CN116849354A - 一种添加植物乳杆菌gl-5的炎症性肠病全营养配方食品 - Google Patents
一种添加植物乳杆菌gl-5的炎症性肠病全营养配方食品 Download PDFInfo
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Abstract
本发明涉及特殊医学用途配方食品技术领域,尤其涉及一种添加植物乳杆菌GL‑5的炎症性肠病全营养配方食品及制备方法。本发明提供的一种添加植物乳杆菌GL‑5的炎症性肠病全营养配方食品,包括以下重量份的组分:碳水化合物66~68份、蛋白质12~14份、脂肪18~22份;所述全营养配方食品中还包括微量元素和植物乳杆菌GL‑5。本发明研制了一款针对UC患者的特医食品,在提供营养支持之余,又能有效辅助改善患者临床症状,不仅具有现实意义,更可对UC的机制研究做出一定的理论贡献。
Description
技术领域
本发明涉及特殊医学用途配方食品技术领域,尤其涉及一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品。
背景技术
炎症性肠病(inflammatorybowel disease,IBD)是一种特发为回肠、结肠、直肠的慢性、复发性、炎症性疾病,常见症状为腹泻、腹痛、血便,主要特征是肠粘膜结构破坏、肠道菌群改变。近十年来,IBD患病率从79.5/10万上升到84.3/10万,其中北美地区IBD患病率最高,美国的IBD患病率可达到464.5/10万,已经发展为一种世界关注的疾病。
溃疡性结肠炎(ulcerative colitis,UC)作为IBD中的一种,以排便急、里急后重、带血腹泻、腹痛、无法根治、病程反复、并发症较多、营养不良为特征性表现,主要是肠粘膜结构和肠道菌群的改变。UC的致病机理并不是很清楚,是由肠道粘膜屏障、肠道菌群和免疫系统之间一系列复杂的相互作用导致。同时,在宿主内源性和外源性多种因素的影响下,饮食成为影响肠道微生物群落结构和功能的关键因素,通过将饮食信号引入宿主-微生物群之间的联系,营养有助于维持体内平衡或降低疾病的易感性。因此研究肠粘膜屏障、肠道菌群、肠内营养和免疫系统在UC发生中的作用和机制,对UC的防治具有重要意义。
参考UC的国外临床治疗,如欧洲和英国临床指南,一般轻到中度UC使用5-氨基水杨酸(5-amino salicylic acid,5-ASA,又名美沙拉嗪),而对于类固醇性或5-ASA不耐受性UC则推荐使用硫嘌呤类药物,对于中到重度UC及常规治疗无效的患者,推荐使用抗肿瘤坏死因子(tumornecrosis factor,TNF)药物【ECCO Guidelines on Therapeutics inUlcerative Colitis:Medical Treatment】。考虑到目前的治疗措施所带来的副作用,如产生肾损伤,电解质紊乱,增加易感染性,诱发感染或增强免疫抑制等,使得UC的CAM疗法(Complementary andAlternative Medicine,补充替代药物)也就随之而发展起来,这里面包括益生菌、益生元、合生元、草药、粪菌移植和针灸等疗法,其中益生菌对UC的辅助治疗效果已有很多研究为其证实。
特殊医学用途配方食品(Food for Special Medical Purpose以下简称FSMP或特医食品)的使用正是临床治疗时必备的营养干预之一。特医食品指为了满足进食受限、消化吸收障碍、代谢紊乱或特定疾病状态人群对营养或膳食的特殊需要,专门加工配制而成的配方食品。它可作为患者的全部或部分营养来源使用,但必须在医学监督下单独或者配合其他食品进行食用。有临床研究表明,对于存在营养不良的风险的内科住院患者,给予规范化、个体化的营养支持干预,包括使用FSMP进行营养补充或管饲,并根据监测情况调节营养治疗方案,能够有效降低死亡率和降低并发症的风险。通过FSMP对UC的患者可以提供营养支持,但是对于其是否能提供针对UC的治疗和预防尚且还是一个未知数。
特医食品可按年龄段分两大类,即特殊医学用途婴儿配方食品(1岁以下)和特殊医学用途配方食品(1岁以上)。其中特殊医学用途配方食品(1岁以上)又分为3类:一是全营养配方食品,可当其作为长期单一营养来源时,能够满足目标人群的每天营养需求;二是特定全营养配方食品,这类食品针对脏器功能不全或衰竭、代谢障碍、机体对某营养素的需求增加或限制的特殊营养代谢状况的患者而设计,可当其作为单一营养来源不仅满足目标人群每天营养需要,而且能满足由于某种疾病和医学状况而产生的对某些营养素或日常食物特殊要求;三是非全营养配方食品,可以满足目标人群部分营养需要。针对第二类食品在《特殊医学用途配方食品通则》的附录A中早已有了规定,指包括第8种炎性肠病全营养配方食品在内的13种特定全营养配方食品,但截止到目前,特定全营养类暂未有产品获得批准。因此基于发明人前期针对UC的益生菌研究,研制一款炎性肠病全营养配方食品具有一定的现实意义。
