CN114644718A - 柚子多糖提取物、提取方法、石斑鱼饲料及制备方法 - Google Patents
柚子多糖提取物、提取方法、石斑鱼饲料及制备方法 Download PDFInfo
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- CN114644718A CN114644718A CN202011491974.6A CN202011491974A CN114644718A CN 114644718 A CN114644718 A CN 114644718A CN 202011491974 A CN202011491974 A CN 202011491974A CN 114644718 A CN114644718 A CN 114644718A
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Abstract
本发明公开了一种柚子多糖提取物、提取方法、石斑鱼饲料及制备方法,其中石斑鱼饲料中包含有柚子多糖提取物以作为饲料添加剂,该石斑鱼饲料具有改善石斑鱼肝脏形态结构,提高抗氧化能力和增强免疫力的作用。在柚子幼果提取物是添加到石斑鱼基础饲料中,根据生长性能指标,其适宜添加量为基础饲料质量的0.3‑0.6g/kg。实验证实,柚子多糖提取物对石斑鱼具有特别的改善肝脏健康状况的作用。本发明实施例公开的柚子多糖提取物,不仅可以提高养殖鱼类的生长性能、提高抗氧化能力和增强免疫力,而且无毒副作用、环境友好。
Description
技术领域
本发明属于水产养殖领域,具体涉及一种柚子多糖提取物、提取方法、石斑鱼饲料及制备方法。
背景技术
目前,由于环境污染,霉变饲料的使用、化学合成药品、抗生素和杀虫剂的滥用等因素引起的鱼类肝脏疾病在全国许多地区频繁发生,造成多种鱼类的大面积死亡,给水产养殖业造成极大的经济损失。
肝脏是鱼体内最主要的代谢器官及最大的消化腺。鱼类肝病已成为当前水产养殖中一种常见而危害很大的疾病,引起了广泛的重视。因此,研究具有保肝护肝作用的饲料添加剂,对于水产品质量安全和品质的提升具有重要的现实意义。
植物中含有多糖、生物碱、皂甙、挥发油、蒽类和有机酸等,具有免疫调节作用。植物提取物在鱼类养殖中具有广泛的应用,主要用于鱼病的防治,具有抗菌、抗病毒、抗氧化及促进免疫功能的有利改善,提高机体抗病能力等作用,因其副作用少,毒性低,不产生抗药性等特点,具有化学药物不可比拟的优势。
植物提取物可以调节鱼类的免疫***,提高养殖鱼类的抗应激能力和免疫机能。同时,也可以作为饲料添加剂促进鱼体的生长,增强抗病力,提高抗氧化能力。因此,植物提取物作为水产养殖动物的饲料添加剂具有广阔的意义前景。
柚子幼果为芸香科柑橘属植物果实,我国柚子的种植面积广,产量高,但同时由于柑橘属座果率低和人工疏花疏果的需要而产生大量的幼果。目前幼果违背得到有效利用,被丢弃在田间。因此,柚子幼果的有效利用将会给柚子行业和环境带来巨大的益处。
石斑鱼,属于鲈形目、鮨科、石斑鱼属,主要分布于热带、亚热带暖水海域,是最重要的海洋鱼类之一。在中国2019年的年产量达到183127吨,比前一年增长了14.76%。石斑鱼是褐点石斑鱼(母本)和鞍带石斑鱼(父本)杂交的一种新兴的海水养殖鱼类新品种,具有生长快速、肉质鲜美、营养价值高、抗病能力强等优点,在我国沿海地区得到了快速发展。但由于养殖规模的扩大,水环境的恶化,疾病防控不足,抗生素等化学物质的滥用,导致鱼类疾病爆发频繁,造成了大量鱼类死亡以及经济损失。
有鉴于此,寻找无污染纯天然无公害饲料添加剂以应用于石斑鱼的养殖以增强其免疫功能具有重要意义。
发明内容
1、要解决的问题
针对上述现有技术存在的问题,本发明提供一种柚子多糖提取物、提取方法、含该柚子多糖提取物的石斑鱼饲料及制备方法,含该多糖提取物的石斑鱼饲料具有特别的改善肝脏免疫功能,提高抗氧化能力和保肝护肝的作用。
2、技术方案
为解决上述问题,本发明采用如下的技术方案。
一种柚子多糖提取物,其糖链的主链为→2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→4) -α-D-GalAp-(1,支链为→5)-α-L-Araf-(1→5)-α-L-Araf-(1→3,6)-β-D-Galp-(1→6)-β-D-Gal p-(1→。
进一步的,所述柚子多糖提取物的分子结构式为:
