CN113068837B - 一株可清除壬基酚并缓解其导致的中毒症状的长双歧杆菌 - Google Patents
一株可清除壬基酚并缓解其导致的中毒症状的长双歧杆菌 Download PDFInfo
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- CN113068837B CN113068837B CN202110367862.8A CN202110367862A CN113068837B CN 113068837 B CN113068837 B CN 113068837B CN 202110367862 A CN202110367862 A CN 202110367862A CN 113068837 B CN113068837 B CN 113068837B
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- bifidobacterium longum
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Abstract
本发明公开了一株可清除壬基酚并缓解其导致的中毒症状的长双歧杆菌,属于微生物技术领域以及医药技术领域。本发明首次发现并研究了长双歧杆菌长亚种CCFM1077在清除壬基酚及缓解壬基酚中毒症状中的应用,具体体现在:将此长双歧杆菌长亚种CCFM1077添加至壬基酚溶液中培养6h,即可使壬基酚溶液中壬基酚的清除率达60.56%。并基于其优良的壬基酚清除能力开展后续大鼠动物实验,长双歧杆菌长亚种CCFM1077对壬基酚造成的大鼠肠道损伤表现出明显的保护作用。可见,长双歧杆菌长亚种CCFM1077在制备壬基酚清除剂和制备预防和/或治疗壬基酚暴露的产品中具有巨大的应用前景。
Description
技术领域
本发明涉及一株可清除壬基酚并缓解其导致的中毒症状的长双歧杆菌,属于微生物技术领域以及医药技术领域。
背景技术
壬基酚(Nonyl Phenol,简称NP)是一种有机化合物,分子式为C15H24O,是重要的精细化工原料和中间体。壬基酚外观在常温下为无色或淡黄色液体,略带苯酚气味,不溶于水,溶于丙酮。主要用于生产表面活性剂、也用于抗氧剂、纺织印染助剂、润滑油添加剂、农药乳化剂、树脂改性剂、树脂及橡胶稳定剂等领域。壬基酚属有机污染物,有“精子杀手”之称。
壬基酚NP是雌激素类典型的内分泌干扰物,具有一定的致癌性,可对生物的存活、发育、生长造成重要影响,并随着食物链由低向高传递从而对人体产生一定的内分泌干扰作用(参见戴媛媛,牛海凤,董玉波,等.壬基酚对水生生物的毒性研究进展[J].环境与健康杂志,2012,29(10):948-951)。
目前报道的动物实验中,可以明显的获知壬基酚NP对动物造成严重的伤害;例如,单伟等研究表明NP可损伤大鼠胰腺胰岛β细胞,可刺激胰岛α细胞增生,并且血糖浓度明显增高,同时,NP可造成胰腺内分泌功能减低(参见单伟,秦书俭,李德华.壬基酚对大鼠胰腺内分泌功能的影响[J].毒理学杂志,2010,24(5):405-408.);吴伟等研究表明NP对鱼类具有一定的雌激素活性,且NP的生物累积性和雌激素活性在产生时间和活性数值方面均是同步和呈正相关的(参见吴伟,瞿建宏.壬基酚在鱼体组织中的累积及对鱼类性腺的影响[J].中国环境科学,2005,25(4):420-423.);NP对小鼠以及鲫鱼都具有潜在的免疫毒性;有研究表明,小鼠用1‰的NP喂食30d后,其腹腔巨噬细胞内酸性磷酸酶的活力下降,吞噬功能减弱。由于酸性磷酸酶是巨噬细胞激活的可靠指标之一,由此推断NP可以抑制小鼠腹腔巨噬细胞的激活,进而抑制其免疫系统功能(参见马全祥,范雪晖,毛泽善.壬基酚对小鼠免疫功能的影响[J].中国公共卫生,2004,20(2):201-202.)。壬基酚可通过皮肤、黏膜、呼吸道、胃肠道等进入人体。由于壬基酚具有神经毒性、免疫毒性、生殖毒性和致癌性,这些被吸收的壬基酚通过血液循环系统广泛分布于人体各个组织时,壬基酚因其脂溶性,易在脂肪含量比较高的组织中积累,会对人体造成严重的损害,因此,为保持健康,人群需避免过量的摄入壬基酚。
但是,在生活中,壬基酚在环境中是广泛存在的,壬基酚扩散在河流、土壤中后经由食物链最终在人类组织中富集。可见,人们在日常生活中几乎无法避免的会暴露于存在壬基酚的环境中。人类对于壬基酚的摄入主要分为体内摄入和体外摄入,一方面是人体存在于含有壬基酚的空气中,过多的通过呼吸道、皮肤、黏膜摄入到体内,另一方面是食用了壬基酚含量超标的食品导致壬基酚中毒。