发明人从青海省果洛藏族自治州的牦牛酸奶中分离出的植物乳杆菌GL-5,已列入国家卫生管理部门颁布规定的《可用于食品的菌种名单》和《可用于婴幼儿食品的菌种名单》中,具有较好的过氧化氢耐受性,其发酵清液检测出高活性的SOD酶(Superoxidedismutase,超氧化物歧化酶),以及较高水平的羟自由基清除能力;且对金黄色葡萄球菌和大肠杆菌具有较强的抑菌作用;相比于益生菌株鼠李糖乳杆菌(L.rhamnosus),植物乳杆菌GL-5具有较强的人工胃肠液存活能力。并通过前期实验证实其对葡聚糖硫酸钠(DextranSulfate Sodium Salt,DSS)诱导的溃疡性结肠炎具有有效的缓解效果,但针对UC将GL-5用于特医食品目前尚无研究。
发明内容
本发明的目的在于提供一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,使其在提供营养支持之余,又能有效辅助改善患者的临床症状,这不仅具有现实意义,更对UC的机制研究可做出一定的理论贡献。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,包括以下重量份的组分:碳水化合物66~68份、蛋白质12~14份、脂肪18~22份;
所述全营养配方食品中还包括微量元素和植物乳杆菌GL-5。
优选的,所述碳水化合物为葡萄糖。
优选的,所述蛋白质为酪蛋白、乳清蛋白和大豆分离蛋白;所述酪蛋白、乳清蛋白和大豆分离蛋白的质量比为0.5~1.5:0.5~1.5:0.5~1.5。
优选的,所述脂肪为食用甘油、亚油酸粉剂、二十碳五烯酸和中链甘油三酯粉剂;所述食用甘油、亚油酸粉剂、二十碳五烯酸和中链甘油三酯粉剂的质量比为1.8~2.2:0.8~1.2:0.8~1.2:3.8~4.2。
优选的,所述微量元素为包括维生素A、维生素D、维生素E、维生素K1、维生素B1、维生素B2、维生素B6、维生素B12、烟酰胺、叶酸、泛酸、维生素C、生物素、钠、钾、铜、镁、铁、锌、锰、钙、磷、碘、氯、硒;每100g全营养配方食品中,含有视黄醇当量566~570μg的维生素A、7~9μg的维生素D、α-生育酚当量3~4mg的维生素E、17~20μg的维生素K1、0.2~0.4mg的维生素B1、0.2~0.4mg的维生素B2、0.2~0.4mg的维生素B6、0.4~0.6μg的维生素B12、0.7~0.9mg的烟酰胺、94~96μg的叶酸、0.9~1.1mg的泛酸、23~25mg的维生素C、8~10μg的生物素、355~359mg的钠、476~480mg的钾、1170~1172μg的铜、77~79mg的镁、6~7mg的铁、4~6mg的锌、1368~1370μg的锰、240~242mg的钙、171~173mg的磷、27~29μg的碘、468~470mg的氯、54~56μg的硒。
优选的,所述植物乳杆菌GL-5的菌浓度为2800~3200亿CFU/g;每100g全营养配方食品中,所述植物乳杆菌GL-5的用量为15~17mg。
优选的,所述全营养配方食品中还包括铬、钼、氟、胆碱、肌醇、牛磺酸、左旋肉碱、核苷酸和膳食纤维;每100g全营养配方食品中,含有铬122~124μg、钼118~120μg、氟0.3~0.5mg、胆碱405~407mg、肌醇309~311mg、牛磺酸42~44mg、左旋肉碱4~6mg、核苷酸7~9mg和膳食纤维5~7g。
本发明基于植物乳杆菌GL-5益生菌,并根据《炎症性肠病全营养配方食品(征求意见稿)》(GB29922-2013)研制一款针对UC患者,在提供营养支持之余,又能有效辅助改善患者临床症状的特医食品,不仅具有现实意义,更对UC的机制研究可做出一定的理论贡献。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1为实施例1制备的配方食品成品状态图;
图2为实施例1制备的配方食品放置10min后的状态图;
图3为实施例2造模7天时小鼠肛门部便血图;
图4为实施例2造模7天时小鼠上端结肠HE染色切片(×100);
图5为小鼠给药干预后体重变化曲线;
图6为小鼠给药干预后各阶段体重柱形图;
图7为小鼠给药干预后G、A、R三组摄食量变化曲线;
图8为小鼠给药干预期间G、A、R三组摄食量柱状图;
图9为小鼠给药干预期间饮水量变化曲线;
图10为小鼠给药干预期间各组饮水量图;
图11为小鼠给药干预期间累计死亡风险图;
图12为DAI指数的变化图;
图13为小鼠近端结肠切片染色图;
图14为小鼠近端结肠病理学评分结果;
