白磷酸肽10份，低聚果糖2份，大豆卵磷脂1.5份，异麦芽酮糖1份，维生素C0.45份，维生素E0.05份。
如上所述的功能性组合物的制备方法，将原料按比例混合均匀，即得。
进一步地，各原料混合后制成胶囊剂、片剂或颗粒剂。
如上所述的功能性组合物在制备功能性食品中的应用。
本发明取得的优点和积极效果为：
1、本发明的功能性组合物，溶解性佳，易于吸收。
2、本发明功能性组合物有良好的免疫调节作用，研究发现乳清蛋白和酪蛋白磷酸肽组合使用相对于仅用一种具有更好的效果，能够缓解免疫抑制小鼠体重增长缓慢的情况，增强免疫抑制小鼠脾脏和小肠谷胱甘肽和超氧化物歧化酶的含量和活性，增强机体抗氧化能力，改善免疫抑制小鼠脾脏和小肠的组织病理损伤。同时，本发明功能性组合物通过抑制脾脏TLR4/MyD88/NF-κB炎症通路的活化以及炎症因子(IL-6、IL-1β、TNF-α)的产生来减轻机体炎症，还可以维持免疫抑制小鼠的肝肾功能，显著降低血清中谷草转氨酶、谷丙转氨酶、尿素氮、肌酐的水平，从而实现对免疫抑制小鼠的有益调控。该发明功能性组合物具有较高的免疫增强活性，可应用于制备增强免疫力的药物或保健品中，对于维持机体健康具有重要的意义，同时也为乳清蛋白在食品和医药领域的健康指导和开发利用奠定理论基础。
3、本发明功能性组合物营养搭配合理，蛋白质含量高。
附图说明
图1为本发明中乳清蛋白功能性组合物对免疫抑制小鼠体重变化的影响图；
图2为本发明中乳清蛋白功能性组合物对免疫抑制小鼠脾脏指数的影响图；
图3为本发明中乳清蛋白功能性组合物对免疫抑制小鼠脾脏和小肠中谷胱甘肽含量的影响图；
图4为本发明中乳清蛋白功能性组合物对免疫抑制小鼠脾脏和小肠中超氧化物歧化酶活力的影响图；
图5为本发明中乳清蛋白功能性组合物对免疫抑制小鼠脾脏和小肠组织病理影响图；
图6为本发明中乳清蛋白功能性组合物对免疫抑制小鼠肝肾功能影响图；
图7为本发明中乳清蛋白功能性组合物对免疫抑制小鼠脾脏炎症因子表达的影响图。
具体实施方式
下面结合实施例，对本发明进一步说明；下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。
实施例1
一种具有增强免疫力功能的功能性组合物，原料重量配比如下：
低聚果糖2份，大豆卵磷脂1.5份，异麦芽酮糖1份，维生素C0.45份，维生素E0.05份。
实施例2
一种具有增强免疫力功能的功能性组合物，原料重量配比如下：
酪蛋白磷酸肽95份，低聚果糖2份，大豆卵磷脂1.5份，异麦芽酮糖1份，维生素C0.45份，维生素E0.05份。
实施例3
一种具有增强免疫力功能的功能性组合物，原料重量配比如下：
乳清蛋白95份，低聚果糖2份，大豆卵磷脂1.5份，异麦芽酮糖1份，维生素C0.45份，维生素E0.05份。
实施例4
一种具有增强免疫力功能的功能性组合物，原料重量配比如下：
乳清蛋白85份，酪蛋白磷酸肽10份，低聚果糖2份，大豆卵磷脂1.5份，异麦芽酮糖1份，维生素C0.45份，维生素E0.05份。
效果验证实验：
1.溶解时间测定
分别称取1g实施例1-4，加入25℃、50mL纯净水溶解，玻璃棒搅拌至完全溶解，记录所用时间。
2.实验动物分组及受试物给予方式
