CN109549195A - 具有抗肥胖活性的含有发酵香橙渣的食物成分 - Google Patents
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Abstract
本发明重点研究从利用植物乳杆菌GAVOL‑07发酵的香橙渣乳酸发酵物(YP)中发现新功能特性及其在肥胖大鼠体内与体外的抗肥胖效果。同时,对所述抗肥胖化合物进行了纯化。
Description
技术领域
本发明涉及一种具有抗肥胖活性的食物成分,它含有利用植物乳杆菌GAVOL-07发酵而成的香橙渣及其纯化活性成分。
背景技术
肥胖是由于能量摄入过度和低能量消耗造成的。医学上被定义为由于机体能量平衡失调引起整个体内脂肪细胞和脂肪组织蓄积过多的状态。肥胖通常会导致各种代谢性疾病,如糖尿病、心血管疾病、高脂血症、高血压、非酒精性脂肪肝和某些癌症。肥胖是全球最大的健康问题之一,是现代人面临的一个重要的问题。
针对肥胖的增加,控制体重的药物和功能性食品越来越受到人们的重视。但是这些不仅昂贵而且不是很有效。
最近关于肥胖治疗的研究集中在植物提取物和水果对控制肥胖方面的潜在作用。将其用于治疗肥胖,不仅是优先考虑开发更安全的药物替代品,而且还考虑到对健康有益。特别是使用天然食品,如发酵食品和益生菌,被认为对肥胖的管理更为安全。
各种柑橘类水果包括香橼(香橙)是一个很好的植物化学物质的来源,它含有维生素、矿物质、纤维,另外还有丰富的酚类物质,包括酚酸、黄酮类化合物,这些都具有促进健康的作用。目前已经进行了许多抗肥胖的研究。柑橘类水果皮可以避免肥胖,可预防生活方式相关的疾病,可有效降低体重。
香橼(香橙)的产地和制造地在韩国。柑橘的产量约1亿3000万吨,用作果汁加工的原料。然而,大量柑橘渣作为果汁生产的主要废弃物,每年产生量高达水果投入量的50%,造成资源浪费,并产生环境问题。
它是类黄酮、酚类化合物和精油等生物活性成分的良好来源。许多研究已经尝试利用它们作为膳食纤维(Sudha,M.L.,Baskaran,V.,&Leelavathi,K.(2007);苹果渣作为膳食纤维和多酚的来源及其对流变特性和蛋糕制作的影响;食品化学,104(2),686~692)和多酚(Li,B.B.,Smith,B.,&Hossain,M.M.(2006);柑桔皮中酚类物质的提取;分离纯化技术,48(2),182~188)的来源。
发明内容
一些研究人员研究了香橙的抗肥胖作用,但利用微生物对香橼(香橙)的渣进行发酵的研究尚未进行过。
本发明的目的是,提供一种具有抗肥胖活性食品成分,它含有利用植物乳杆菌GAVOL-07发酵的香橙渣及其纯化的活性成分。
与现有技术相比,本发明的有益效果如下:
本发明的实验结果表明,香橙渣发酵物(YP)抑制脂质堆积和成脂转录因子的表达,并增加甘油释放量。YP显示出抗肥胖作用。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1显示不同浓度YP对胰脂肪酶活性的抑制作用;
图2显示YP对3T3-L1的脂肪细胞活力的影响;
图3代表YP在3T3-L1细胞内对脂肪细胞分化的影响;其中,图3A为不同YP浓度下前脂肪细胞形态学观察结果;图3B为不同YP浓度下3T3-L1脂肪细胞的油红O染色胞内油红O染色的脂质堆积;
图4代表YP对3T3-L1细胞的成脂酶表达的影响;
图5表示DIO大鼠的体重和器官重量的各组对比结果;其中,图5a为体重增长图;图5b为各器官中脂肪重量增长图;ND:正常饮食,HFD:高脂饮食,YP:香橙渣发酵物(YP)(2ml/kg/day);