图15为小鼠肠道微生物代谢物三组差异代谢物数目;
图16为小鼠肠道微生物代谢物R组与M组差异代谢物数目;
图17为小鼠肠道微生物代谢物差异分子热图;
图18为小鼠肠道微生物代谢物富集条形图;
图19为Chao1指数图;
图20为Chao1指数稀释曲线图;
图21为Shannon指数图;
图22为Shannon指数稀释曲线图;
图23为Pielou指数图;
图24为Pielou指数稀释曲线图;
图25为丰度等级曲线图;
图26为加权PCoA分析图;
图27为NMDS分析图;
图28为UPGMA聚类分析图;
图29为组间差异显著性分析图;
图30为ASV/OTU的花瓣图;
图31为物种丰度差异图(a(左图)为门水平上的物种丰度差异;b(右图)为属水平上的物种丰度差异);
图32为物种聚类的属水平物种组成热图;
图33为小鼠休息次数图;
图34为小鼠休息时长图;
图35为小鼠运动次数图;
图36为小鼠运动时长图;
图37为小鼠平均速度图;
图38为小鼠活跃度比较图;
图39为小鼠在棒时间图;
图40为小鼠转棒速度图。
生物保藏说明
植物乳杆菌GL-5,拉丁文名为Lactobacillusplantarum;
该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号;保藏时间为2019.11.21;保藏编号为CGMCC No.18988。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
本发明结合《特殊医学用途配方食品通则》(GB29922-2013)和2018年发布的《食品安全国家标准炎性肠病全营养配方食品》(征求意见稿),针对炎性肠病全营养配方食品的规定总结了以下几点规律:
(1)我国IBD患者平均体重为53.5kg,配方的总能量按照每天能量需要量1800kcal计算;
(2)每100mL(液态产品或可冲调为液体的产品在即食状态下)或每100g(直接食用的非液态产品)所含有的能量应不低于295kJ(70kcal);
(3)蛋白质含量应≥0.84g/100kJ(3.5g/100kcal),其中优质蛋白质≥50%。可以选用整蛋白、食物蛋白质水解物、肽类和/或氨基酸作为蛋白质的来源;此时蛋白质按照平均体重为53.5kg来算是1.2g/kg/d,推荐的是1.2-1.5g/kg/d,而中国居民膳食指南推荐的是1.16g/kg/d;
(4)脂肪供能比应≤35%,亚油酸供能比应≥2.0%,α-亚麻酸供能比应≥0.5%。其中中链甘油三酯(Medium chain triglycerides,MCT)含量应不低于总脂肪的40%;
(5)微量营养素的含量范围以推荐摄入量(Recommended nutrient intakes,RNI)或适宜摄入量(Adequate intakes,AI)的最低值为依据,计算每100kcal或每100g的最小值;以可耐受最高摄入量(Tolerable upper intake levels,UL)的最低值为依据,计算每100kcal或每100g的最大值。
配方中不应含有乳糖、果糖、麸质、氢化油脂、卡拉胶、黄原胶、羧甲基纤维素钠、木糖醇、三氯蔗糖、阿斯巴甜。
对于IBD而言,国内外目前尚未有特定的饮食推荐,而且饮食干预实验的局限性,比如缺乏对照组,难以获得准确的饮食摄入量,食物间的相互作用以及接受药物治疗的患者无法体现出饮食试验的显著差异等,导致患者开始从非医学资源领域寻求饮食指导。因此对于当前研究中所有关于UC的饮食推荐目前都缺乏可靠的数据支持,只能从单一或者常见的膳食结构以及获得成功的饮食个案中获得线索。
结合征求意见稿中及当前研究进展,可以暂将UC患者的膳食原则总结为:高精制碳水化合物,适量蛋白质,低脂肪,低膳食纤维,高维生素和微量元素(RNI~UL)。而在《中国居民膳食营养素参考摄入量》(2013版)中推荐的健康成人的膳食能量需要量依次是:膳食中碳水化合物提供的能量应占总能量的50%~65%、脂肪占20%~30%、蛋白质占10%~15%为宜。因此,此次配方设计在推荐的范围内做出一定调整,暂将碳水化合物、蛋白质和脂肪的总能量占比依次定为67%、13%、20%。其余微量元素按照食品安全国家标准炎性肠病全营养配方食品》(征求意见稿)中的推荐进行计算。
1.配方中各原料成分的选择及含量计算
1.1碳水化合物的选择及含量计算
本实施例选择葡萄糖(购自河北百优生物科技有限公司)作为配方的碳水化合物来源,根据每天每人(53.5kg)总能量及葡萄糖能量计算含量如下:
葡萄糖:1800×0.67×4.184÷1715×100≈294(g)
1.2蛋白质的选择及含量计算