在2007年中国总饮食研究(TDS)的食物样品中测量了双酚A(BPA)和壬基酚(NP)的浓度。其中,144个样品中的143个均检测到了NP,其浓度为30ng/kg至1268μg/kg。果蔬及其制品、肉类、油类等中均含有壬基酚,其中牛奶污染最为严重。在中国,0至6岁儿童的NP摄入范围为0.3至17μg/kg体重(bw)/天,并且0至1岁婴儿的平均每日摄入量已超过欧洲食品安全局(EFSA)设定的每日耐受摄入量5μg/kg体重(bw)/天。而成年人的NP平均摄入量为520ng/kg(bw/day)。
目前对壬基酚的清除技术中的生物降解手段采用的液全是非食用菌,不可用于食品。例如,公开号为CN110819353B的中国发明专利当中,公开了一种布氏白僵菌SB010在壬基苯酚生物降解中的应用,在4-n-NP初始浓度为25、50和100mg/L下,培养168h后降解率分别达到100%,98.2%和93.2%,虽然降解效率很高,但是其为非食用菌株,并不能用于食品领域当中去除壬基苯酚。公开号为112522131A的中国发明专利共公开了一种根瘤菌,NPLJ-2在好氧条件下对水体中初始浓度为100mg/L的壬基酚的生物降解率可达到80%以上。但这些非食用菌并不能用于被壬基酚污染的食品,且也无法清除已经被摄入人或动物体内的壬基酚,也就无法对壬基酚造成的毒性起到保护作用。
而针对可辅助治疗壬基酚中毒的药物研究少之又少。一些研究发现褪黑素、组氨酸、桑葚提取液等抗氧化物质可通过抗氧化的途径缓解神经毒性或者肝损伤,但首先这些保护手段都是发生在已经壬基酚中毒之后,且并不能同时保护整个机体,是治标不治本的方法。
因此,急需找到一种可有效减除环境尤其是食品中壬基酚的产品,在壬基酚被肠道吸收前就清除掉壬基酚,以此来减缓人群过量摄入壬基酚带来的伤害。
发明内容
[技术问题]
本发明要解决的技术问题是:提供一种可有效清除环境中,特别是食品环境中的壬基酚含量的方法,并且提供一种能够有效缓解壬基酚中毒症状的产品及其应用。
[技术方案]
为解决上述问题,本发明提供了长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum)CCFM1077在降解环境中壬基酚方面的应用,所述环境为非生物体内的环境。
在本发明的一种实施方式中,所述长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum)CCFM1077,其于2019年09月05日保藏于广东省微生物菌种保藏中心,地址为,广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,保藏编号为GDMCCNo.60769。所述长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1077记载于公开号为CN111073828A的中国发明专利申请文本中。
在本发明的一种实施方式中,所述体外环境包括食品加工环境、食品酿造环境、乳制品环境、果蔬制品环境。
本发明还提供了一种可降解环境中壬基酚的产品,所述产品中包含长双歧杆菌长亚种CCFM1077。
在本发明的一种实施方式中,所述产品包含食品、药品或化学品。
本发明还提供了一种可缓解壬基酚中毒症状的产品,所述产品中包含长双歧杆菌长亚种CCFM1077。
在本发明的一种实施方式中,所述产品包含食品、药品或保健品。
本发明还提供了上述长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum)CCFM1077在制备可缓解壬基酚中毒症状的的产品中的应用。
本发明的一种实施方式中,所述长双歧杆菌长亚种CCFM1077在产品中添加的活菌数为不低于1×106CFU/mL或1×106CFU/g。
在本发明的一种实施方式中,所述产品包含食品、药品或壬基酚清除剂。
在本发明的一种实施方式中,所述食品包括发酵果蔬、发酵乳、乳酪、含乳饮料、乳粉或其他含有长双歧杆菌长亚种CCFM1077的食品。
本发明的一种实施方式中,所述食品为保健食品;或所述食品为使用含有上述长双歧杆菌长亚种CCFM1077的发酵剂生产得到的乳制品、豆制品或果蔬制品;或所述食品为含有上述长双歧杆菌长亚种CCFM1077的饮料或零食。
本发明的一种实施方式中,所述发酵剂的制备方法为将上述长双歧杆菌长亚种CCFM1077接种到培养基中在厌氧环境下进行培养,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水或缓冲液清洗后用冻干保护剂重悬,得到重悬液;将重悬液采用真空冷冻法进行冻干,得到发酵剂。