图6代表具有抗肥胖的化合物A、B、C的薄层色谱图;其中,第1道为:甲醇沉淀步骤中的甲醇可溶组分,第2道为:酸碱分离步骤中的羧酸组分,第3道为:硅胶色谱中的化合物A,第4道为:硅胶色谱中的化合物B,第5道为:硅胶色谱中的化合物C;
图7表示化合物A结构的核磁共振谱(1HNMR Spectrum)结果;
图8表示化合物B结构的核磁共振谱(1HNMR Spectrum)结果;
图9表示化合物C结构的核磁共振谱(1HNMR Spectrum)结果;
图10表示化合物A和化合物B的结构式;
图11表示YP的活性化合物的纯化步骤示意图。
具体实施方式
以下本发明的详细说明,通过本发明可以付诸实践的特定实施例来阐述,通过充分详细地说明,使得本行业的人能够将本发明付诸实施。本发明的各种实施例应当被理解为可以彼此不同,但彼此没必要互相排斥。因此下面描述的详细说明,并不应被解释为局限的含义,本发明的范围应被理解为专利权利请求范围内记载的内容和与其同等的所有范围。
本发明重点研究从利用植物乳杆菌GAVOL-07乳酸发酵(Lactate Fermentate:LE)的香橙渣中找到新的功能特性,以此开发新资源。
本发明旨在探讨LC抗脂肪细胞的影响。LC抗肥胖的可能性,通过脂肪细胞分化测定,并通过测量饮食诱导肥胖大鼠其脂肪细胞内的脂肪积累、甘油释放、对脂肪酶的表达以及血脂来判定。
YP对3T3-L1脂肪细胞和肥胖大鼠具有抗肥胖作用。利用YP对分化的3T3-L1脂肪细胞进行处理,使甘油释放显著增加并使成脂转录因子PPARγ和C/EBPα的蛋白质表达减少。在动物模型中,肥胖大鼠被高脂饮食人工诱导10周。在接下来的8周,实验组用YP(2ml/kg/天)填喂。实验结束时,两组大鼠的体重进行对比,填喂YP组的体重与高脂饮食组(HFD)相比减少13%。同时与HFD组相比,降低肝(180%)、附睾(35%)的脂肪重量。YP防止饮食引起的血清浓度水平的升高,与HFD组相比,TC(37%)、TG(11.6%)、LDL(50%)降低。然而,HDL-C的水平,YP组(22%)比HFD组高。从YP(100g)提取的抗肥胖活性化合物A、B、C用甲醇沉淀净化,酸碱分离(0.25g),用硅胶柱色谱和核磁共振分析。化合物A、B和C被证实为与吡喃葡萄糖酯结合的绿原酸。这些化合物未曾作为关于柑橘报告的一个对象被报告过。这一结果表明,这些化合物是经乳酸菌发酵的新的生物转化化合物。
实施例
1.材料与方法
1.1准备香橙渣发酵制品
在以往的研究中发现,用于香橙渣发酵物(YP)的乳酸菌产生较多纤维素酶和果胶酶,该乳酸菌被命名为植物乳杆菌GAVOL-07(Lactobacillus Plantarum GAVOL-07)。植物乳杆菌GAVOL-07于2016年6月29日被保存在韩国微生物保藏中心(KCCM)[保存编号:KCCM11852P]。
香橙渣发酵前,分离的菌株新芽在37℃温度下、乳酸杆菌MRS培养基(Difco-BD,Sparks,MD,USA)里,厌氧无菌条件下被反复传代培养48小时。
香橙渣的发酵根据实验室优化程序进行。香橙渣,从GAVO农场有限公司购买,糖挑选后压缩。在香橙渣(100g)中加入30ml蒸馏水,然后接种108(CFU)/ml的植物乳杆菌GAVOL-07,在35℃厌氧条件下,发酵48小时。发酵后,通过滤纸过滤。
1.2胰脂肪酶(Pancreatic Lipase:PL)活性的定量研究
PL活性根据Bustanji法[参考Y.Bustanji,I.AlMasri,M.Mohammad,M.Hudaib,K.Tawaha,H.Tarazi和H.AlKhatib,“银杏的萜内酯对胰脂肪酶的抑制活动”,酶抑制作用与药物化学报Vol.26,No.4,2011,pp.453-459],通过比色测定即通过测量对硝基苯酚(p-nitrophenol)的释放量来定量。