本实施例对几种动物蛋白和植物蛋白进行营养学评分,以经消化率修正的氨基酸评分为指标,评分结果如表1所示,再结合征求意见稿中优质蛋白质≥50%,考虑该类蛋白质食物中蛋白质的含量及能量,故选择酪蛋白、乳清蛋白和大豆分离蛋白作为配方的蛋白质来源(均购自江苏仟润生物科技有限公司),三种蛋白按照1:1:1的质量比例,根据总能量和各自的能量计算含量如下:
酪蛋白:1800×0.13×4.184÷3÷1581×100≈21(g);
乳清蛋白:1800×0.13×4.184÷3÷1569×100≈21(g);
大豆分离蛋白:1800×0.13×4.184÷3÷1563×100≈21(g)
表1几种食物蛋白质经消化率修正的氨基酸评分(PDCAAS)表
食物蛋白 | PDCAAS | 食物蛋白 | PDCAAS |
酪蛋白 | 1.00 | 菜豆 | 0.68 |
鸡蛋蛋白 | 1.00 | 燕麦粉 | 0.57 |
大豆分离蛋白 | 0.99 | 花生粉 | 0.52 |
牛肉 | 0.92 | 小扁豆 | 0.52 |
豌豆粉 | 0.69 | 全麦 | 0.40 |
1.3脂肪的选择及含量计算
本实施例选择食用甘油(购自河北百优生物科技有限公司)、亚油酸粉剂(购自陕西新天域生物科技有限公司)、EPA(购自青岛海维森生物科技有限公司)和中链甘油三酯粉剂(购自青岛海维森生物科技有限公司)作为配方的脂肪来源,四者的质量比例为2:1:1:4,根据总能量和各自的能量计算含量如下:
食用甘油:1800×0.2×4.184÷8×2÷1808×100≈21(g)
亚油酸粉剂:1800×0.2÷8×1÷619×100≈7(g)
EPA:1800×0.2÷8×1÷740×100≈6(g)
中链甘油三酯粉剂:1800×0.2÷8×4÷654×100≈27(g)
1.4微量元素的选择及含量计算
微量元素添加量计算结果如表2所示。
表2维生素和矿物质指标(10岁以上人群)
注:aRE为视黄醇当量。1μgRE=3.33IU维生素A=1μg全反式视黄醇(维生素A)。维生素A只包括预先形成的视黄醇,在计算和声称维生素A活性时不包括任何的类胡萝卜素组分。
b钙化醇,1μg维生素D=40IU维生素D。
c1mgα-TE(α-生育酚当量)=1mgd-α-生育酚。
d烟酸不包括前体形式。
eN.S.为没有特别说明。
在本实施例制备的配方食品中仅选择添加表2中的维生素A、维生素D、烟酰胺、维生素B6、维生素B12、钙、铁、硒。含量根据1800kcal在最小值(RNI)和最大值(UL)之间选择中位数进行计算(若最高值为N.S时按照最低值计算,若最低值为N.S时按照最高值以一半计算)。所以,维生素A的添加量(RE当量)为(1800÷100)×[(39+225)/2]=2376μg;同理,维生素D为35μg;烟酰胺为3.6mg;维生素B6为1.26mg;维生素B12为2.34ug;钙为1008mg;铁为28mg;硒为229.5μg(购置的植物活性硒片一片中硒含量为200μg,故直接添加一片即可)。
其中维生素A、维生素D、烟酰胺、维生素B6、维生素B12、柠檬酸钙、焦磷酸铁购自江苏仟润生物科技有限公司。植物活性硒片购自普丽普莱。
1.5可选择成分选择和含量计算
可选择成分的添加量计算结果如表3所示。
表3可选择成分指标(10岁以上人群)
注:a氟的化合物来源为氟化钠和氟化钾,核苷酸和膳食纤维来源参考GB 14880表C.2中允许使用的来源,其他成分的化合物来源参考GB 14880。
bN.S.为没有特别说明。
在本实施例的食品配方中选择添加肌醇和牛磺酸,按照1.4的计算方法计算添加量,其中肌醇添加量为1297mg,牛磺酸添加量为180mg。均购自江苏仟润生物科技有限公司。
1.6益生菌含量计算
本实施例提供的配方食品中添加的益生菌为植物乳杆菌GL-5,其菌粉规格为3000亿CFU/g,按照每天每人(53.5kg)摄入2×1010CFU的摄入量,添加0.07g。
将上述原料按照各自质量精确称取混合,即得到一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品。成品如图1和图2所示,图1为刚制备成丸时的状态,图2为放置10min后的状态。
根据《感官分析食品感官质量控制导则》(GB/T 29605-2013)和《感官分析建立感官特性参比样的一般导则》(GB/T 29604-2013)建立适合该食品的感官评价表,表4,选3人进行评价。感官评价结果如表5所示。
表4感官评价表
注:打分标准从左往右依次为-2,-1,0,1,2。
表5感官评分结果
指标 | ① | ② | ③ |
色泽 | 1 | 0 | 1 |
杂质 | 2 | 2 | 2 |
气味 | 0 | 1 | 1 |
冲调性 | 2 | 2 | 2 |
合计 | 5 | 5 | 6 |
注:打分标准从左往右依次为-2,-1,0,1,2。