在本发明的一种实施方式中,所述冻干保护剂包含100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
本发明的一种实施方式中,所述药品含有上述长双歧杆菌长亚种CCFM1077、药物载体和/或药用辅料。
本发明的一种实施方式中,所述药物载体包含微囊、微球、纳米粒和/或脂质体。
本发明的一种实施方式中,所述药用辅料包含赋形剂和/或附加剂。
本发明的一种实施方式中,所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂。
本发明的一种实施方式中,所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
本发明的一种实施方式中,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
有益效果
(1)本发明提供了一株长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum)CCFM1077在清除壬基酚中的应用,具体体现在:将此长双歧杆菌长亚种CCFM1077添加至含有壬基酚的溶液中厌氧培养6h,即可使壬基酚溶液中壬基酚的清除率达60.56%,可见,长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1077在制备壬基酚清除剂中具有巨大的应用前景。
(2)采用本发明提供的方法,基于长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum)CCFM1077优良的壬基酚清除能力开展大鼠动物实验,长双歧杆菌长亚种CCFM1077对壬基酚造成的大鼠肠道损伤表现出明显的保护作用,因此,长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1077在制备预防和/或治疗壬基酚暴露的产品(如食品或药品等)中具有巨大的应用前景。
附图说明
图1:不同长双歧杆菌清除水溶液中壬基酚的清除率。
图2:长双歧杆菌长亚种CCFM1077清除发酵果汁中壬基酚的清除率。
图3:长双歧杆菌长亚种CCFM1077对壬基酚中毒大鼠结肠IL-1β表达的保护效果。
图4:长双歧杆菌长亚种CCFM1077对壬基酚中毒大鼠结肠TNF-α表达的保护效果。
图5:长双歧杆菌长亚种CCFM1077对壬基酚中毒大鼠粪便丁酸含量的影响。
具体实施方式
下述实施例中涉及的脱脂奶粉购自纽瑞兹食品有限公司;下述实施例中涉及的壬基酚标准品购自德国Dr.Ehrenstorfer公司;
下述实施例中所涉及的短双歧杆菌FSHMX3M8、短双歧杆菌FBJHD5M2、短双歧杆菌FBJCP1M6、短双歧杆菌FHuN-CS-6-M1、短双歧杆菌GuXi-2016 6-7、短双歧杆菌CJ-6-5-3、短双歧杆菌FHNFQ49M1、长双歧杆菌FJSNT19M1、长双歧杆菌FSCDJY15M2、长双歧杆菌RG23、长双歧杆菌FHeNJZ28M10、长双歧杆菌FGDLZ19M5、长双歧杆菌FXZDX24M5-1、长双歧杆菌FSCREG6M5-3、长双歧杆菌FJSNT39M10、长双歧杆菌FHuBZX30M2、长双歧杆菌FGDLZ63M1来自于江南大学食品生物技术中心菌种保藏中心。
下述实施例中所涉及的短双歧杆菌CCFM1078记载于公开号为CN112546074A的中国发明专利申请文本中,长双歧杆菌CCFM752记载于公开号为CN111979161A的中国发明专利申请文本中。
下述实施例中涉及的培养基如下:
LFMATA固体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、酵母粉5g/L、吐温1mL/L、磷酸氢二钾3g/L、乙酸钠2g/L、柠檬酸二铵2g/L、七水硫酸镁0.1g/L、一水硫酸锰0.05g/L、碳源20g/L、万古霉素20×10-3g/L、链霉素0.256g/L、庆大霉素6.4×10-2g/L、L-半胱氨酸0.5g/L、琼脂18g/L。
MRS固体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、琼脂20g/L、半胱氨酸氨酸盐0.5g/L,pH为6.8。