猪胰脂肪酶II型(Sigma,美国,EC 3.1.1.3)悬浮在Tris-HCl缓冲液(2.5mmol,pH值7.4的2.5mmol NaCl)使浓度达到5mg/ml(200unit/ml),用搅拌机混合15分钟,然后将溶液在1500g离心分离10分钟,回收上清液。0.10ml的PL溶液用不同浓度(0~20%)的YP和选定的化合物在37℃下预培育5分钟,然后加入PNPB培养基(10mM,在乙腈里)。在用分光光度法在410nm处测量溶液的吸光度前,用Tris-HCl缓冲液使体积增加到1ml。对硝基苯酚的释放量通过测量在410nm处的吸光度相比于空白变性酶吸光度的增加量来确定。PL活性与对硝基苯酚释放率有关。通过减去空白的吸光度来校正样本的吸光度。样本的脂肪酶抑制活性(%)确定公式如下:
脂肪酶抑制活性(%)=(1-A/B)×100
A=样本的吸光度,B=对照的吸光度
1.3 3T3-L1细胞培养及细胞毒性
3T3-L1细胞购自ATCC(马纳萨斯,弗吉尼亚洲,美国),培养在高糖杜尔伯科改良伊格尔培养基(DMEM:Gibco BRL,Grand Isand,纽约,美国),补充10%小牛血清,1%青霉素链霉素抗生素(Gibco BRL,Grand Isand,纽约,美国),存放在37℃,5%二氧化碳细胞培养箱里。3T3-L1前脂肪细胞在6孔板一直生长直到细胞达到汇合。成脂分化通过在DMEM培养基加入10μg/ml胰岛素、0.5mM的3-异丁基-1-甲基黄嘌呤(3-isobuthyl-l-methylxanthine:IBMX)和0.25μm的地塞米松(DEX)与10%胎牛血清(FBS)来诱导。为了检测经乳酸发酵的香橙渣的抗脂肪细胞活性,它们分别以0.1和0.25mg/ml浓度被添加到分化培养基里。48小时后,培养基被去除,含10%胎牛血清的DMEM培养基被取代。每2天更换一次培养基,保持6天。采用油红O染色检测细胞内脂肪细胞 分化的程度。脂肪染色强度在520nm处用吸光度测量。
本发明进行了细微修改的细胞活力MTT检测[参考Niu DL,Harada H,Wang LS,Zhang YJ,Yang CR.红雪茶综合体(地衣共生菌,梅衣科)的化学分类研究.地衣2011;43:213-23].在无菌条件下,在24孔板的每一口井中添加100μL的MTT(0.5mg/mL)。在37℃培育4小时后,未转化MTT通过抽吸除去,和甲产物溶解在二甲亚砜(DMSO)。用酶标仪(UVM340;Biochrom公司,剑桥,英国)在540nm处测定吸光度值来确定细胞活力。细胞毒性可以从裂解细胞分泌的乳酸脱氢酶(LDH)和细胞中产生的ssDNA来确定。通过非放射性细胞毒性试验(Promega公司,菲奇堡,威斯康辛,美国)和酶联免疫吸附测定(ELISA)及检测细胞凋亡(Enzo生命科学国际公司,普利茅斯会议,宾汐法尼亚,美国)试剂,分别评估低密度脂蛋白活性和进行ssDNA定量分析。
1.4油红O染色
3T3-L1脂肪细胞进行油红O染色,基本按照Ramirez Zacarias等人所描述的方法。[参照Ramírez-Zacarías JL,Castro-F,Kuri-Harcuch W.通过对胞浆内脂肪组织进行油红O染色量化脂肪转换和甘油三酸酯,组织化学,1992;97:493-7]。