ICR小鼠，雄性，18±2g，SPF级，购自北京斯贝福实验动物责任限公司。适应性喂养后，称量初始体重，按体重随机分为5组，每组12只。实验第1-3天，除空白组对照组外，其余各组每天腹腔注射0.1mL,80mg/(kg·bw)环磷酰胺，建立免疫抑制小鼠模型，空白对照组每天注射等量生理盐水。依据小鼠体重重量分配原则，分别用纯净水配置实施例1-4溶液。第4天起，取实施例1组分，按照每天0.05g/(kg·bw)灌胃方式(0.2mL)给予实施例1组小鼠。取实施例2组分，按照每天0.1g/(kg·bw)灌胃方式(0.2mL)给予实施例2组小鼠。取实施例3组分，按照每天0.85g/(kg·bw)灌胃方式(0.2mL)给予实施例3组小鼠。取实施例4组分，按照每天1g/(kg·bw)灌胃方式(0.2mL)给予实施例4组小鼠。空白组和环磷酰胺模型组每日灌胃等体积纯净水，保持手术操作一致。连续灌胃14d后，禁食24h解剖，取脾脏、小肠，1500rpm,10min分离血清。
3.小鼠体重变化率
从环磷酰胺造模第一天起，每隔1天记录小鼠体重。计算小鼠体重变化率。
4.小鼠脾脏指数的测定
分离脾脏后，用生理盐水冲洗表面血液，再用滤纸吸去表面水分，称脾脏重量，根据公式：脏器指数(mg/g)＝脾脏重量/体重，计算得到。
5.组织病理学观察
将脾脏和小肠组织，在10％甲醛中固定，后经过流水冲洗、脱水、浸蜡、包埋等操作制作石蜡切片。然后除去石蜡，利用苏木精-伊红染色进行组织病理学观察，利用苏木精染细胞核、红染细胞质。
6.脾脏中炎症因子的测定
利用Trizol法提取脾脏组织RNA，经过紫外分光光度计检验纯度和浓度后，利用天根生化科技(北京)有限公司FastKingRTKit(KR116)反转录试剂盒进行cDNA合成，再使用天根生化科技(北京)有限公司SuperRealPreMixPlus(SYBRGreen)(FP205)实时定量试剂盒进行基因表达分析。β-acitin基因作为内参基因，用2-ΔΔCt法计算各组mRNA水平。引物序列为：TNF-α：上游5’-TCTTCTCATTCCTGCTTGTGGC-3’，下游5’-CACTGGTGGTTTGCTACGACG-3’；IL-1β：上游5’-GGCAGCTACCTGTGTCTTTCCC-3’，下游5’-ATATGGGTCCGACAGCACGAG-3’；IL-6上游5’-CTGCAAGAGACTTCCATCCAG-3’，下游5’-AGTGGTATAGACAGGTCTGTTGG-3’；β-actin上游5’-GATCGATGCCGGTGCTAAGA-3’，下游5’-TCCTATGGGAGAACGGCAGA-3’。
7.蛋白质免疫印迹技术检测TLR4/MyD88/NF-κB炎症通路的活化
样品用十二烷基硫酸钠聚丙烯酰胺凝胶分离，转移到硝化纤维素膜上。用针对目标蛋白的一抗检测，然后用二抗检测抗体-抗原复合物。通过凝胶成像和分析系统收集信号。
8.血清生化指标的检测
使用南京建成生物工程研究所有限公司市售谷丙转氨酶试剂盒(C009-2-1)、谷草转氨酶试剂盒(C010-2-1)、尿素氮试剂盒(C013-2-1)以及肌酐试剂盒(C011-2-1)，对小鼠血清谷丙转氨酶、谷草转氨酶、尿素氮和肌酐含量进行检测。
9.小鼠氧化防御系统指标测定
使用南京建成生物工程研究所有限公司市超氧化物歧化酶试剂盒(A001-1)和谷胱甘肽试剂盒(A006-2-1)对小鼠脾脏和小肠中的超氧化物歧化酶和谷胱甘肽水平进行检测。
本发明能提高乳清蛋白粉溶解性以及增强免疫抑制小鼠的免疫力的相关检测结果如下：
1.产品溶解时间越短则溶解性越好。如表1所示，实施例4的溶解性最佳，说明促溶成分有效促进了活性成分的溶解。
表1产品的溶解性
| 组别 | 溶解时间(s) |
| 实施例2 | 63.8±5.7 |
| 实施例3 | 102.6±7.8 |
| 实施例4 | 38.5±2.2 |
2.为了探究本发明功能性组合物对环磷酰胺所致免疫抑制小鼠体重变化的影响，记录了小鼠的体重变化，如图1所示。与空白组相比，模型组小鼠体重变化率显著低于空白组小鼠体重变化率，表明其生长速度缓慢。与模型组相比，经过实施例1-4的保护，实施例1-4均能一定程度上缓解小鼠体重变化率的降低。其中实施例4的处理对小鼠体重变化率的改变最为明显，说明实施例4可以有效的保护小鼠免于环磷酰胺所致免疫抑制情况下体重变化率的降低。