MRS液体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L,pH为6.8。
下述实施例中涉及的检测方法如下:
活菌数的检测方法:采用国标《GB 4789.35-2016食品安全国家标准食品微生物学检测乳酸菌检测》。
下述实施例中所涉及的体内相关基因表达量的检测方法:
(1)提取总RNA
结肠、海马体和前额皮质总RNA的提取参照诺维赞公司的RNA提取试剂盒的方法。
(2)反转录
反转录方法参考诺维赞反转录试剂盒的说明书。
(3)q-PCR
设计引物序列与合成,以GAPDH作为内参基因,引物序列见表1:
表1:引物序列
用八连管配置PCR反应体系,具体体系如表2:
表2:反应体系
配好体系后上机进行荧光定量反应,反应条件:50℃,2min;95,2min。40个循环:95℃,15s;60℃,1min。之后设置溶解曲线65℃-95℃,0.5℃/循环。
下述实施例中所涉及的粪便短链脂肪酸的检测方法:
(1)标准溶液配制
标准液1的配制:每种SCFAs(乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸)(在D510黄色铁皮防爆柜里)各取10μL,用无水乙醚补充至1mL;
标准液2的配制:取标准液1的混合物100μL,用无水乙醚补充至1mL;
不同浓度标曲的配制:分别标准液2的混合物200μL、150μL、100μL、75μL、50μL、25μL、15μL、用无水乙醚补充至1mL,上机检测。
(2)粪便短链脂肪酸的提取
称取20-50mg样品放置于2mL EP管中,记录粪便重量;在EP管中加入500μL饱和NaCl溶液,振荡至无明显块状物;均质后,加入40μL硫酸酸化;加入硫酸后,用漩涡振荡仪振荡30s,混匀;加入1000μL的乙醚用以萃取短链脂肪酸,用漩涡振荡仪振荡30s;振荡后以12000rpm,15min,4℃的条件离心;
离心后取上清,将上清液加入到装有0.25g的无水硫酸钠的2mL EP管中静置15min,再次以同样条件离心,离心后将液体加入气相小瓶,上机分析。
(3)短链脂肪酸检测
采用Rtx-Wax柱对各短链脂肪酸进行分离,初始柱温为100℃,以7.5℃/min的升温速率升至140℃,再以60℃/min的速率分升至200℃,在200℃下保持3min。
采用全扫模式(质荷比扫描范围33-110)检测各短链脂肪酸,选取各分析物标准品的特征离子进行定量分析。
下述实施例中涉及的双歧杆菌菌体的制备方法如下:
将双歧杆菌菌液划线于MRS固体培养基上,37℃厌氧条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于MRS液体培养基中,37℃条件下厌氧培养18h,得到菌液;将菌液经6000g离心15min,得到双歧杆菌菌体。
实施例1:长双歧杆菌长亚种CCFM1077对水溶液中壬基酚的清除作用
具体步骤如下:
(1)菌液的制备:
取冻存甘油管里的双歧杆菌在MRS固体培养基上划线,37℃厌氧培养48小时后,用无菌接种环挑取单菌落接入MRS液体培养基中厌氧培养18-24小时进行2代活化,得到活化液;
然后以1%(v/v)的接种量将活化液接入液体培养基活化得到第3代;当双歧杆菌培养至菌浓度为3×109cfu/mL时,在6000g条件下离心15分钟获取菌体,无菌环境中用生理盐水清洗三遍后重悬得到菌液,此时,菌液的浓度为:3×109cfu/mL。
按照上述方法分别制备得到短双歧杆菌CCFM1078菌液、短双歧杆菌FSHMX3M8菌液、短双歧杆菌FBJHD5M2菌液、短双歧杆菌FBJCP1M6菌液、短双歧杆菌FHuN-CS-6-M1菌液、短双歧杆菌GuXi-2016 6-7菌液、短双歧杆菌CJ-6-5-3菌液、短双歧杆菌FHNFQ49M1菌液、长双歧杆菌长亚种CCFM1077菌液、长双歧杆菌CCFM752菌液、长双歧杆菌FSCCD3M1菌液、长双歧杆菌FSCDJY15M2菌液、长双歧杆菌RG23菌液、长双歧杆菌FHeNJZ28M10菌液、长双歧杆菌FGDLZ19M5菌液、长双歧杆菌FXZDX24M5-1菌液、长双歧杆菌FSCREG6M5-3菌液、长双歧杆菌FJSNT39M10菌液、长双歧杆菌FHuBZX30M2菌液、长双歧杆菌FGDLZ63M1菌液。
(2)壬基酚标准品母液的制备:
准确称取10mg的壬基酚标准品预先溶于1mL二甲亚砜中,充分混匀溶解后使用0.22μm微孔滤膜进行过滤除菌,得到浓度为10mg/mL的壬基酚标准品母液;
(3)双歧杆菌CCFM1077对壬基酚的清除
