CN115518080A - 乳双歧杆菌BLa80在改善和缓解过敏反应上的应用 - Google Patents
乳双歧杆菌BLa80在改善和缓解过敏反应上的应用 Download PDFInfo
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- CN115518080A CN115518080A CN202211285560.7A CN202211285560A CN115518080A CN 115518080 A CN115518080 A CN 115518080A CN 202211285560 A CN202211285560 A CN 202211285560A CN 115518080 A CN115518080 A CN 115518080A
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- bla80
- bifidobacterium lactis
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Abstract
本发明属于微生物技术领域，涉及乳双歧杆菌BLa80在改善和缓解过敏反应上的应用。本发明首先提供乳双歧杆菌BLa80在制备改善和缓解过敏反应的药物上的应用。本发明的实验证明，BLa80能够极显著降低小鼠搔抓行为及背部皮损炎症评分，显著降低行为观察评分及耳朵肿胀程度。乳双歧杆菌BLa80极显著降低模型组血清中IgE水平，显著提高IFN‑γ水平，显著降低IL‑4水平，乳双歧杆菌BLa80可极显著提高IFN‑γ/IL‑4比值，表明乳双歧杆菌BLa80能够调节Th1/Th2的平衡。本发明还提供了乳双歧杆菌BLa80在制备改善和缓解过敏的保健食品和化妆品中的应用。
Description
技术领域
本发明属于微生物技术领域，尤其是涉及乳双歧杆菌BLa80在改善和缓解过敏反应上的应用。
背景技术
过敏又称特应性反应或变态反应，是指对无害且普遍存在的环境过敏原产生特异性的异常倾向，可能发生在肠道、皮肤和呼吸道，主要有腹痛腹泻、特应性皮炎、变应性鼻炎、哮喘等临床表现。其中特应性皮炎(Atopic Dermatitis，AD)又称“异位性皮炎”“遗传过敏性皮炎”“异位性湿疹”“特应性湿疹或皮炎综合征(atopiceczema dermatitissyndrome，AEDS)”等，是一种与遗传过敏性体质有关的慢性、复发性、炎症性皮肤病。近年来认为AD是一种双相性炎症皮肤疾病，Thl、Th2型细胞因子的交互关系与AD病情的变化密切相关，免疫炎症反应表现为Thl/Th2细胞亚群动态失衡。Th2细胞分泌过多白介素-4(IL-4)等细胞因子，刺激B细胞生成过量IgE抗体。一般认为IgE水平或含量在AD发病中起多重重要作用，由于AD患者体内Th2细胞的高反应性，刺激机体免疫细胞分泌了大量的IgE，通过识别并结合外界某些过敏原，从而触发后续过敏反应，引起了一系列的系统免疫调节异常。
皮肤屏障是人体的第一道防线，当皮肤屏障功能受损，外界的刺激物更容易经表皮进入，使机体体内的抗原呈递细胞与过敏原相结合，刺激免疫细胞释放相关介质组胺和P物质等，引起患者剧烈瘙痒，而瘙痒又诱发患者出现搔抓，进一步破坏了皮肤屏障的完整性，由此形成“受损-搔抓-受损”恶性循环。
本发明所述乳双歧杆菌BLa80能够有效的缓解和改善特应性皮炎的过敏反应，提高特应性皮炎患者的生活质量。
发明内容
基于本领域的上述需求，本发明提供乳双歧杆菌BLa80菌株在改善和缓解过敏反应方面的应用。
本发明首先提供乳双歧杆菌BLa80在制备改善和缓解过敏反应的药物上的应用。所述药物包括但不限于微生态制剂、包括但不限于外用药物。