根据配方成品及评价结果来看,目前的产品的色泽一般,粉末尚可,但刚冲调之后色泽一般,于水中充分溶剂,无杂质,无团块,且伴随淡淡奶味(来源于乳清蛋白),可于后期添加其他成分或更换成分进行调整。
实施例2
本实施例利用实施例1配制的适用于溃疡性结肠炎患者的特医食品,对溃结小鼠进行10天的干预。
选择SPF级雌性C57BL/6雌性小鼠84只,并评估每只小鼠的健康状况,小鼠垫料及鼠笼一周更换两次,其中小鼠垫料为杨木创花,对其进行辐照灭菌。适应性饲养7天后,用于实验。适应期间小鼠肢体力量正常,尾悬后躯干与尾部成钝角甚至直角,尤其后肢弹跳力尚可,可原地弹跳约20~30cm;不喜抱团,对陌生环境的探索欲望较强,于鼠笼内皆可直立向上探索;笼内垫料干燥,无异味,粪便干燥、成型,黄棕/棕黑色,质地较硬,新鲜粪便表面光滑水润,呈细长/椭圆型;每日饲料和饮水摄入的变化幅度较小。适应良好。
随机将小鼠分为6组,每组14只,一组分两笼进行饲养。六组小鼠依次为正常组(N组)、模型组(D组),GL-5配方组(G组),安素组(A组),美沙拉嗪组(M组),联合治疗组(R组)。其中,GL-5配方为实施例1制备的配方食品,安素为已上市的安素全营养配方食品,联合治疗组为GL-5配方和美沙拉嗪结合。除正常组(N组)给予纯化水,其余各组给予3.5%DSS水溶液造模7天。造模期小鼠前三天仅表现为饮水增加,尿量增多,笼内湿润垫料面积增大,个别小鼠体重下降,肛门部正常,活动强度下降;第4天组内一半小鼠出现体重下降,约1g左右,其余体重与前一日持平,肛门部隐隐便血,个别小鼠便血,出现粪便稀软可拾取,红棕色,成型;第5天全部造模小鼠体重开始下降,出现鲜红色垫料,个别小鼠便血;第7天小鼠体重继续下降,多半便血,肛门部可见明显便血或血迹,粪便红棕色,细短柱状,不可拾取,部分小鼠伴随轻微震颤和间歇性拱背,与其他小鼠互动较少,独立于笼内角落;造模7天小鼠肛门部便血情况见图3。造模结束时各组随机挑选一只小鼠颈椎脱臼处死,取其上端结肠1cm制作病理切片。造模小鼠结肠出现不同程度变短,如图4所示,切片镜下观察出现不同程度炎症,病变深度可达肌层甚至浆膜层,隐窝出现不同程度的结构改变和脓肿,如隐窝扭曲,萎缩,表面不规则等,杯状细胞粘液减少。因此造模成功,可以用于后续实验。造模结束后按照表6进行为期10d的干预。
表6配方食品缓解小鼠结肠炎的实验分组及给药
小鼠每日给予纯化水150mL和饲料100g,次日记录后添加至相同的量。自适应期第1天始对每日饲料量、饮水量和体重进行记录,并对小鼠适应期、造模期和干预期的精神、活动、肌肉力量的一般情况进行描述。此试验中不良事件主要指干预期小鼠的死亡率。
小鼠体重经干预后第1天G组和A组开始回弹,其次分别是R组,M组和D组(见图5)。且对比干预后D组体重,G组和A组有统计学差异(P<0.05)。R组和M组之间尽管没有统计学差异,但最终体重及干预阶段体重变化均高于M组(19.32±1.27>19.03±0.99g,0.99±1.20g>0.97±0.97g)(见图6)。值得一提的是,根据以往经验当小鼠体重在15g左右时会处于病危状态,且在之后的1~2天便会死亡,死亡时体重在13~14g之间,而G组的一只小鼠造模结束时体重为15.0g,但从干预后第一天开始的13.9g→13.0g→12.1g→12.2g→13.0g→14.8g,直至最后恢复体重恢复正常(16.8g)。在摄食量方面,G组,A组,R组尽管已经给予了营养干预,但并未限制其普通饲料的摄入,但从图7中可看到,即使给予了满足小鼠一天能量的营养,对于普通饲料的摄入,G组也是率先增加,且到实验终点时,G组和R组的摄食量都比A组高;而从整个干预期的摄食量来看,G组>A组>R组,且G组和R组有统计学差异(*P<0.05)(见图8)。当小鼠患有结肠炎时,会出现喝得多,排得多情况,而在干预之后,G组和R组呈现出与D组截然不同的恢复(***P<0.01和**P<0.001)(见图9和图10)。
各组在干预期内的不良事件(即死亡率)如下:N组:0/13=0;D组:2/13=15.38%;G组:0/13=0;A组:0/13=0;M组:6/13=46.15%;R组:4/13=30.77%。从结果来看,G组的死亡率低于M组(P<0.05),但R组低于M组却不具有统计学差异(P=0.2658>0.05)。观察小鼠的累计死亡风险图,可以看到M组、D组率先出现死亡,且实验结束时M组死亡风险较高(见图11)。
干预期结束后眼球采取外周血于1.5mL EP管。在EP管中室温静置一小时,以3500rpm离心10分钟,其中离心机为MPW-380R、英国ATS,上清即为血清,使用200μl加样枪将血清抽取于另一1.5mL EP管中,放置在-20°冰箱保存,用于进行安全性评价。
