CN117562258B - 动物双歧杆菌动物亚种在促进人参提取物缓解肝损伤功效发挥上的应用 - Google Patents
动物双歧杆菌动物亚种在促进人参提取物缓解肝损伤功效发挥上的应用 Download PDFInfo
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- CN117562258B CN117562258B CN202311184003.0A CN202311184003A CN117562258B CN 117562258 B CN117562258 B CN 117562258B CN 202311184003 A CN202311184003 A CN 202311184003A CN 117562258 B CN117562258 B CN 117562258B
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- ginseng extract
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- bifidobacterium animalis
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Abstract
本发明公开了动物双歧杆菌动物亚种在促进人参提取物缓解肝损伤功效发挥上的应用。本发明的动物双歧杆菌动物亚种CCFM1274可在富含人参提取物的发酵基料中生长,并且该菌株发酵人参提取物的发酵液能提升苷元型黄酮和短链脂肪酸等活性物质含量,可促进人参提取物功效发挥,包括提高人参提取物缓解非酒精肝损伤的能力,以及降低肝细胞内脂肪堆积的能力。利用该菌种和人参提取物可生产缓解非酒精肝损伤功效提升的产品,且生产工艺具有安全、高效、成本低廉、反应温和等优点,适合规模化工业生产。因此,使用动物双歧杆菌CCFM1274应用至含人参提取物产品具有巨大的应用价值。
Description
技术领域
本发明属于微生物技术领域。更具体地,涉及动物双歧杆菌动物亚种CCFM1274在促进人参提取物功效发挥上的应用,包括在促进人参提取物缓解非酒精代谢肝损伤功效发挥上的应用。
背景技术
随着人们生活条件的改善,不良的饮食习惯和久坐不动的生活方式,非酒精性脂肪性肝病(NAFLD)患者的数量逐渐增加,NAFLD是一种慢性代谢性疾病,其临床症状为过多脂质积聚在肝脏中。其疾病谱包括:非酒精性单纯性脂肪肝,非酒精性脂肪性肝炎,肝硬化和肝癌。西方国家的患病率约占一般人群的20%-30%。近些年,NAFLD在中国也有增长趋势,并且极大的增加了NAFLD患者患糖尿病、动脉粥样硬化性等心血管疾病及肝损伤的几率,严重的可发展为肝硬化,肝癌和肝功能衰竭。
目前,临床上广泛使用贝特类、他汀类和选择性TC吸收抑制剂等降脂药,其能有效调节脂质代谢异常,促进TG的转化,使TC水解,降低TG和TC的含量。均有很好的临床疗效,但长期服用后有停药反跳、肝肾功能损害等不良反应。为了扩大NAFLD的治疗方案,近年来研究趋势集中在可抑制肝细胞脂质积聚和炎症的中草药提取物及活性成分上。
人参在我国医药史上具有极其特殊的地位,被誉为“百草之王”,是传统中药中的标志性药材。人参已经大量应用到药品、保健品和化妆品当中。有研究显示,人参提取物能发挥一定的缓解NAFLD功效,具备开发为缓解NAFLD的相关产品的广泛价值。
但是,直接使用人参提取物很难获得直接的效果,在食用含人参提取物时,功效发挥作用差。因此,如何促进人参提取物的功效发挥是影响人参提取物开发为缓解NAFLD相关产品的关键。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和不足,提供动物双歧杆菌动物亚种CCFM1274在促进人参提取物缓解非酒精代谢肝损伤功效发挥上的应用。
本发明的目的是提供动物双歧杆菌动物亚种(Bifidobacterium animalissubsp.lactis)CCFM1274在制备可提升人参提取物缓解非酒精性脂肪性肝病功效的产品中的应用。
本发明另一目的是提供一种缓解非酒精性脂肪性肝病功效提升的发酵人参提取物。
本发明再一目的是提供一种缓解非酒精性脂肪性肝病的产品。
本发明再一目的是提供一种促进人参提取物中苷元型黄酮和/或短链脂肪酸含量增加的方法。