本发明涉及的乳双歧杆菌BLa80，其分类命名为动物双歧杆菌乳亚种(Bifidobacterium animalis subsp.lactis)，该菌株于2021年05月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.22547。该菌株首先公开于中国专利申请CN 113564078 A《一株降胆固醇的乳双歧杆菌BLa80及其应用》，其公开日为2021.10.29，申请日为2021.07.29。
本发明的体外实验证明，本发明所述的乳双歧杆菌BLa80，具有较好胃肠道耐受能力，对大肠杆菌、沙门氏菌和金黄色葡萄球菌具有较强的抑制作用。
所述的乳双歧杆菌BLa80能够极显著降低小鼠搔抓行为及背部皮损炎症评分，显著降低行为观察评分及耳朵肿胀程度。
乳双歧杆菌BLa80极显著降低模型组血清中IgE水平，显著提高IFN-γ水平，显著降低IL-4水平，乳双歧杆菌BLa80可极显著提高IFN-γ/IL-4比值，表明乳双歧杆菌BLa80能够调节Th1/Th2的平衡。乳双歧杆菌BLa80可有效改善和缓解特应性皮炎小鼠的过敏反应。
乳双歧杆菌BLa80能够有效改善和缓解特应性皮炎患者的皮肤状况等过敏反应。乳双歧杆菌BLa80能够改善和提高特应性皮炎患者的生活质量。
本发明还提供了乳双歧杆菌BLa80在制备改善和缓解过敏的保健食品中的应用。
所述保健食品，包括压片糖果、发酵饮料、软糖、调制乳粉、发酵乳、或固体饮料。
另外的，本发明还提供了乳双歧杆菌BLa80在制备化妆品中的应用。化妆品在使用过程中，不可避免的接触和刺激到皮肤，BLa80可以在化妆品中起到改善和缓解过敏的作用。
所述化妆品，包括洗剂、乳液、膏霜及喷雾剂。
本发明所采用的乳双歧杆菌BLa80来源于母乳，安全无致病性，在国家公认的可用于普通食品的名单中。通过动物试验、人体试验证明其具有改善和缓解过敏功效。将其广泛应用到食品领域、日用品领域，增大消费者消费的可能性，通过日常摄入就可以达到改善和缓解过敏的目的。当然，所采用的乳双歧杆菌BLa80也可以用于制备治疗和缓解过敏的药物中。
具体实施方式
以下由特定的具体实验例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。以下实验例用于说明本发明，但不用来限制本发明的范围。
实施例1乳双歧杆菌BLa80通过胃肠道的环境评价
模拟人工胃液：配制0.5％NaCl溶液，加入0.3％胃蛋白酶，用1mol/LHCL调节pH值至2.5，然后充分溶解后用0.22μm微孔滤膜过滤除菌备用。
模拟人工肠液：配制0.5％NaCl溶液，加入0.1％胰蛋白酶，用0.1mol/LNaOH调节pH值至8.0，然后充分溶解后用0.22μm微孔滤膜过滤除菌备用。
乳双歧杆菌BLa80在厌氧条件下活化培养2代。将活化后的乳双歧杆菌BLa80菌液离心，收集菌体。取0.4mL菌体悬液分别接入1.6mL配制好的pH2.5的模拟人工胃液和pH8.0的模拟人工肠液中混匀，37℃条件下消化，同时分别取0h和3h的消化液检测活菌数，计算存活率，结果见下表。其中，菌株存活率(％)＝Nt/N0×100％，式中N0表示菌株0h的活菌数(CFU/mL)，Nt表示菌株3h的活菌数(CFU/mL)。
表1乳双歧杆菌BLa80模拟消化道环境实验数据表
乳双歧杆菌Bla80在pH2.5的人工胃液中3h存活率为96.7％，在pH8.0的人工肠液中3h存活率为93.6％。实验说明，该乳双歧杆菌BLa80具备较强的耐受胃肠道环境的能力(见表1)。
实验例2、乳双歧杆菌BLa80的抑菌活性评价
取拮抗菌株按2％(V/V)接种至含有0.1％的L-半胱氨酸盐酸盐MRS液体培养基的厌氧玻璃管中，37℃恒温静置培养12h。将致病菌株大肠杆菌、沙门氏菌、金黄色葡萄球菌分别接种于液体牛肉膏蛋白胨培养基中，37℃、转数250rpm、恒温摇床过夜培养，然后制备病原菌悬液。将MRS固体培养基冷却至55℃左右，与病原菌悬液按一定比例混匀，使体系病原菌活菌数在106CFU/mL数量级，然后迅速倾注于预先放置牛津杯的平板中，待培养基冷却凝固后，取出牛津杯，于每孔注入200μL拮抗菌株菌液，将平皿轻盖后正置于37℃恒温培养箱，培养适宜时间后观察，并用游标卡尺测量抑菌圈直径。