准确称取10mg/mL的壬基酚标准品母液100μL用无菌水定容至100mL,得到浓度为10mg/L的壬基酚标准品母液;
以不添加长双歧杆菌长亚种的浓度为10mg/L的壬基酚标准品母液作为空白对照,分别将上述制备得到的菌液依次分别与2mL浓度为10μg/mL的壬基酚标准品母液混合后于37℃、厌氧的条件下共培养6h,得到培养液1~29;
将培养液1~29 12000rpm离心10min,取上清用0.22μm微孔滤膜进行过滤除菌,得到滤液1~29。
检测滤液1~29中壬基酚的含量,并根据公式:
清除率(%)=[(空白对照中壬基酚的含量-滤液中壬基酚的含量)/空白对照中壬基酚的含量]×100%,计算滤液1~29中壬基酚的清除率,检测结果见表3和图1。
表3:双歧杆菌对壬基酚的减除量
菌株 | 减除量(μg) | 菌株 | 减除量(μg) |
CCFM1078 | 7.16 | FSCCD3M1 | 7.20 |
FSHMX3M8 | 3.10 | FSCDJY15M2 | 8.00 |
FBJHD5M2 | 7.67 | RG23 | 6.06 |
FBJCP1M6 | 7.21 | FHeNJZ28M10 | 5.59 |
FHuN-CS-6-M1 | 2.42 | FGDLZ19M5 | 7.00 |
GuXi-2016 6-7 | 6.36 | FXZDX24M5-1 | 8.75 |
CJ-6-5-3 | 4.42 | FSCREG6M5-3 | 6.03 |
FHNFQ49M1 | 7.54 | FJSNT39M10 | 5.32 |
CCFM1077 | 12.11 | FHuBZX30M2 | 5.67 |
CCFM752 | 7.38 | FGDLZ63M1 | 5.80 |
由表3、图1可知,壬基酚初始量20μg,壬基酚减除量最高达12.11μg。由图1可知,壬基酚的清除率最高(高达60.56%)。可见,长双歧杆菌(Bifidobacterium longum)CCFM1077可高效清除水溶液中的壬基酚。
实施例2:长双歧杆菌长亚种CCFM1077对苹果汁中壬基酚的清除作用
具体步骤如下:
(1)苹果汁的制备
苹果经清洗后,由榨汁机榨汁加工。然后,布过滤器过滤果汁后,5000g下离心,收集上清液于-20℃下储存用于后续实验。
(2)长双歧杆菌长亚种CCFM1077菌悬液的制备
取冻存甘油管里的双歧杆菌在MRS固体培养基上划线,37℃厌氧培养48小时后,用无菌接种环挑取单菌落接入MRS液体培养基中厌氧培养18-24小时进行2代活化,得到活化液;
然后以1%(v/v)的接种量将活化液接入液体培养基活化得到第3代;当双歧杆菌培养至菌浓度为3×109cfu/mL时,在6000g条件下离心15分钟获取菌体,无菌环境中用生理盐水清洗三遍后重悬得到菌液,此时,菌液的浓度为:3×109cfu/mL。
(3)长双歧杆菌长亚种CCFM1077对苹果汁中壬基酚的清除
将步骤(1)得到的苹果汁与一定量的壬基酚标准物质混合(壬基酚终浓度10mg/L),制备得到生苹果汁;
将步骤(2)制备得到的长双歧杆菌长亚种CCFM1077菌悬液以2%(v/v)的接种量接种到50mL生苹果汁中,在37℃下厌氧条件下孵育24h,每6h取一次样。样品经6000g离心30min后,收起上清液,得到发酵果汁;
参照吕岱竹《超声波提取-液相荧光法测定水果中壬基酚聚氧乙烯醚及其降解产物》中的前处理方法对发酵果汁进行前处理后,用高效液相-荧光检测器对壬基酚进行检测。
实验结果如图2所示,长双歧杆菌长亚种CCFM1077对苹果汁中的壬基酚的清除率为68.00%。与壬基酚标准物质混合后得到的生苹果汁中初始壬基酚含量为500μg,而添加长双歧杆菌长亚种CCFM1077菌悬液后,壬基酚在苹果汁中减除量为340μg。
实施例3:长双歧杆菌长亚种(Bifidobacterium longum subsp.longum)CCFM1077能缓解壬基酚对大鼠造成的毒性
具体步骤如下:
自北京维通利华购买雄性幼年Wsitar大鼠(3周)。遵循12h/12h的明暗循环中,将大鼠饲养在适宜温度(22℃±1℃)和湿度(55%±10%)的笼中,自由进食进水。大鼠进驻江南大学动物实验中心后,经过一周的适应期后,将32只4周龄的大鼠随机分成4组,实验周期为42天。
将实验分为:载体对照组(简称对照组),壬基酚处理组(简称模型组),双歧杆菌CCFM1077+壬基酚处理组(简称CCFM1077组),双歧杆菌FHuNCS6M1+壬基酚处理组(简称FHuNCS6M1组);实验分组具体如表4:
表4:不同组大鼠的实验处理
(2)长双歧杆菌长亚种CCFM1077对壬基酚诱导大鼠炎症反应的缓解作用
参照体内相关基因表达量检测中的方法,提取结肠和大脑前额叶质层组织中的mRNA,设计炎症因子相关引物,引物序列如下表5所示,然后进行q-PCR。
表5炎症因子相关引物
结果如图3~4所示,结果显示,经过长双歧杆菌CCFM1077灌胃之后的CCFM1077组中结肠IL-1βmRNA的相对表达量为0.64,相对于模型组(结肠IL-1β的相对表达量为1.45)降低了55.86%,对照组的结肠IL-1β的相对表达量分别为0.97。
经过长双歧杆菌CCFM1077灌胃之后的CCFM1077组中前额叶质层TNF-α的表达量为1.32,相对于模型组(额叶质层TNF-α的表达量为2.41)降低了45.23%,对照组的前额叶质层TNF-α的表达量为1.01。
结果表明,结肠和前额叶质层的炎症反应表达表现出明显的缓解作用。
(3)长双歧杆菌CCFM1077对壬基酚中毒大鼠粪便短链脂肪酸含量的改善
按照粪便短链脂肪酸的检测方法,提取并检测短链脂肪酸的含量。
结果显示,经过长双歧杆菌CCFM1077灌胃之后的CCFM1077组中中毒大鼠粪便丁酸的含量为22.66μmol/g粪便,相对于模型组(中毒大鼠粪便丁酸的含量为16.05μmol/g)增加了41.18%,对照组中毒大鼠粪便丁酸的含量为21.65μmol/g。
如图5所示,长双歧杆菌CCFM1077对壬基酚导致的丁酸含量异常表现出明显的改善作用
实施例4:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备菌粉,菌粉的具体制备过程如下:
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下厌氧培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到长双歧杆菌长亚种CCFM1077菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
实施例5:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备牛乳,发酵乳的具体制备过程如下:
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到长双歧杆菌长亚种CCFM1077菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将脱脂奶在95℃热杀菌20min后冷却至4℃,得到原料;在原料中添加长双歧杆菌长亚种CCFM1077菌粉至浓度为不低于1×106CFU/mL,得到牛乳。
实施例6:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备豆奶,豆奶的具体制备过程如下:
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到长双歧杆菌长亚种CCFM1077菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将大豆在温度80℃下浸泡2h后去除大豆皮,得到去皮大豆;将去皮大豆沥去浸泡水后加沸水磨浆,得到豆浆;将豆浆在高于80℃的温度条件下保温12min,得到熟豆浆;将熟豆浆用150目筛网过滤后离心分离,得到粗豆奶;将粗豆奶加热到温度140~150℃后迅速导入真空冷却室进行抽真空,使得粗豆奶中的异味物质随着水蒸汽迅速排出,得到熟豆奶;将熟豆奶降温至约37℃后在熟豆奶中添加长双歧杆菌长亚种CCFM1077菌粉至浓度为不低于1×106CFU/mL,得到豆奶。
实施例7:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备果蔬饮料,果蔬饮料的具体制备过程如下:
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到长双歧杆菌长亚种CCFM1077菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将新鲜水果和蔬菜洗净后榨汁,得到果蔬汁;将果蔬汁在温度140℃下高温热杀菌2秒,得到杀菌后的果蔬汁;将杀菌后的果蔬汁降温至约37℃后在杀菌后的果蔬汁中添加长双歧杆菌长亚种CCFM1077菌粉至浓度为不低于1×106CFU/mL,得到果蔬饮料。