本发明上述目的通过以下技术方案实现:
动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.lactis)CCFM1274在改良MRS培养基上培养48h后菌落呈圆形、凸面或透镜状,微白,不透明,有平滑至粘液状的柔软表面。该菌于2022年09月15日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo:62798。利用该菌发酵人参提取物可显著提升苷元型黄酮和短链脂肪酸(乙酸、丁酸),可促进人参提取物功效发挥。具体体现在:(1)动物双歧杆菌CCFM1274与人参提取物复合发酵液能提高HepG2非酒精性细胞损伤的细胞增殖能力并降低肝细胞脂肪堆积;(2)人参提取物与菌株复合发酵液预处理肝细胞可提高其抑制肝细胞脂质积聚的能力;(3)该动物双歧杆菌CCFM1274能显著提高人参提取物抑制肝细胞脂质积聚的能力。因此:
本发明提供动物双歧杆菌动物亚种CCFM1274在制备可促进人参提取物非酒精性脂肪性肝病功效的产品中的应用。
本发明还提供动物双歧杆菌动物亚种CCFM1274和人参提取物组合在制备可缓解非酒精性脂肪性肝病的产品中的应用。
本发明还提供一种缓解非酒精性脂肪性肝病功效提升的发酵人参提取物,其由动物双歧杆菌动物亚种CCFM1274发酵人参提取物得到。
具体可选择地,发酵方法,包括如下步骤:
(1)将动物双歧杆菌动物亚种CCFM1274活化,得菌液;
(2)在含人参提取物的发酵基料中,接入(1)的菌液,进行发酵;
(3)发酵完成后,离心,取发酵上清液,即得。
在本发明的一种实施方式中,步骤(1)是将动物双歧杆菌动物亚种CCFM1274在MRS固体培养基上划线,平板倒置培养(优选37℃倒置培养48h),然后挑取单菌落接入MRS液体培养基(优选5mL)中培养(优选37℃培养48h),得菌液。
在本发明的一种实施方式中,步骤(2)所述含人参提取物的发酵基料中人参提取物浓度为2-10mg/mL(优选5mg/mL)。
在本发明的一种实施方式中,步骤(2)中菌液接种量为1-8%(v/v)。
在本发明的一种实施方式中,步骤(2)所述发酵是30-40℃恒温发酵24-72h(优选37℃恒温发酵48h)。
在本发明的一种实施方式中,步骤(3)中离心条件为6000-10000r/min离心10-20min(优选为8000r/min离心15min)。
本发明还提供所述发酵人参提取物在制备可缓解非酒精性脂肪性肝病的产品中的应用。
在本发明的一种实施方式中,所述产品为食品、药品或保健品。
另外,所述肝损伤包括非酒精代谢肝损伤,所述脂肪肝包括非酒精性脂肪肝。
在上述应用中,所述产品包括如下至少一种功能:
(1)提高肝细胞增殖活力;
(2)减少肝细胞内红色脂滴数量;
(3)提高肝细胞内谷草转氨酶与谷丙转氨酶的含量;
(4)提高TG清除率;
(5)抑制肝细胞脂质积聚。
本发明还提供动物双歧杆菌动物亚种CCFM1274在促进人参提取物中苷元型黄酮和/或短链脂肪酸(乙酸、丁酸)含量增加方面的应用。以及在制备苷元型黄酮和/或短链脂肪酸(乙酸、丁酸)含量增加的人参提取物产品中的应用。
本发明还提供一种促进人参提取物中苷元型黄酮和/或短链脂肪酸(乙酸、丁酸)含量增加的方法,是向含有人参提取物中的体系中添加动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.lactis)CCFM1274。
本发明还提供一种缓解非酒精性脂肪性肝病的产品,含有动物双歧杆菌动物亚种CCFM1274和人参提取物。
一种缓解非酒精性脂肪性肝病的产品,含有上述的发酵人参提取物。
在本发明的一种实施方式中,所述产品中,所述动物双歧杆菌动物亚种CCFM1274的添加量至少为:1×109CFU/ml。
在本发明的一种实施方式中,所述药品含有动物双歧杆菌动物亚种CCFM1274、药物载体和/或药用辅料。
在本发明的一种实施方式中,所述保健品含有动物双歧杆菌动物亚种CCFM1274、载体和/或辅料。
基于上述内容,本发明还提供了一种促进人参提取物功效发挥的方法,所述方法为向含有人参提取物中的体系中添加动物双歧杆菌动物亚种CCFM1274。所述方法为非疾病诊断治疗目的。具体方法为:向含有人参提取物中的体系中添加动物双歧杆菌动物亚种CCFM1274后,进行发酵,发酵方法如上文所述。
本发明具有以下有益效果:
本发明的动物双歧杆菌动物亚种CCFM1274可在富含人参提取物的发酵基料中生长,并且该菌株发酵人参提取物的发酵液能提升苷元型黄酮和短链脂肪酸(乙酸、丁酸)等活性物质含量,可促进人参提取物功效发挥,包括提高人参提取物缓解非酒精肝损伤的能力,以及降低肝细胞内脂肪堆积。
利用动物双歧杆菌动物亚种CCFM1274和人参提取物可生产缓解非酒精肝损伤功效提升的产品,生产工艺能够以人参总皂苷提取物为原料,促进人参提取物在功效上的发挥,该工艺具有安全、高效、成本低廉、反应温和等优点,该工艺适合规模化工业生产。
而且,动物双歧杆菌是常见的肠道微生物,它在维持肠道平衡,抑制肠道内有害微生物生长繁殖,抵抗病原体感染,增强机体免疫力等方面发挥重要作用。过往的研究已经表明动物双歧杆菌对人体健康有诸多益处。动物双歧杆菌也是如今应用在食品工业中最多的双歧杆菌菌种之一,尤其是乳制品行业,如动物双歧杆菌BB-12、动物双歧杆菌HN019和动物双歧杆菌bI-04等。
因此,使用动物双歧杆菌CCFM1274应用至含人参提取物产品具有巨大的应用前景和应用基础。
附图说明
图1为各组别细胞活力比;图注中,标注不同字母表示差异显著(p<0.05),标注相同字母表示差异不显著(p>0.05)。
图2为各组别油红浓度比。
图3各组别油红染色图;图注中,标注不同字母表示差异显著(p<0.05),标注相同字母表示差异不显著(p>0.05)。
图4为各组别相对ALT、AST比;图注中,标注不同字母表示差异显著(p<0.05),标注相同字母表示差异不显著(p>0.05)。
图5各组别相对TG比;图注中,标注不同字母表示差异显著(p<0.05),标注相同字母表示差异不显著(p>0.05)。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
本发明所用动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.lactis)CCFM1274,已于2022年09月15日保藏于广东省微生物菌种保藏中心(GDMCC),保藏编号为GDMCC No:62798,保藏地址为广州市先烈中路100号大院59号楼5楼。
下述实施例中所涉及的短双歧杆菌(Bifidobacterium breve)CCFM1025的保藏编号为GDMCC No.60386,记载于公开号为CN114533765A的专利申请文本中;短双歧杆菌(Bifidobacterium breve)CCFM683的保藏编号为CGMCC No.11828,记载于公开号为CN115624572A的专利申请文本中;动物双歧杆菌(Bifidobacterium animalis)CCFM1155的保藏编号为GDMCC No.61495,记载于公开号为CN113088473A的专利文本中;动物双歧杆菌(Bifidobacterium animalis)CCFM1160的保藏编号为GDMCC No.61500,记载于公开号为CN113897300A的专利文本中。
下述实施例中涉及的人参提取物购自广州无限极公司;RPMI-1640培养液与胎牛血清购自gibco公司;无水乙醇购自国药集团化学试剂有限公司;谷草转氨酶试剂盒、谷丙转氨酶试剂盒、TG试剂盒均购自南京建成公司;乙酸为分析纯,乙腈为色谱纯均购自国药集团化学试剂有限公司;油酸(美国Sigma公司,批号:026K13971V);油红O(美国Sigma公司,批号:O0625)。
下述实施例中所涉及的HepG2细胞来自:江南大学食品学院生物技术中心细胞库。
下述实施例中涉及的培养基如下:
改良MRS液体培养基(g/L):蛋白胨10g/L、酵母提取物5g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.58g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、蒸馏水1000g/L、半胱氨酸盐酸盐0.5g/L。
改良MRS固体培养基(g/L):蛋白胨10g/L、酵母提取物5g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.58g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、琼脂20g/L、蒸馏水1000g/L、半胱氨酸盐酸盐0.5g/L。