表2乳双歧杆菌BLa80对病原菌的抑制作用
如表2所示，乳双歧杆菌BLa80对大肠杆菌、伤寒沙门氏菌和金黄色葡萄球菌拮抗直径分别是42mm、41mm、27mm。表现出对肠道致病菌极强的抑制作用，尤其对大肠杆菌和沙门氏菌的抑制能力。
实验例3、乳双歧杆菌BLa80对HT-29细胞的粘附能力
菌种活化：乳双歧杆菌BLa80按2％接种量接种于含有0.1％的L-半胱氨酸盐酸盐MRS液体培养基的厌氧玻璃管中，37℃培养14h，取发酵液离心、收集菌体，用PBS洗涤3次后，将菌体重悬于不含双抗的DMEM培养液中，调节菌悬液的浓度为108CFU/mL。
HT-29细胞培养：在细胞培养瓶中，加入5mL DMEM完全培养液(含10％胎牛血清，1％青霉素和链霉素溶液)，于5％CO2恒温培养箱中37℃孵育，每天换1次培养液，待细胞状态良好后(70％～80％融合)，用0.2％消化液(胰酶-EDTA)进行消化传代。
黏附性试验：将消化的HT-29细胞调整浓度到105cell/mL，接种到12孔细胞培养板中，每孔1mL，于5％CO2浓度的培养箱中孵育至细胞长至单层，用无菌PBS洗涤两次，其中1孔用胰酶消化，用血球计数板进行细胞计数；分别2孔加入BLa80菌悬液和对照菌鼠李糖乳杆菌LGG菌悬液各1mL，于5％CO2培养箱中37℃孵育2h后，用无菌PBS洗涤细胞5次，除去未黏附的菌悬液，每孔加入0.2mL胰酶-EDTA缓冲液消化细胞5min，消化完后加入0.8mL PBS吹打均匀，取菌液进行稀释活菌计数。上述细胞黏附性实验以鼠李糖乳杆菌LGG作为对比菌株，每个实验做3个平行，鼠李糖乳杆菌LGG是行业内公认的黏附性较好的商业菌株，通过黏附实验对菌株对HT-29细胞的黏附性进行检测，结果如表3。
表3乳双歧杆菌BLa80黏附特性测试结果
由表3结果可知，本发明的乳双歧杆菌BLa80菌株黏附性能有明显优势。
实验例4、乳双歧杆菌BLa80缓解和改善小鼠特应性皮炎
实验动物及分组：C57BL/6雌性小鼠40只，SPF级，6-7周龄，保持昼夜节律各12h。自由进食和饮水，饲养环境温度18-24℃，相对湿度50％-70％。随机分为4组，分别为空白组(Blank)、模型组(Model)、乳双歧杆菌BB12组、乳双歧杆菌BLa80组。小鼠适应一周后，第二周开始灌胃至实验结束。乳双歧杆菌BB12组、乳双歧杆菌BLa80组分别灌胃含相应菌株(1×109亿CFU/mL)的PBS溶液0.2mL，空白组、模型组灌胃等量的PBS溶液。从第三周开始造模，造模前一天在小鼠背部去毛，大约为2.5cm×1.5cm。实验当日除空白组，其他三组小鼠背部外涂0.5％DNFB(2,4-二硝基氟苯)溶液25μL致敏，第5天、第8天、第14天在小鼠右耳背面、背部外涂0.2％的DNFB溶液20μL诱发皮炎，左耳外涂20μL丙酮/橄榄油基质溶液作为对照。
小鼠搔抓行为评价:实验结束前后，将小鼠单独放置，观察10min内小鼠前爪搔抓耳部、头部，后爪搔抓躯干、背部，嘴啃咬全身各部位的次数，连续搔抓算作1次。
小鼠行为观察评价：实验结束前后，参照以下评分标准对小鼠行为进行评价。
表4小鼠行为观察评价标准
小鼠背部皮损评价：参照临床表现标准评价背部皮损炎症；红斑、水肿/丘疹、表皮剥脱/抓痕、鳞屑(干燥)，分为无、轻、中、重四个等级，分别记为0、1、2、3分。其中无症状均记为0分；红斑：隐约可见，呈粉红色(1分)；清晰可见，呈暗红色(2分)；深红色或火红色(3分)。水肿/丘疹；几乎察觉不到离出皮面(1分)；清晰可见高出皮面但不显著(2分)；显著离出皮面(3分)。表皮剥脱/抓痕：少许较浅的表皮剥脱(1分)；许多表浅的和/或一些较深的表皮剥脱(2分)；弥漫性表浅的和/或许多深在性的表皮剥脱(3分)。鳞屑(干燥)：糠状脱屑(1分)；片状鳞屑稀薄覆盖(2分)；厚鳞屑覆盖(3分)