分化3T3-L1细胞用不同浓度的YP进行处理。细胞用PBS洗涤两次,然后用10%福尔马林固定20分钟,取出10%福尔马林后,每个井加入丙二醇3分钟后,用PBS彻底冲洗。然后将细胞与油红O工作液培育3小时。在3T3-L1脂肪细胞中的脂滴上的染色用蒸馏水冲洗三次。细胞的染色染料用异丙醇提取,通过分光光度法在510nm处使用多孔板测定吸光度。
1.5免疫印迹分析
用免疫印迹分析法定量分析AMPK磷酸化蛋白。分化的3T3-L1细胞用不同浓度的YP处理。72小时后,收集细胞,用PBS清洗后,在冰冷的裂解缓冲液(20mM Tris,pH 8,150mMNaCl,10mM磷酸钠,100μM钒酸钠,100μM钼酸铵、10%甘油、0.1%NONIDET P-40,0.1%SDS,1×蛋白酶和磷酸酶抑制剂)储备溶解15分钟后,12,000rpm条件下离心分离20分钟,将分离的蛋白转移到300mA的Hybond-c硝酸纤维素膜(Amersham Biosciences公司,Piscataway,纽泽西,美国)90分钟。用5%脱脂牛奶封堵2个小时后,每个膜用1:1000稀释的特定抗体(大鼠单克隆抗体PPARγ、C/EBPα、SREBP-1或β-actin)穿刺,用辣根过氧化物酶共轭的抗大鼠IgG抗体培育12小时。免疫活性蛋白的表达可通 过增强的化学发光(Amersham,ArlingtonHeights伊利诺,美国)直观看到并通过使用4000R图像站(柯达,纽黑文,康乃狄克,美国)的信号可检测到。抗β-actin抗体是用来验证每个泳道被装入等量的蛋白质。
1.6动物及饮食
六周龄的雄性斯泼累格·多雷(sprague_dawley:SD)大鼠体重(b.w.)180g,从东方生物(城南、韩国)购买并饲养在自动控制空气条件即温度(21±2℃)、湿度(50-60%)和照明(12:12小时亮/暗周期)的笼中。正常饮食对照组的大鼠随意喂食商业颗粒饲料(AIN-76A鼠精饲料、东方生物)和水。高脂饮食诱导肥胖组大鼠喂饲60%高脂饲料(60%高脂鼠精饲料,东方生物)。东信国立大学实验动物护理和使用委员会批准该科学实验报告(批准号:2016-10-02),动物的照顾依据所述大学建立的“动物实验指南”进行。
大鼠适应环境1周后随机分为四组:正常饮食组(ND,n_6);高脂饮食诱导肥胖组(HFD,n_6),由60%高脂饮食喂养的大鼠组成;YP处理组(正控制)(YP,n_6),由60%高脂饮食喂养和以2mL/kg/天的量摄入YP的大鼠组成。
1.7血清和血浆生化参数分析
在牺牲前12小时取出饲料。用眼眶静脉穿刺收集每只大鼠的血液样本冷冻培育1小时。全血在3,000rpm,4℃条件下离心分离20分钟后保持-80℃直到进行检测。取出肝脏、肾脏、脾脏、睾丸和脂肪组织,用PBS冲洗,用纸巾擦拭,快速称重,液氮冷冻,保存在-80℃条件下直到进行检测。
血清WBC、RBC、Hb、HCT、PLT通过血细胞计数器(HEMAVRT 950FS,德鲁科学,美国)测定。血清总胆固醇(TCHO)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)通过生化分析仪测定((DRI-CHEM 4000i,fujiflim公司,日本)。
1.8YP的活性化合物的纯化
YP的活性化合物的纯化步骤如图11所示,通过培养离心得到的上清液加入两倍量冷甲醇后,混合物在黑暗中保存24小时以沉淀不溶性物质。然后将沉淀物质从甲醇中分离出来,加入少量甲醇使沉淀悬浮,然后通过蒸发浓缩悬浮液。在加热中溶解少量的水后,在浓缩液中加入等量的甲醇,以分离甲醇不溶性和可溶性的组分。各组分均用旋转蒸发器浓缩,冻干得到活性组分,甲醇可溶组分(MSF)。甲醇可溶组分(MSF)执行酸碱分离。测定各组分的PL抑制活性,显示PL的抑制活性的活性组分(羧酸)在溶剂系统中 进行硅胶柱色谱分析(甲苯:乙酸乙酯:甲酸=5:4:1)。在TLC中显示PL抑制活性的活性组分被称为化合物A、B、C。