3.脏器指数是生物医学中用于衡量器官组织的受损状况的基本指标，可以对机体损伤程度进行综合的衡量。在正常状态下，动物体内各组织器官的质量/体重的比值维持相对恒定，受损后，脏器指数将发生改变。脾脏是机体重要的免疫器官，脾脏指数在一定程度上可以反映机体免疫功能情况。为了探究本发明功能性组合物对环磷酰胺免疫抑制小鼠机体免疫功能的影响，我们测定了脾脏指数，如图2所示。与空白组相比，使用环磷酰胺后，模型组小鼠脾脏指数显著降低，表明机体免疫功能失调。经过实施组的保护，可见，各实施组小鼠的脏器指数呈现升高趋势，其中，实施组4较实施组1-3脾脏指数恢复效果最优，可能是多种成分协同发挥促进作用，表明该配方下的乳清蛋白粉具有缓解环磷酰胺对小鼠的免疫抑制作用。
4.谷胱甘肽(GSH)能帮助保持正常的免疫系统功能，并具有抗氧化作用、整合解毒作用，参加机体氧化还原过程，能够清除过量的自由基，减弱脂质过氧化程度。如图3A脾脏中GSH含量结果可知，模型组小鼠脾脏中GSH的含量相比于空白组显著下降了60.9％，脾脏内GSH含量的下降说明环磷酰胺的处理造成了脾脏内抵抗氧化能力物质含量的下降。与模型组相比，实施例1组脾脏GSH值升高了2.2％，实施例2组脾脏GSH值升高了16.3％，实施例3组脾脏GSH值升高了111.2％，实施例4组脾脏GSH值升高了143.6％。其中，实施例4组对脾脏的氧化防御系统的保护作用尤其显著。
小肠也是人体重要的免疫器官之一，参与全身的免疫系统调节，小肠免疫功能的失调或缺陷，可延展至全身各处。我们检测了小肠中GSH含量，由图3B可知，与空白组相比，模型组小鼠小肠GSH含量下降了65.2％。与模型组相比，实施例1组小肠GSH值升高了6.2％，实施例2组小肠GSH值升高了15.1％，实施3组小肠GSH值升高了60％，实施例4组小肠GSH值升高了119.3％。其中，实施例4组对小肠的氧化防御系统的保护作用尤其显著。
5.体内外复杂的环境影响使之不断产生自由基，参与免疫抑制的生理过程。机体有一系列防御体系清除自由基，例如超氧化物歧化酶(SOD)。SOD被认为是防止氧化损伤的第一道防线，与超氧阴离子结合清除体内过多的活性氧，降低体内脂质过氧化程度。同时，研究和临床上都证实了用SOD治疗自身免疫性疾病有一定疗效。由图4可知，环磷酰胺显著降低了小鼠脾脏和小肠组织中的SOD活力。而经过实施例1-4处理后，脾脏中SOD活力依次升高2.2％，16.3％，111.2％，143.6％；小肠中SOD活力依次升高2.4％，13.6％，22.2％，29.8％。其中，实施组4处理对脾脏和小肠组织中SOD的影响最为明显，进一步证明复配粉使机体抵抗氧化损伤的能力增强是由多种成分协同介导的。
6.谷草转氨酶(AST)和谷丙转氨酶(ALT)的释放可以作为肝功能损害的重要指标。当肝细胞受到损伤时，会引起细胞膜通透性的改变，胞内的转氨酶会被放入到血液中，血液中转氨酶的活力会出现上升。如图5A-B所示，模型组的血清AST活力和ALT较空白组分别升高了120.4％和334.5％。与模型组相比，实施例1-4组血清AST活力分别下降了7.7％，18.1％，36.4％，47.5％；血清ALT活力分别下降了25.8％，25.2％，38.3％，62.7％。其中，实施例4组对免疫抑制小鼠的肝功能维护效果最好。