改良MRS液体培养基(g/L):蛋白胨10g/L、酵母提取物5g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.58g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、蒸馏水1000g/L、半胱氨酸盐酸盐0.5g/L。
改良MRS固体培养基(g/L):蛋白胨10g/L、酵母提取物5g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.58g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、琼脂20g/L、蒸馏水1000g/L、半胱氨酸盐酸盐0.5g/L。
含人参提取物的发酵基料(g/L):人参提取物5mg/mL,蛋白胨10g/L、酵母提取物5g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O2.6g/L、MgSO4·7H2O 0.58g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、蒸馏水1000g/L、半胱氨酸盐酸盐0.5g/L。
其中人参提取物为人参水提物,市购即可,规格为5:1的人参比例水提物(原药材和提取浓缩干燥后获得的提取物的比例为5:1,即1g人参提取物相当于5g人参药材)。
RPMI-1640培养基:购自gibco公司。
实施例1:菌株CCFM1274的分离筛选、鉴定及保藏
1、菌株分离筛选
将采自内蒙古的健康成人粪便样本用无菌生理盐水稀释后,在改良MRS固体培养基上进行平板涂布、培养、分离出形态一致的纯的单菌落。将不同的单菌落分别接种于含有人参提取物的改良MRS液体培养基中培养,通过分析培养发酵后培养基中稀有人参皂苷量的变化,筛选出转化人参提取物最强的菌株,记为菌株CCFM1274。
2、菌株鉴定
对上述得到的菌株CCFM1274进行形态学与16S rDNA分子鉴定(测序由苏州金唯智生物科技有限公司进行,比对在NCBI中进行核苷酸序列比对),鉴定结果菌株CCFM1274属于动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.lactis)。
3、菌种的保存
将动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.lactis)CCFM1274接种于5mL MRS液体培养基中,37℃培养24h;取1mL菌液于无菌离心管中,8000r/min离心3min后弃去上层培养基,菌泥重悬于30%甘油溶液中置于-80℃中保存。
4、菌株保藏
于2022年09月15日将上述获得的动物双歧杆菌动物亚种(Bifidobacteriumanimalis subsp.lactis)CCFM1274保藏于广东省微生物菌种保藏中心(GDMCC),保藏编号为GDMCC No:62798,保藏地址为广州市先烈中路100号大院59号楼5楼。
实施例2:动物双歧杆菌动物亚种CCFM1274发酵人参提取物复合发酵液
1、发酵方法:
(1)将实施例1中的动物双歧杆菌动物亚种CCFM1274在MRS固体培养基上划线,平板于37℃倒置培养48h;挑取单菌落接入5mL的MRS液体培养基中于37℃培养48h,制备得到种子液。
(2)在含人参提取物的发酵基料中,加入5%(v/v)该动物双歧杆菌动物亚种CCFM1274的种子液,37℃恒温发酵48h,发酵结束后,得到发酵液,发酵液中的菌液浓度为1×109CFU/mL。
(3)将发酵液以8000r/min离心20min,收集上清,调节pH至7.2~7.4;0.22μm一次性滤器过滤除菌,得到动物双歧杆菌动物亚种CCFM1274与人参提取物复合发酵液,分装后于-20℃冰箱保存。
2、分析上述获得的动物双歧杆菌动物亚种CCFM1274与人参提取物复合发酵液的成分,与发酵前人参提取物成分对比。如表1所示:
表1 CCFM1274菌株发酵人参提取物前后功能物质对比
备注:*代表的丁酸为发酵后新增成分。
由上表数据可知,本发明的动物双歧杆菌CCFM1274发酵后的人参提取物培养基中苷元型黄酮与短链脂肪酸(乙酸、丁酸)含量显著增加,苷元型黄酮与短链脂肪酸具有改善非酒精性脂肪肝的效果。
对比例:
将实施例2的发酵方法中的菌种分别替换为短双歧杆菌(Bifidobacteriumbreve)CCFM1025、短双歧杆菌(Bifidobacterium breve)CCFM683、动物双歧杆菌(Bifidobacterium animalis)CCFM1155、动物双歧杆菌(Bifidobacterium animalis)CCFM1160,分别制备得到短双歧杆菌CCFM1025与人参提取物复合发酵液、短双歧杆菌CCFM683与人参提取物复合发酵液、动物双歧杆菌CCFM1155与人参提取物复合发酵液、动物双歧杆菌CCFM1160与人参提取物复合发酵液。
实验例:发酵液缓解非酒精性代谢肝损伤的作用实验
1、FFA造模液制备:
(1)30%牛血清白蛋白(BSA)溶液的制备
无菌PBS缓冲液预热至55℃,精确称取3.6g不含脂肪酸的牛血清白蛋白粉末,加入至50mL离心管内,并加入12mL PBS溶液,室温条件下8000r离心20min,得30% BSA溶液,外观黄色澄清。
(2)棕榈酸(PA)溶液的制备
吸取6mL 0.1mol/L NaOH溶液于50mL离心管内,加入0.0614g棕榈酸颗粒,置于75℃水浴30min,充分皂化后呈澄清透明状,保持75℃,迅速加入6mL 30%BSA溶液,混匀后,置于55℃继续溶解30min,得20mM棕榈酸PA溶液,呈棕黄澄清状,性状稳定,置于-20℃冰箱冻存。
(3)40mM油酸钠溶液的制备
取6mL 0.1mol/L NaOH溶液和500μL油酸溶液(1g至3.54mL超纯水,1000mmol/L)加入离心管中,置于75℃水浴锅,皂化30min,皂化完成后呈无色透明,保持温度情况下加入6mL 30%BSA溶液充分混匀,冻存于-20℃冰箱。
(4)使用时将20mM PA溶液、40mM油酸钠溶液(体积比1:1)混以普通RPMI-1640培养基(体积比1:1)得到FFA造模液。
2、制备油红O染色液:
取油红O颗粒0.5g,溶于100mL异丙醇,于水浴锅中缓慢加热直至完全溶解,冷却至室温后,滤纸过滤,避光、4℃保存。用前2h取油红O原液以3:2比例加蒸馏水混合,用0.45μm有机滤膜过滤除渣。
3、动物双歧杆菌动物亚种CCFM1274发酵液制备:
将活化完成的动物双歧杆菌动物亚种CCFM1274接入改良MRS液体培养基中,于37℃厌氧培养24h。以5%(v/v)接种量接入改良MRS液体培养基中,37℃恒温厌氧发酵48h,菌液浓度为1×109CFU/mL,以8000r/min离心20min,收集上清,调节pH至7.2~7.4;0.22μm一次性滤器过滤除菌,得到动物双歧杆菌动物亚种CCFM1274发酵液,分装后于-20℃冰箱保存。
4、具体造模实验
将HepG2细胞接种在含10%胎牛血清和1%青链霉素合剂的RPMI-1640培养基,于37℃、5%CO2的培养箱中以1:3的比例传代培养。待细胞密度达到80%时,进行不同分组处理。具体分组数据如下:
空白对照组:普通RPMI-1640培养基培养48h。
模型组:普通RPMI-1640培养基培养24h后,再以1.0mmol/L FFA造模液培养24h。
人参提取物组:普通RPMI-1640培养基培养24h后,以浓度为5mg/ml的人参提取物与1.0mmol/L FFA造模液培养24h。
CCFM1274组(即动物双歧杆菌动物亚种CCFM1274发酵液组):普通RPMI-1640培养基培养24h后,以上述制备的菌液浓度为1×109CFU/ml的动物双歧杆菌动物亚种CCFM1274发酵液与1.0mmol/L FFA造模液进行培养24h。
CCFM1274+人参提取物组:普通RPMI-1640培养基培养24h,以实施例2制备得到的动物双歧杆菌动物亚种CCFM1274与人参提取物复合发酵液与1.0mmol/L FFA造模液干预培养24h。
CCFM1025+人参提取物组:普通RPMI-1640培养基培养24h,以对比例制备得到的短双歧杆菌CCFM1025与人参提取物复合发酵液与1.0mmol/L FFA造模液干预培养24h。
CCFM683+人参提取物组:普通RPMI-1640培养基培养24h,以对比例制备得到的短双歧杆菌CCFM683与人参提取物复合发酵液与1.0mmol/L FFA造模液干预培养24h。
CCFM1155+人参提取物组:普通RPMI-1640培养基培养24h,以对比例制备得到的动物双歧杆菌CCFM1155与人参提取物复合发酵液与1.0mmol/L FFA造模液干预培养24h。