进一步的,柚子多糖提取物的糖链的主链中1,5-linked Araf的含量为22.10-22.90%;1,4 -linked Glcp的含量为14.35-14.50%;1,4-linked Galp的含量分别为17.10-17.90%。
进一步的,柚子多糖提取物的糖链的主链中1,5-linked Araf的含量为22.40%;1,4-linked Glcp的含量为14.42%;1,4-linked Galp的含量分别为17.68%。
一种柚子多糖提取物的提取方法,包括如下步骤:
S1:柚子幼果干燥后制成粉末,按照料液比为1:30加入去离子水,用果胶酶和纤维素酶 (2%w/v,pH6.0,50℃)发酵1.5-3h;
S2:对步骤S1获取的产物灭酶后,通过超声波破壁萃取;
S3:超声破壁萃取后,对萃取液进行离心和浓缩,采用三氯乙酸脱蛋白,用四倍体积的乙醇在4℃条件下沉淀过夜分层,取沉淀;
S4:将下层的沉淀溶于去离子水中,并注入DEAE-Sepharose快速流动色谱柱中,加入超纯水和不同浓度的梯度NaCI溶液(0.2,0.5,2mol/L,pH7.0),在5mL/min下洗脱柱;收集成分后浓缩、透析(4℃,48h)和冻干后,将馏分(1mg/mL)应用于SephadexG-200柱上进行纯化。
一种柚子多糖提取物作为饲料添加剂的应用。
一种石斑鱼饲料,包括柚子多糖提取物和基础饲料,其中柚子多糖提取物占基础饲料质量的0.03-0.06%。
进一步的,所述的基础饲料,其成分组成为:粗蛋白44.52-44.83%、粗脂肪11.58-11.91%、粗灰分11.28-11.89%、水分6.36-6.71%。
进一步的,所述石斑鱼饲料包括以下按重量份数计的原料:
一种石斑鱼饲料的制备方法,包括如下步骤:
步骤1:取鱼粉、豆粕、面粉并混合均匀;
步骤2:往步骤1中的混合物中加入充分混匀的复合维生素矿物盐、氯化胆碱、维生素C、磷酸二氢钙,并搅拌均匀;
步骤3:往步骤S2中获得的混合物中加入鱼油、豆油和卵磷脂,搅拌均匀以形成混合物物;
步骤4:准备步骤3中混合物质量总和25-35%的水,将柚子多糖提取物与啤酒酵母混合均匀后充分溶解在水中充分搅拌以形成混合溶液,其中水的温度控制在70-80℃之间;
步骤5:步骤4中的混合溶液添加到步骤3中的混合物中的混合溶液中并充分混合,制成颗粒料后晾干。
3、有益效果
相比于现有技术,本发明实施例公开了从柚子幼果中提取了多糖提取物,对其进行分离纯化,对结构进行了表征,得率与纯度较高,该多糖提取物具有理想的免疫调节作用;
本发明实施例将柚子多糖提取物添加到饲料后,作为功能性生态饲料具有增强石斑鱼生长性能及免疫功能的作用,不仅可以提高养殖鱼类的生长性能、饲料效率和免疫力以及改善肝脏结构、脂肪沉积;
本发明实施例采用柚子多糖提取物作为添加剂形成的石斑鱼饲料,无毒副作用、环境友好,减少水体的污染、药物的残留,进而降低对水中生物的耐药性、免疫***受抑制及肝损伤造成的损伤;
本发明实施例采用柚子作为原料能对废弃资源进行再次利用,充分实现资源的循环利用。
附图说明
图1为饲喂添加柚子多糖提取物(YZ-0.5)的石斑鱼饲料8周的石斑鱼肝组织的变化(H E染色,400×);(A)饲喂0mg/kg YZ-0.5的肝组织(YZ-0.5);(B)饲喂150mg/kg Y Z-0.5的肝组织(YZ-0.5-150);(C)饲喂300mg/kg YZ-0.5的肝组织(YZ-0.5-300);(D)饲喂600mg/kgYZ-0.5的肝组织(YZ-0.5-600);(E)饲喂1200mg/kg YZ-0.5的肝组织(YZ-0. 5-1200);
图2为饲喂添加柚子多糖提取物(YZ-0.5)饲料8周的石斑鱼肝脏组织化学的变化(油红O染色,400×);(A)饲喂0mg/kg YZ-0.5的肝组织(YZ-0.5);(B)饲喂150mg/k g YZ-0.5的肝组织(YZ-0.5-150);(C)饲喂300mg/kg YZ-0.5的肝组织(YZ-0.5-300);(D) 饲喂600mg/kg YZ-0.5的肝组织(YZ-0.5-600);(E)饲喂1200mg/kg YZ-0.5的肝组织(YZ -0.5-1200);
图3为饲料中添加不同含量的柚子多糖提取物(YZ-0.5)对石斑鱼肝脏脂代谢相关基因表达的影响;其中LPL:脂蛋白脂肪酶;ACO1:酰基-CoA氧化酶1;PPARα:过氧化氢酶体增殖物激活受体α;
图4为柚子多糖提取物(YZ-0.5)对石斑鱼原代肝细胞的影响;(A)(B)(C)肝细胞用YZ-0.5(0,37.5,75,150,300和600μg/ml)分别孵育24,48,72h;(D)用2ml/L 20%的脂肪乳(LE)诱导肝细胞48h,再加入不同浓度的YZ-0.5(75,150和300μg/ml)处理24h;