小鼠耳朵肿胀程度评价：造模第0天和造模结束后，分别用游标卡尺测量小鼠双侧耳朵中部的厚度。
血清细胞因子检测：小鼠处死前取血清，采用ELISA法检测血清中IgE、IL-4、IFN-γ、IL-10含量。
造模后小鼠行为观察评分显著低于对照组小鼠，搔抓行为评分、背部皮损炎症评分以及耳朵肿胀均与对照组呈差异极显著，表明采用DNFB诱导的特应性皮炎小鼠模型构建成功。
乳双歧杆菌BLa80能够减少小鼠搔抓次数，且效果极显著，并明显优于BB12组。
乳双歧杆菌BLa80显著提高特应性皮炎小鼠的行为观察评分，并使其基本恢复至对照组评分，且优于BB12组。
乳双歧杆菌BLa80改善小鼠背部皮损炎症，并且与模型组比较具有极显著差异，效果优于BB12组。
乳双歧杆菌BLa80显著改善小鼠耳朵肿胀，与模型组具有显著性差异，改善效果优于BB12组。
表5乳双歧杆菌缓解和改善特应性皮炎小鼠的过敏反应
注：与对照组比较：#p＜0.05，##p＜0.01；与模型组比较：*p＜0.05，**p＜0.01。
由表5可知，乳双歧杆菌BLa80能够极显著降低小鼠搔抓行为及背部皮损炎症评分，显著降低行为观察评分及耳朵肿胀程度，对照菌株乳双歧杆菌BB12可显著降低小鼠搔抓行为、行为观察评分和背部皮损炎症评分。乳双歧杆菌BLa80的缓解和改善特应性皮炎小鼠的过敏反应效果优于乳双歧杆菌BB12。
表6乳双歧杆菌BLa80对特应性皮炎小鼠的细胞因子表达水平影响
注：与对照组比较：#p＜0.05，##p＜0.01；与模型组比较：*p＜0.05，**p＜0.01。
由表1可知，乳双歧杆菌BLa80极显著降低模型组IgE水平，显著提高IFN-γ水平，显著降低IL-4水平，乳双歧杆菌BLa80可极显著提高IFN-γ/IL-4比值。IFN-γ和IL-4分别做为Th1和Th2细胞分泌的特征性细胞因子，两者之间的平衡即比值间接反应了促炎和抗炎的平衡情况。BLa80组的IFN-γ/IL-4比值提升至与对照组无显著性差异的水平。可见乳双歧杆菌BLa80可有效改善和缓解特应性皮炎小鼠的过敏反应。
实验例5、乳双歧杆菌BLa80菌粉制备
1)乳双歧杆菌BLa80菌粉发酵工艺：
将保存于甘油管中的乳双歧杆菌菌株接入10mL的L-MRS培养基中，37℃静置厌氧培养18h即为一级种子；将一级种子菌液以2％(V/V)的接种量再接种于L-MRS培养基中，在37℃、厌氧条件下培养12h，得到二级种子，再以2％的接种量接入L-MRS培养基中，于37℃、厌氧条件下培养12h后作为三级种子。
发酵培养基配方为：乳糖20/L、牛肉浸粉10/L、酵母浸粉10/L、牛骨蛋白胨10g/L、磷酸氢二钾3g/L、柠檬酸氢铵3g/L、乙酸钠5g/L、硫酸镁0.5g/L、硫酸锰0.1g/L、吐温-801mL/L、L-半胱氨酸盐酸盐1g/L。
发酵：配制10L发酵培养基加到15L厌氧发酵罐中，121℃灭菌20分钟，降温后用NaOH调发酵培养基pH至7。将乳双歧杆菌三级种子液200mL接入到发酵培养基中，在温度37℃、转速100rpm的条件下发酵。发酵过程中自动流加25％氨水以维持发酵液pH值为5.5-6.0，发酵全程通入氮气。发酵16h时，当发酵液的OD600不再增加时，终止发酵。
2)所述的乳双歧杆菌BLa80菌粉制备工艺：
(1)将通过上述乳双歧杆菌高密度发酵的方法得到的发酵液离心，去上清，收集菌泥。所述的离心的方法优选为4℃、8000rpm离心10min。
(2)将离心收集的菌泥与冻干保护剂按照质量比1:1混合均匀，置于冷冻干燥机内，-40℃预冻2h，再于10Pa条件下4h升温至-10℃冷冻干燥15h，后于20Pa条件下2h升温至25℃冷冻干燥10h，即可得到乳双歧杆菌冻干菌粉。