N组和G组血清经全血分析仪检测后生化指标如表7,G组的碱性磷酸酶较N组的位于参考值范围内,除碱性磷酸酶和总蛋白之外的其他指标均无显著性差异(P>0.05)。表明本发明提供的配方食品安全。
表7血清生理生化指标
注:在P<0.05的水平上具有统计学差异。
在小鼠造模期结束和干预期结束后按照表4对各小鼠进行两次疾病活动指数(Disease activity index,DAI)评价,以此评价配方食品对疾病的治愈程度。
表8DAI评分细则
小鼠于造模和干预结束进行两次DAI评价。从图12中可见,干预后各组DAI指数均较造模期降低不少;在干预期DAI方面,与D组相比,G组和A组呈现显著差异(P<0.01),而R组有统计学差异(P<0.05)。
干预期结束后颈椎脱臼处死小鼠,取小鼠近端结肠病变组织1cm,放入10%福尔马林中固定过夜。依次经过脱水、透明、浸蜡、包埋、切片、展片、捞片和烤片、脱蜡复水,然后经过HE染色、阿尔新蓝染色以及Massion染色,最后显微镜观察组织形态学。
(1)HE染色:
A.石蜡切片脱蜡至水;依次将切片放入二甲苯Ⅰ10min~二甲苯Ⅱ10min~无水乙醇Ⅰ5min~无水乙醇Ⅱ5min~95%酒精5min~90%酒精5min-80%酒精5min~70%酒精5min~蒸馏水洗;
B.苏木素染细胞核;切片入Harris苏木素染3~8min,自来水洗,1%的盐酸酒精分化30s,自来水冲洗,0.6%氨水返蓝,流水冲洗;
C.伊红染细胞质;切片入伊红染液中染色1~3min;
D.脱水封片;将切片依次放入95%酒精I 5min~95%酒精II 5min~无水乙醇Ⅰ5min~无水乙醇Ⅱ5min~二甲苯Ⅰ5min~二甲苯Ⅱ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片;
E.显微镜镜检,图像采集分析;采用表5的Fedorak组织学损伤评分细则对切片进行组织学评分。
表9 Fedorak组织学损伤评分细则
评分 | 炎症 | 病变深度 | 隐窝破坏 | 病变范围 |
0 | 无 | 无 | 无 | \ |
1 | 轻度 | 粘膜下层 | 1/3隐窝破坏 | 1%-25% |
2 | 重度 | 肌层 | 2/3隐窝破坏 | 26%-50% |
3 | \ | 浆膜层 | 仅有完整表面上皮 | 51%-75% |
4 | \ | \ | 全部隐窝和上皮被破坏 | 76%-100% |
(2)阿尔新蓝染色:
A.烘片、脱蜡;65℃烘片30min;依次经二甲苯Ⅰ、二甲苯Ⅱ脱蜡;
B.依次经无水乙醇Ⅰ、95%Ⅰ、85%、75%、自来水水化;
C.染色;依次入Alcian酸化液、Alcian染色液染色浸泡,流水冲洗:入核固红染色液复染,流水冲洗。其中Alcian酸化液、Alcian染色液购置于Servicebio的阿利新蓝染液套装;
D.脱水;依次经85%、95%Ⅰ、95%Ⅱ各10秒,无水乙醇Ⅰ30s、无水乙醇Ⅱ1min;
E.透明;经二甲苯Ⅰ1min、二甲苯Ⅱ2min封片。组织上方滴加中性树胶,玻片稍倾斜,用手将盖片顺玻片斜面轻放,防止气泡产生;
F.20g儿甲绿复染10~20minG.显微镜下观察拍照。
(3)Massion染色:
A.10%甲醛液固定组织,石蜡切片,常规脱蜡至水。
B.苏木素染液5~10min;
C.流水稍洗,1%盐酸分化;
D.流水冲洗数分钟;
E.Masson复合染色液5~10min;
F.蒸馏水稍冲洗;
G.1%磷钨酸液处理约5min;
H.不用水洗,直接用亮绿染色液(或苯胺蓝液)复染5min;
I.1%冰醋酸水处理1min;
J.95%酒精脱水多次;
K.无水乙醇脱水,二甲苯透明,中性树胶封固。
从图13中可以看到,对比干预前后的小鼠近端结肠的HE染色切片,除N组和D组外,其余各组的结肠粘膜都呈现一定程度恢复,从造模结束和干预结束的HE染色可以看到隐窝的恢复及隐窝间隙的缩小;阿尔新蓝染色可以看到黏膜上杯状细胞的恢复,masson染色可以看到各组粘膜肌肉层的恢复,综合三种染色,可以看出G组,A组,R组粘膜恢复较好。对各组HE染色切片依据表5进行结肠组织损伤评分可以看出,G组,R组与D组的评分呈统计学差异(*P<0.05),G组,R组与M组的评分也呈统计学差异(*P<0.05),且R组与M组之间呈显著性差异(**P<0.01),如图14所示。
干预期结束后小鼠颈椎脱臼处死,在取结肠时取结肠内容物于冻存管中放置干冰保存,送样至诺米代谢公司进行非靶向代谢物及微生物多样性分析。