肌酐(CR)和尿素氮(BUN)是临床上用于评估肾功能的两个指标。一旦肾功能出现损伤，二者无法经过肾小球滤过而被直接释放到血液中，血清CR和BUN的升高意味着肾功能的损害。如图5C-D所示，模型组小鼠血清CR和BUN含量较空白组相比显著升高132.8％和62.7％，表明小鼠肾功能受损。与模型组相比，实施例1-4组血清CR含量分别下降了6.7％，16.0％，33.1％，51.3％；血清BUN含量分别下降了14.0％，17.2％，24.3％，30.6％。其中，实施例4组对免疫抑制小鼠肾脏功能的保护效果最佳。
7.如图6A所示的脾脏组织病理学染色结果，空白组小鼠脾脏结构清晰，无病变、坏死细胞出现。模型组小鼠脾脏组织结构重度异常，视野内脾小结结构散乱，红白髓分界不清晰，淋巴细胞胞核圆润，局部排列较疏松，如红色箭头所示；红髓内多核巨噬细胞大量增生，如黑色箭头所示。经过实施例1处理后，小鼠脾脏组织仍存在细胞间质充血、红细胞破裂的现象。经过实施例2处理后，小鼠脾脏组织轻度松散且伴有红髓内多核巨噬细胞轻度增生。实施例3保护后，小鼠脾脏组织细胞间质充血情况明显减少。经过实施例4保护后，小鼠脾脏组织状态较为良好，并且实施例4对免疫抑制小鼠的脾脏保护效果最好。
如图6B所示的脾脏组织病理学染色结果，空白组小鼠小肠结构清晰，粘膜层上皮细胞排列整齐，无病变。模型组小鼠小肠组织结构异常固有层可见少量炎症细胞浸润，如红色箭头所示；隐窝上皮胞浆淡染，气球样变，可见明显的水肿，如黄色箭头所示；粘膜下层水肿，致使粘膜层和肌层出现较大间隙，如蓝色箭头所示。经过实施例1和2的保护后，小鼠免疫小肠组织肠绒毛结构较为完整，但粘膜层和肌层之间仍存在间隙。经过实施例3保护后，小鼠小肠结构排列稍有松散，但整体病变程度明显减轻。经过实施例4保护后，小鼠小肠结构较为完整，排列较为整齐，表明实施4对免疫抑制小鼠小肠有良好的保护作用。
8.Toll样受体(TLR)是参与非特异性免疫(天然免疫)的一类重要蛋白质分子，能够识别病原体分子，刺激下游信号转导分子，诱导效应细胞炎性介质表达，从而影响免疫应答，也是连接非特异性免疫和特异性免疫的桥梁。TLR4表达水平升高激活下游蛋白MyD88以及NF-κB信号的反应。如图7所示，与空白组相比，模型组TLR4、MyD88、NF-κB的表达显著升高，分别为空白组的0.9倍、1.3倍、1.1倍。经过实施例1-4保护后，炎症蛋白的表达均出现不同程度的下降，其中，实施例4对抑制TLR4/MyD88/NF-κB信号通路的激活有良好的作用。一旦TLR4/MyD88/NF-κB信号被激活，则会诱导靶基因转录产生炎症因子。TNF-α是炎症反应过程中重要的炎性介质，能激活中性粒细胞和淋巴细胞，使血管内皮细胞通透性增加，调节其他组织代谢活性并促使其他细胞因子的合成和释放。IL-1β的激活是介导促炎反应的中心环节，导致继发性炎症介质(包括IL-6)的激活。IL-6能诱导B细胞分化和产生抗体，并诱导T细胞活化增殖、分化，参与机体的免疫应答，是炎性反应的促发剂。如图7所示，模型组小鼠脾脏中炎症因子的表达较空白组显著升高，经过实施例1-4保护后，炎症因子的表达均出现不同程度的下降，其中，相比于实施例1-3组，实施例4对环磷酰胺所致小鼠脾脏中炎症因子的表达有较好的抑制作用，以此缓解了免疫抑制小鼠的脾脏炎症。
综上所述，本发明功能性组合物能够有效减轻免疫抑制小鼠的机体炎症、增加抗氧化酶活性从而达到免疫调节的作用。同时，本发明功能性组合物相比于其他市售产品而言，有更好的溶解性，解决了乳清蛋白粉不易溶解的问题。
序列表
<110> 天津科技大学，优能康（天津）医疗科技有限公司
<120> 一种具有免疫调节作用的功能性组合物及其制备方法和应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> TNF-α：上游(Unknown)