CCFM1160+人参提取物组:普通RPMI-1640培养基培养24h,以对比例制备得到的动物双歧杆菌CCFM1160与人参提取物复合发酵液与1.0mmol/L FFA造模液干预培养24h。
具体操作如下:
(1)将生长密度接近80%的HepG2细胞进行消化处理,以1×104个/孔浓度接种于96孔板,每孔100μL细胞悬液,将96孔板置于培养箱内培养24h,使细胞80%贴壁后,吸出培养液,用无菌PBS溶液洗去死细胞;
按照空白对照组、模型组、人参提取物组、CCFM1274组、CCFM1274+人参提取物组、CCFM1025+人参提取物组、CCFM683+人参提取物组、CCFM1155+人参提取物组、CCFM1160+人参提取物组加入成分,每组均设置3个平行孔,继续培养24h后,用CCK8法检测细胞存活率:将CCK8和1640培养液1:10混匀,小心吸出96孔板内培养液,用PBS溶液轻柔清洗一遍,每孔避光加入110μL CCK8-1640培养基混合液,放入培养箱继续培养2h,通过酶标仪测定450nm波长下各孔吸光度值。
细胞存活率的计算方法:细胞活力(%)=[(As-Ab)/(Ac-Ab)]×100%。
As:具有细胞、CCK溶液和给药溶液孔的吸光值;
Ab:具有培养基、CCK溶液而没有细胞孔的吸光值;
Ac:具有细胞、CCK溶液而没有给药溶液孔的吸光值;
结果如图1所示,结果显示:当复合发酵液浓度为20μg/ml时,对细胞的影响最小,故选择以20μg/ml作为后续实验浓度。
(2)以1×105个/孔将HepG2细胞接种于6孔板,当细胞密度接近70%时,根据分组:空白对照组、模型组、人参提取物组、CCFM1274组、CCFM1274+人参提取物组、CCFM1025+人参提取物组、CCFM683+人参提取物组、CCFM1155+人参提取物组、CCFM1160+人参提取物组,接入不同成分对细胞进行干预,24h后进行油红O染色。
染色步骤:染色液37℃预热。吸出12孔板中培养液,PBS溶液清洗两次,吸去PBS溶液;4%多聚甲醛固定细胞30min,吸除;60%异丙醇洗涤3min,便于油红着色;油红O避光染色20min;60%异丙醇洗涤10s;超纯水洗涤1min,洗去多余染色液,于倒置显微镜下观察并拍照。观察后用异丙醇溶解染色的脂滴来定量脂滴的蓄积,在500nm处测量吸光度。
(3)将HepG2细胞以1×105个/孔接种于12孔板上,待24h细胞贴壁后,分为对照组、模型组、人参提取物组、CCFM1274组、CCFM1274+人参提取物组、CCFM1025+人参提取物组、CCFM683+人参提取物组、CCFM1155+人参提取物组、CCFM1160+人参提取物组,继续培养24h后,收集各孔细胞上清液,按照ALT、AST测试盒说明书测定细胞上清液ALT、AST含量。细胞使用IP裂解液裂解后,按照BCA蛋白浓度测定试剂盒和TG测试盒说明书进行测定胞内TG含量。
5、实验结果(图2~5所示):
结果显示:
(1)空白对照组:油红吸光度为0.45,相对油红浓度为0.34,ALT含量为16.84U/L,AST含量为7.66U/L,TG含量为211mmol/L。
(2)模型组:油红吸光度为1.65,相对油红浓度为1.99,ALT含量为32.42U/L,AST含量为18.53U/L,TG含量为596mmol/L。
(3)动物双歧杆菌动物亚种CCFM1274+人参提取物组:
油红染色面积均较其他组别低,而油红吸光度只有0.9,相对油红浓度只有0.73;也远远小于其他组别。
CCFM1274+人参提取物组的ALT含量为17.12U/L,AST含量为8.75U/L,TG含量为315mmol/L。
(4)人参提取物组:油红染色面积略微降低,而油红吸光度为1.29,相对油红浓度为1.22;
ALT含量为25.04U/L,AST含量为15.98U/L,TG含量为408mmol/L。
(5)动物双歧杆菌动物亚种CCFM1274组:
该组油红染色面积略微降低,而油红吸光度为1.5,相对油红浓度为1.58;
ALT含量为30.14U/L,AST含量为17.72U/L,TG含量为514mmol/L。
(6)短双歧杆菌CCFM1025+人参提取物组:
油红染色面积略微降低,而油红吸光度为1.26,相对油红浓度为1.19;