通过液质联用(LC-MS)非靶向代谢组技术检测C57BL/6雌性小鼠经干预后肠道菌群代谢物的动态变化,了解肠道菌群在宿主中的代谢状态,直观地研究肠道菌群与疾病发生发展的关系,为疾病的预防和治疗,调高宿主的健康水平提供新的思路。从样本一级物质列表中寻找差异代谢物,以统计检验中预设的P value和VIP阈值进行筛选。由图15和图16可以观察到M组和R组经干预后分别上调和下降的代谢物数量,上调36种,下降112种,总计148种差异谢物。根据差异分子热图可以了解到,在R组中分子量相对表达较多的有异丙烯内酯,丁香酸,花生四烯酸,甘氨酸,表达较少的为塞来昔布,吡哆醇,环己酰亚胺图17。根据图18可得到本实施例中所受差异代谢物比较显著的前五个代谢通路依次为过氧化物酶体增殖物激活受体,花生四烯酸代谢,亚油酸代谢,ABC转运蛋白,β-丙氨酸代谢。
在菌群多样性分析的α多样性评价中,Chao1是用Chao1算法估计样品中所含OTU数目的指数,在生态学中常用来估计物种总数。正如图19和图20所示的,G组和R组的物种总数较D组恢复的比较良好。Shannon指数是综合物种数量和丰度两个层面的结果来估算样品中微生物多样性指数之一。Shannon值越大,说明群落多样性越高。图21和图22显示M组和D组之间有统计学差异(P<0.05),G组虽然与D组之间没有统计学差异,但在各干预组之间呈最高水平。Pielou指数是衡量物种均匀度的指标,用于衡量生态系统中不同物种之间数量的差异度。对比D组,G组,M组有统计学差异(P<0.05),G组和R组之间有统计学差异(P<0.05)(见图23和图24)。丰度等级曲线将每个样本中的ASV/OTU按其丰度大小沿横坐标依次排列,并以各自的丰度值为纵坐标,用折线或曲线将各ASV/OTU互相连接,从而反映各样本中ASV/OTU丰度的分布规律;每条折线代表一个样本(组),折线在横轴上的长度反映了该样本在该丰度中的ASV/OTU数。折线的平缓程度,反映了群落组成的均匀度,折线越平缓,则群落中各ASV/OTU间的丰度差异越小,群落组成的均匀度越高,折线越陡峭,则均匀度越低。从图25中可以看到G组,A组,R组的曲线较为平缓。
在β多样性分析中,主坐标分析(Principal coordinates analysis,PCoA)通过将样本距离矩阵经过投影后,在低维度空间进行展开,并最大限度地保留原始样本的距离关系;由于NMDS采用等级排序,因此可近似认为两点之间的距离越近(远),表明两个样本中微生物群落的差异越小(大)。图26和图27的加权PCoA分析和NMDS分析中皆显示G组和R组与N组的面积重合较多,距离较近。β多样性聚类分析中多采用层次聚类(Hierarchicalclustering)的分析方法,以等级树的形式展示样本间的相似度,通过聚类树的分枝长度衡量聚类效果的好坏。样本根据彼此之间的相似度聚类,样本间的分枝长度越短,两样本越相似。图28表明N组,G组,R组的菌群相似度较高。本实施例采用anosim组间差异显著性分析,图29表明G组与D组之间有统计学差异(P<0.05)。
在物种差异分析中,使用ASV/OTU丰度表制作韦恩图,根据其在各组间的有无情况分别统计各个集合的成员数,也就是各组独有的,以及组间共有的ASV/OTU的个数(不是丰度值)。当组数小于等于5时,绘制韦恩图;当组数大于等于5时,绘制花瓣图(图30)。为进一步探究花瓣图中各个区域的物种丰度组成,统计每个区域相应ASV/OTU的丰度,并使用柱状图进行展示在门和属水平上的物种丰度差异。在门水平上,各组中最丰富的是厚壁菌门和拟杆菌门。厚壁菌门丰度经干预后丰度上升,在组间排序为M>G>R>A>N>D;拟杆菌门经干预后丰度下降,在其组间排序为G<M<R<A<N<D。拟杆菌门和厚壁菌门两者做比后顺序为G<M<R<A<N<D。其余菌门,变形菌门,放线菌门,疣微菌门,候选门辐射均在干预后丰度下降,如图31的左图所示。在属水平,各组最丰富的菌属为颤螺菌属,乳杆菌属,副拟杆菌属,阿洛巴氏菌属,瘤胃球菌属,拟杆菌属,另枝菌属。其中颤螺菌属丰度在组间的排序为M>G>A>N>R>D,瘤胃球菌属丰度在组间排序为G<A<N<R<D<M,如图31的右图所示。对照属水平的物种组成热图可以看到,G组中阿克曼氏菌丰度的增加尤为突出,韦荣氏球菌属丰度的降低最为明显;R组中副拟杆菌属丰度的增加尤为突出,梭菌属丰度的降低最为明显;与此同时,D组中显著增加的孪生球菌属,志贺氏菌属,螺杆菌属在其余各组中均有不同程度的下降,其中节肢/关节念珠菌在N组也有一定程度富集,但在G组,R组和A组有了一定的减少;M组显著降低的异杆菌属在R组,G组有着不同程度的增加,如图32所示。可得知,在菌群方面有益菌丰度增加、有害菌丰度降低、微生物多样性增加、肠道菌群更加丰富健康、代谢方面:抑制炎症、促进营养代谢的小分子表达增加、促进炎症降低代谢的小分子表达减少。
造模期和干预期结束后分别进行2次以下的神经行为学评价。
(1)测试焦虑、抑郁状态的自发活动实验:运用自发行为视屏分析系统(购自北京智鼠多宝生物科技有限公司,型号:DB018),设置好电脑参数,共2个观察室,2只小鼠一批,一批测试时长为5min,各组随机选取6只小鼠依次放至观察室内进行测试,对休息次数、休息时长、平均速度、活跃度等数据进行统计。