1.9核磁共振谱
采用JEOL-FX100核磁共振波谱测定核磁共振谱,基于脉冲傅里叶变换的方法,如TMS[四甲基硅烷:(CH3)4Si)]作为气体参照物,7~10mg的试剂按5~20%(W/V)溶解在甲醇里。
2结果与讨论
2.1胰脂肪酶(PL)活性的定量研究
通过胰脂肪酶把甘油三酯水解为脂肪酸和2-单酰甘油,膳食脂肪从肠道被吸收。它是作为确定抗肥胖剂的天然产物和民族医药植物潜在疗效的最常用的机制之一。[参考S.Habtemariam,“Sigmoidin A的抗肥胖潜力”,医药生物,50卷,2012年12月,1519-1522页;C.D.Zheng,Y.Q.Duan,J.M.Gao and Z.G.Ruan,“37种中药的抗脂肪酶性能筛选”中国医学协会期刊,73卷,2010年6月,319-324页.29,36]。显示未加工YP的胰脂肪酶抑制活性的图表如图1所示。YP以剂量依赖的方式呈现抑制PL活性。如图1所示,YP的IC50值从612μg/ml增加到1000μg/ml时,观察到78%的活性抑制作用。这表明它类似于Choi etal的报告,他发现只有剂量超过100ppm,异黄酮苷的抑制胰脂肪酶的活性才能达30%。同时,Ananya和Nattapong已经发表,齿叶赛金莲木和印楝提取物抑制胰脂肪酶大于30%。
2.2 YP对3T3-L1脂肪细胞活力的影响
检测细胞内毒性,3T3-L1细胞使用一系列浓度(200、400、600、800、1000μg/mL)的YP处理10天,采用MTT比色法测定细胞活力,如图2所示。从图2中可知,直到YP的浓度为1000μg/mL,仍未显示对3T3-L1细胞活力的显著影响。
2.3 YP对3T3-L1脂肪细胞的脂质堆积的影响
CA对3T3-L1脂肪细胞内脂滴的积累的影响,如图3所示。相比于对照细胞内脂质堆积,YP浓度500μg/ml和1000μg/ml条件下细胞内油红O染色的脂质堆积值分别显著下降了19.1%和33.4%,表明经过处理的细胞内脂质堆积降低(P<0.05)。细胞TG被定量分析,用1μM和10μM的CA处理的脂肪细胞与对照组相比,TG含量分别降低了29.5%和53.1%(p<0.05)。
2.4 YP对3T3-L1脂肪细胞的大成脂转录因子的表达的影响
为了定量分析脂肪生成中在3T3-L1细胞内YP对主要成脂转录因子的表达的影响,3T3-L1细胞用不同剂量(200、400、600、800和1000μg/ml)YP处理。10天后,进行RT-PCR和免疫印迹分析。
如图4a和4b所示,YP以依赖剂量的方式降低3T3-L1细胞内主要成脂转录因子,如PPARγ、C/EBPα和SREBP-1的蛋白质水平。特别是,与对照组相比,这些表达水平,除了HSL、LPL、C/EBPα、SREBP-1在200μg/ml(15.2%,P<0.05)的初始浓度外都明显降低。脂肪生成的复杂过程由生产PPARγ开始,这是由C/EBPα和SREBP-1控制和激活的。其中,PPARγ起到一个关键的转录因子的作用,触发上调脂肪细胞特异性基因如在脂肪细胞分化过程中负责TG堆积的脂肪型脂肪酸结合蛋白(A-FABP)的随后表达。[参考,Jeon T,Hwang SG,Hirai S等人,红曲米提取物通过下调3T3-L1细胞中成脂转录因子和基因表达来抑制脂肪细胞分化,生命科学。2004;75(26):3195–3203。][Rosen ED Spiegelman BM。脂肪细胞分化的分子调控。