所述的冻干保护剂包含如下按质量百分比计的组分：海藻糖6％、麦芽糊精8％、蔗糖2％、维生素C 0.02％、甘油3％、L-精氨酸0.5-0.8％，余量为水。
实施例6、乳双歧杆菌BLa80改善和缓解特应性皮炎的人体试食试验
本次人体试食试验共招募124人，被随机分为2组：治疗组(70人)每天服用1条益生菌乳双歧杆菌BLa80产品(含1×109CFU/条)，安慰剂组(46人)服用等量麦芽糊精。服用方法：可直接服用或温水(不超过37℃)冲服服用。干预时间为4周，实验结束后2周继续随访特应性皮炎患者，观察是否有不良反应产生。
纳入标准：①瘙痒；②典型的形态和部位(屈侧皮炎)或不典型的形态和部位同时伴发干皮症；③慢性或慢性复发性病程。同时具备以上3条即可诊断AD。典型的形态和部位(屈侧皮炎)包括面部和肢端受累。非典型的形态和部位包括:①典型的湿疹样皮疹，发生在非屈侧部位(头皮皮炎、眼睑湿疹、乳头湿疹、外阴湿疹、钱币状湿疹、指尖湿疹、非特异性手部或足部皮炎/特应性冬季足、甲或甲周湿疹和身体其他部位的湿疹样皮疹)；②非典型湿疹样皮疹，单纯糠疹、唇炎、耳下和耳后/鼻下裂隙、痒疹、汗疱疹、丘疹性苔藓样变异。
排除标准：①伴心、肾、脑等重要器官严重疾病以及入选前7天内有应用抗组胺类药品患者。②长期食用酸奶、益生菌饮料的患者。
告知志愿者试验性质和内容，签订知情同意书。试食4周后，统计原始调查问卷数据，失访者8人。试食前后，使用特应性皮炎的标准化严重性指数(SCORAD指数)及皮肤病生活质量指数(DLQI)量表对志愿者的情况进行调查、记录。
表7试食前后两组SCORAD评分
注：试食后两组比较：#p＜0.05，##p＜0.01；试食后与试食前比较：*p＜0.05，**p＜0.01。
试食前，BLa80组与安慰剂组SCORAD评分无显著性差异，服用乳双歧杆菌BLa80后SCORAD评分与试食前相比较，极显著降低，同时，试食后，BLa80组SCORAD评分与安慰剂组差异极显著。表明乳双歧杆菌BLa80能够有效改善和缓解特应性皮炎患者的皮肤状况等过敏反应。
表8试食前后两组DLQI评分
注：试食后两组比较：#p＜0.05，##p＜0.01；试食后与试食前比较：*p＜0.05，**p＜0.01。
试食前，BLa80组与安慰剂组DLQI评分无显著性差异，服用乳双歧杆菌BLa80后DLQI评分与试食前比极显著降低，同时，试食后，BLa80组DLQI评分与安慰剂组差异极显著。表明乳双歧杆菌BLa80能够改善和提高特应性皮炎患者的生活质量。
Claims (7)
1.乳双歧杆菌BLa80在制备改善和缓解过敏反应的药物上的应用，所述乳双歧杆菌BLa80，于2021年05月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.22547。
2.根据权利要求1所述的应用，其特征在于，所述药物包括但不限于微生态制剂、外用药物。
3.根据权利要求1或2所述的应用，其特征在于，所述改善和缓解过敏反应的症状包括：
(1)降低搔抓行为及背部皮损炎症评分，显著降低行为观察评分及耳朵肿胀程度；
(2)降低血清中IgE水平，提高IFN-γ水平，降低IL-4水平。
4.乳双歧杆菌BLa80在制备改善和缓解过敏的保健食品中的应用，所述乳双歧杆菌BLa80，于2021年05月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.22547。
5.根据权利要求4所述的应用，其特征在于，所述保健食品，包括压片糖果、发酵饮料、软糖、调制乳粉、发酵乳、或固体饮料。
6.乳双歧杆菌BLa80在制备化妆品中的应用。
7.根据权利要求6所述的应用，其特征在于，所述化妆品，包括洗剂、乳液、膏霜或喷雾剂。
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