测定小鼠抗疲劳的转棒疲劳仪:运用转棒疲劳仪(购自北京智鼠多宝生物科技有限公司,型号:DB094),打开电源设置参数,动物数:6只,运动时长:5min,最大转速:40c/min,加速时间:20s,运动方式:正转,各组随机选取6只小鼠放至转棒上后点击“开始”记录小鼠在棒时间(s)及掉落转速(c/min)。
造模后小鼠在自发行为实验中地表现呈现为休息次数变短、持续时间较长,运动次数下降、持续时间较短,整个过程平均速度和活跃度的下降。干预后各组在自发行为实验中各项指标的排序及比较如下:休息次数:N>R>A>G>M>D;休息时长:D>M>A>R>N>G;运动次数:N>R>A>G>M>D;运动时长:G>N>R>A>M>D,R组和M组有统计学差异(*P<0.05);平均速度:N>G>A>R>D>M,R组和M组有统计学差异(*P<0.05);活跃度:N>G>R>M>A>D,如图33~图38所示。
造模后小鼠在经历抗疲劳实验后呈现在棒时间明显下降,转棒速度增加,干预后各组的在棒时间排序为:G>N>A>R>M>D,且D组和R组之间呈显著性差异(P<0.01),如图39所示;干预后各组的转棒速度排序为:G<N<A<R<D<M,对比D组,G组,R组有统计学差异(*P<0.05),A组呈显著性差异(*P<0.01),如图40所示。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,包括以下重量份的组分:碳水化合物66~68份、蛋白质12~14份、脂肪18~22份;
所述全营养配方食品中还包括微量元素和植物乳杆菌GL-5。
2.根据权利要求1所述的一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,所述碳水化合物为葡萄糖。
3.根据权利要求2所述的一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,所述蛋白质为酪蛋白、乳清蛋白和大豆分离蛋白;
所述酪蛋白、乳清蛋白和大豆分离蛋白的质量比为0.5~1.5:0.5~1.5:0.5~1.5。
4.根据权利要求3所述的一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,所述脂肪为食用甘油、亚油酸粉剂、二十碳五烯酸和中链甘油三酯粉剂;
所述食用甘油、亚油酸粉剂、二十碳五烯酸和中链甘油三酯粉剂的质量比为1.8~2.2:0.8~1.2:0.8~1.2:3.8~4.2。
5.根据权利要求4所述的一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,所述微量元素为包括维生素A、维生素D、维生素E、维生素K1、维生素B1、维生素B2、维生素B6、维生素B12、烟酰胺、叶酸、泛酸、维生素C、生物素、钠、钾、铜、镁、铁、锌、锰、钙、磷、碘、氯、硒;
每100g全营养配方食品中,含有视黄醇当量566~570μg的维生素A、7~9μg的维生素D、α-生育酚当量3~4mg的维生素E、17~20μg的维生素K1、0.2~0.4mg的维生素B1、0.2~0.4mg的维生素B2、0.2~0.4mg的维生素B6、0.4~0.6μg的维生素B12、0.7~0.9mg的烟酰胺、94~96μg的叶酸、0.9~1.1mg的泛酸、23~25mg的维生素C、8~10μg的生物素、355~359mg的钠、476~480mg的钾、1170~1172μg的铜、77~79mg的镁、6~7mg的铁、4~6mg的锌、1368~1370μg的锰、240~242mg的钙、171~173mg的磷、27~29μg的碘、468~470mg的氯、54~56μg的硒。
6.根据权利要求5所述的一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,所述植物乳杆菌GL-5的菌浓度为2800~3200亿CFU/g;
每100g全营养配方食品中,所述植物乳杆菌GL-5的用量为15~17mg。
7.根据权利要求1~6任一项所述的一种添加植物乳杆菌GL-5的炎症性肠病全营养配方食品,其特征在于,所述全营养配方食品中还包括铬、钼、氟、胆碱、肌醇、牛磺酸、左旋肉碱、核苷酸和膳食纤维;
每100g全营养配方食品中,含有铬122~124μg、钼118~120μg、氟0.3~0.5mg、胆碱405~407mg、肌醇309~311mg、牛磺酸42~44mg、左旋肉碱4~6mg、核苷酸7~9mg和膳食纤维5~7g。
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