细胞与发育生物学的年度回顾。2000;16:145–171]。C/EBPα,在脂肪细胞分化过程中的表达较晚,通过交叉调节,与PPARγ协同促进分化[参考,32.Kim JB Spiegelman BM.ADD1/SREBP1促进脂肪细胞分化与脂肪酸代谢的基因表达。基因与发育。1996;10(9):1096-1107]。这些成脂转录因子在3T3-L1细胞中起到脂肪生成和脂肪沉积的主要调节器作用。由于其过度表达可加速脂肪细胞的分化,因此可能成为肥胖治疗和预防的潜在治疗靶点。考虑到这些成脂转录因子的机制和免疫印迹分析结果,它们通过抑制成脂转录因子的表达,一致显示出减少TG和脂肪堆积。
总之,本发明的研究结果表明,由于YP在没有引起明显的细胞毒性的情况下,使细胞内TG含量和脂质堆积以剂量依赖方式显著降低,它可以有效地抑制3T3-L1脂肪细胞的脂肪生成。因此,YP可以作为提供预防和治疗肥胖的一个治疗方法。
2.5 YP对高脂饮食诱导肥胖大鼠的体重与器官重量的影响
本发明人进行动物研究进一步评估YP的体外抗肥胖作用,本发明人采用高脂饮食诱导的肥胖大鼠模型。高脂饮食试验已被广泛使用,也是已被接受的营养实验,它是诱导动物达到超重条件和脂肪沉积的很好的策略[参考,Chun MR,Lee YJ,Kim KH,Kim YW,ParkSY,Lee KM,Kim JY,Park YK.高碳水化合物和高脂饲料对大鼠肌肉胰岛素抵抗 的影响。韩国医学科学杂志。2010;25:1053–1059]。8周后,YP喂养组的最终体重明显低于对照组(图5a)。高脂组与YP组的比较表明,在低浓度的YP下,体重减轻13%。另外,与高脂组比较,YP组肝脏和附睾脂肪重量明显低(图5b)。此外,与HFD组大鼠相比,观察到YP组肝脏(18%)和附睾(35%)脂肪重量显著减少。因此,YP在不影响饮食摄入量的前提下,可以使身体和器官减重。
2.6 YP对高脂饮食诱导肥胖大鼠的血清和血浆生化参数影响
如下表1所示,与HFD组相比,YP组的血清总胆固醇(37%)和低密度脂蛋白胆固醇(50%)显著降低,而血清高密度脂蛋白胆固醇(22%)的水平明显增加。另外,YP所有的等离子体参数均与HFD组相比显著下降。这一结果表明,YP积极改善循环和肝脏脂质水平。表1是血清和血浆生化指标的比较。
表1
2.7化合物A,B、C的构造与纯化
香橙渣发酵物(YP)的上清液(100g),用甲醇沉淀净化,,酸碱分离(0.25g),使用硅胶柱色谱法分析。采用TLC(甲苯:乙酸乙酯:甲酸=5:4:1)合成的化合物A(0.12g)、B(0.04g)和C(0.02g)的纯化如图6显示,然后进行核磁共振分析,结果如图7-9所示。结果为,化合物A、B和C被证实为与吡喃葡萄糖酯结合的绿原酸。化合物A和B的结构式如图10所示。这些化合物未曾作为关于柑橘报告的一个对象被报告过。这一结果表明,这些化合物是经乳酸菌发酵的新型生物转化化合物。
本发明具体应用途径很多,以上所述仅是本发明的优选实施方式。应当指出,以上实施例仅用于说明本发明,而并不用于限制本发明的保护范围。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,这些改进也应视为本发明的保护范围。
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1.一种具有抗肥胖活性的食物成分,其特征在于,它含有利用植物乳杆菌GAVOL-07发酵而成的香橙渣发酵物作为活性成分。
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