ALT含量为22.60U/L,AST含量为15.34U/L,TG含量为407mmol/L。
(7)短双歧杆菌CCFM683+人参提取物组:
油红染色面积略微降低,而油红吸光度为1.22,相对油红浓度为1.15;
ALT含量为22.92U/L,AST含量为14.54U/L,TG含量为419mmol/L。
(8)动物双歧杆菌CCFM1155+人参提取物组:
油红染色面积略微降低,而油红吸光度为1.26,相对油红浓度为1.18;
ALT含量为23.98U/L,AST含量为14.44U/L,TG含量为381mmol/L。
(9)动物双歧杆菌CCFM1160+人参提取物组:
油红染色面积略微降低,而油红吸光度为1.28,相对油红浓度为1.19;
ALT含量为25.05U/L,AST含量为16.12U/L,TG含量为435mmol/L。
以上结果对比分析表明:动物双歧杆菌动物亚种CCFM1274+人参提取物组能显著提高HepG2细胞增殖活力、减少细胞内红色脂滴数量,并提高谷草转氨酶与谷丙转氨酶的含量,提高TG清除率,抑制肝细胞脂质积聚;而且效果显著优于单一菌株CCFM1274、单一人参提取物、短双歧杆菌CCFM1025+人参提取物、短双歧杆菌CCFM683+人参提取物、动物双歧杆菌CCFM1155+人参提取物、动物双歧杆菌CCFM1160+人参提取物组。
综上,本发明的动物双歧杆菌动物亚种CCFM1274与人参提取物复合发酵液能提高HepG2细胞增殖活力、减少细胞内红色脂滴数量,降低谷草转氨酶与谷丙转氨酶的含量,提高TG清除率,进一步抑制肝细胞脂质积聚,从而具备改善、延缓脂肪肝的效果和应用价值。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1. 动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.lactis)CCFM1274在制备可提升人参提取物缓解非酒精性脂肪性肝病功效的产品中的应用,所述动物双歧杆菌动物亚种CCFM1274于2022年09月15日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62798,所述应用的方法为利用动物双歧杆菌动物亚种CCFM1274发酵人参提取物,所述发酵的条件是30-40℃恒温发酵24-72h。
2.权利要求1中所述的动物双歧杆菌动物亚种CCFM1274和人参提取物在制备可缓解非酒精性脂肪性肝病的产品中的应用,所述应用的方法为利用动物双歧杆菌动物亚种CCFM1274发酵人参提取物,所述发酵的条件是30-40℃恒温发酵24-72h。
3.一种缓解非酒精性脂肪性肝病功效提升的发酵人参提取物,其特征在于,由权利要求1中所述的动物双歧杆菌动物亚种CCFM1274发酵人参提取物得到,所述发酵的条件是30-40℃恒温发酵24-72h。
4.权利要求3所述发酵人参提取物在制备可缓解非酒精性脂肪性肝病的产品中的应用。
5.根据权利要求1、2或4所述的应用,其特征在于,所述产品为包括如下至少一种功能的产品:
(1) 提高肝细胞增殖活力;
(2) 减少肝细胞内红色脂滴数量;
(3) 提高肝细胞内谷草转氨酶与谷丙转氨酶的含量;
(4) 提高TG清除率;
(5) 抑制肝细胞脂质积聚。
6.权利要求1中所述的动物双歧杆菌动物亚种CCFM1274在促进人参提取物中苷元型黄酮和/或短链脂肪酸含量增加方面的应用,或在制备苷元型黄酮和/或短链脂肪酸含量增加的人参提取物产品中的应用,所述应用的方法为利用动物双歧杆菌动物亚种CCFM1274发酵人参提取物,所述发酵的条件是30-40℃恒温发酵24-72h。
7.一种促进人参提取物中苷元型黄酮和/或短链脂肪酸含量增加的方法,其特征在于,向含有人参提取物的体系中添加动物双歧杆菌动物亚种CCFM1274,进行发酵,所述发酵的条件是30-40℃恒温发酵24-72h。
8.一种用于制备缓解非酒精性脂肪性肝病产品的组合物,其特征在于,含有权利要求1中所述的动物双歧杆菌动物亚种CCFM1274和人参提取物,所述组合物用于制备缓解非酒精性脂肪性肝病产品的方法为将组合物进行发酵,发酵条件是30-40℃恒温发酵24-72h。
9.一种缓解非酒精性脂肪性肝病的产品,其特征在于,含有权利要求3所述的发酵人参提取物。
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