实施例8:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备胶囊制品,胶囊制品的具体制备过程如下
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;菌悬液添加至浓度为30g/L的海藻酸钠溶液中至浓度为2×109CFU/mL后,充分搅拌,使得长双歧杆菌长亚种CCFM1077的细胞均匀地分散于海藻酸钠溶液中,得到混合液;将混合液挤压到浓度为20g/L的氯化钙溶液中形成胶粒;待形成的胶粒静止固化30min后,过滤收集胶粒;将收集得到的胶粒进行冷冻干燥48h,得到粉剂;将粉剂装入到药用胶囊中,得到胶囊制品;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基。
实施例9:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备发酵乳,发酵乳的具体制备过程如下:
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到长双歧杆菌长亚种CCFM1077菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将长双歧杆菌长亚种CCFM1077菌粉与商业干粉发酵剂保加利亚乳杆菌和商业干粉发酵剂嗜热链球菌按照质量比1:1:1的比例混合,得到发酵剂;将糖添加至鲜奶中至浓度为50g/L,得到混合液;将混合液在65℃、20MPa的条件下进行均质后在95℃下保温杀菌5min,得到发酵原料;将发酵原料降温至35℃后以0.03%(v/v)的接种量将发酵剂接种至发酵原料中,于35℃下保温发酵16h,得到发酵乳;将发酵乳于42℃下放置4h进行凝乳后,在4℃下冷藏24h进行后熟,得到发酵乳成品。
实施例10:长双歧杆菌长亚种CCFM1077的应用
具体步骤如下:
长双歧杆菌长亚种CCFM1077可用于制备片剂,片剂的具体制备过程如下:
长双歧杆菌长亚种CCFM1077划线于MRS固体培养基上,37℃条件下厌氧培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下厌氧培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经6000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到长双歧杆菌长亚种CCFM1077菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
称取长双歧杆菌长亚种CCFM1077菌粉25.7重量份、淀粉55.0重量份、纤维素衍生物4.5重量份、羧甲基淀粉钠12.0重量份、滑石粉0.8重量份、蔗糖1.0重量份与水1.0重量份,得到原材料;将原材料混合,得到湿颗粒;将湿颗粒用中南制药机械厂的压片机进行压片后使用青州市益康中药机械有限公司的小型药物干燥机进行干燥,得到片剂。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (8)
1.长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum)CCFM1077在降解环境中壬基酚方面的应用,其特征在于,所述环境为非生物体内的环境。
2.如权利要求1所述的应用,其特征在于,所述环境为食品加工环境、食品酿造环境、乳制品环境、果蔬制品环境。
3.一种长双歧杆菌长亚种CCFM1077在制备缓解壬基酚中毒症状的产品中的应用。
4.如权利要求3述的应用,其特征在于,所述长双歧杆菌长亚种CCFM1077在产品中添加的活菌数为不低于1×106 CFU/mL或1×106 CFU/g。
5.如权利要求3或4所述的应用,其特征在于,所述产品为食品、药品或壬基酚清除剂。
6.如权利要求5所述的应用,其特征在于,所述食品为发酵果蔬、发酵乳、乳酪、含乳饮料、乳粉或其他含有长双歧杆菌长亚种CCFM1077的食品。
7.如权利要求6所述的应用,其特征在于,所述药品含有长双歧杆菌长亚种CCFM1077、以及药物载体和/或药用辅料。
8.如权利要求7所述的应用,其特征在于,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
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