<400> 1
tcttctcatt cctgcttgtg gc 22
<210> 2
<211> 21
<212> DNA
<213> TNF-α：下游(Unknown)
<400> 2
cactggtggt ttgctacgac g 21
<210> 3
<211> 22
<212> DNA
<213> IL-1β：上游(Unknown)
<400> 3
ggcagctacc tgtgtctttc cc 22
<210> 4
<211> 21
<212> DNA
<213> IL-1β：下游(Unknown)
<400> 4
atatgggtcc gacagcacga g 21
<210> 5
<211> 21
<212> DNA
<213> IL-6 上游(Unknown)
<400> 5
ctgcaagaga cttccatcca g 21
<210> 6
<211> 23
<212> DNA
<213> IL-6 下游(Unknown)
<400> 6
agtggtatag acaggtctgt tgg 23
<210> 7
<211> 20
<212> DNA
<213> β-actin上游(Unknown)
<400> 7
gatcgatgcc ggtgctaaga 20
<210> 8
<211> 20
<212> DNA
<213> β-actin下游(Unknown)
<400> 8
tcctatggga gaacggcaga 20
Claims (5)
1.一种溶解性高的且具有增强免疫力的功能性组合物，其特征在于：原料按重量配比如下：乳清蛋白50-100份；酪蛋白磷酸肽5-15份；促溶成分2.5-7.5份；
所述的促溶成分，原料按重量配比如下：低聚果糖1-3份；大豆卵磷脂0.5-2.5份；异麦芽酮糖0.5-1.5份；维生素C 0.2-1份；维生素E 0.01-0.1份。
2.根据权利要求1所述的功能性组合物，其特征在于：原料按重量配比如下：乳清蛋白85份；酪蛋白磷酸肽10份，低聚果糖2份，大豆卵磷脂1.5份，异麦芽酮糖1份，维生素C 0.45份，维生素E 0.05份。
3.如权利要求1或2所述的功能性组合物的制备方法，其特征在于：将原料按比例混合均匀，即得。
4.根据权利要求3所述的功能性组合物的制备方法，其特征在于：各原料混合后制成胶囊剂、片剂或颗粒。
5.如权利要求1至2任一项所述的功能性组合物在制备功能性食品中的应用。
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| CN105285315A (zh) * | 2015-11-30 | 2016-02-03 | 临夏州华安生物制品有限责任公司 | 清真牦牛乳酪蛋白磷酸肽营养粉 |
| CN109430413A (zh) * | 2018-09-26 | 2019-03-08 | 北京斯利安药业有限公司 | 一种增强免疫力的组合物、功能性食品及其制备方法 |
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| CN105285315A (zh) * | 2015-11-30 | 2016-02-03 | 临夏州华安生物制品有限责任公司 | 清真牦牛乳酪蛋白磷酸肽营养粉 |
| CN109430413A (zh) * | 2018-09-26 | 2019-03-08 | 北京斯利安药业有限公司 | 一种增强免疫力的组合物、功能性食品及其制备方法 |
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