图5为柚子多糖提取物(YZ-0.5)对20%LE诱导的肝细胞形态变化的影响;细胞爬片进行HE染色后,在倒置显微镜下观察细胞形态(200×);(A)对照组:细胞中不加LE 和YZ-0.5处理;(B)模型组:细胞中只加入20%LE(2ml/L)处理72h;(C)自然恢复组:细胞中先加入20%LE(2ml/L)处理48h,换液后再加入新鲜的完全培养基孵育24h。(D)YZ -0.5(75μg/ml)+20%LE(2ml/L);(E)YZ-0.5(150μg/ml)+20%LE(2ml/L);(F)YZ- 0.5(300μg/ml)+20%LE(2ml/L);
图6为柚子多糖提取物(YZ-0.5)降低LE诱导的细胞凋亡;将细胞进行Annexin V-AP C/7AAD双染后,用流式细胞仪检测细胞的凋亡率;Q1:死亡细胞;Q2:晚凋和坏死细胞;Q3:活细胞;Q4:早凋细胞;
图7为柚子多糖提取物(YZ-0.5)对20%LE诱导的肝细胞脂代谢相关基因表达的影响; (A)脂肪合成基因(G6PD和ME1);脂肪分解基因(CPT1,PPARα和ACO1)。G6PD:葡萄糖6-磷酸脱氢酶;ME1:苹果酸酶1;CPT1:肉碱棕榈酰转移酶1;PPARα:过氧化氢酶体增殖物激活受体α;ACO1:酰基CoA氧化酶1。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
一种柚子多糖提取物(YZ-0.5),其糖链的主链为→2,4)-α-L-Rhap-(1→4)-α-D-Gal Ap-(1→4)-α-D-GalAp-(1,支链为→5)-α-L-Araf-(1→5)-α-L-Araf-(1→3,6)-β-D-Galp-(1 →6)-β-D-Galp-(1→。
具体的柚子多糖提取物的分子结构式为:
柚子多糖提取物(YZ-0.5)的糖链的主链中1,5-linked Araf的含量为22.10-22.90%;1,4- linked Glcp的含量为14.35-14.50%;1,4-linked Galp的含量分别为17.10-17.90%。
作为优选的方案,柚子多糖提取物(YZ-0.5)的糖链的主链中1,5-linked Araf的含量为2 2.40%;1,4-linked Glcp的含量为14.42%;1,4-linked Galp的含量分别为17.68%。
在提取柚子多糖提取物(YZ-0.5)后通过傅里叶变换红外光谱、分子量估计、单糖组成、甲基化分析和核磁共振光谱对柚子多糖提取物(YZ-0.5)进行结构表征。
红外光谱结果显示柚子多糖提取物(YZ-0.5)含有-OH、C-H、N-H、C-O、C=O和O-H振动。HPGPC分析得出柚子多糖提取物(YZ-0.5)的分子量约为15,332Da。根据保留时间和HPIEC谱图可知,柚子多糖提取物(YZ-0.5)是由鼠李糖、半乳糖醛酸、葡萄糖、半乳糖和***糖组成的杂多糖。通过GC-MS对柚子多糖提取物(YZ-0.5)进行甲基化分析,结果可知,其糖链类型多达11种,且可知其主链主要含有1,5-linked Araf、1,4-linked Glcp和1, 4-linked Galp,含量分别为:22.40%、14.42%和17.68%。最后通过核磁共振光谱推测YZ-0.5的主链为→2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→4)-α-D-GalAp-(1,支链为→5)-α-L-Araf-(1→5)-α-L-Araf-(1→3,6)-β-D-Galp-(1→6)-β-D-Galp-(1→,最终获得的分子结构式如上式所示。
从柚子中提取上述的柚子多糖提取物的提取方法,包括如下步骤:
S1:柚子幼果干燥后制成粉末,按照料液比为1:30加入去离子水,用果胶酶和纤维素酶 (2%w/v,pH6.0,50℃)发酵1.5-3h;优选的发酵时间为2h。
S2:对步骤S1获取的产物灭酶后,通过超声波破壁萃取;
S3:超声破壁萃取后,对萃取液进行离心和浓缩,采用三氯乙酸脱蛋白,用四倍体积的乙醇在4℃条件下沉淀过夜分层,取沉淀;
S4:将下层的沉淀溶于去离子水中,并注入DEAE-Sepharose快速流动色谱柱中,加入超纯水和不同浓度的梯度NaCI溶液(0.2,0.5,2mol/L,pH7.0),在5mL/min下洗脱柱;收集成分后浓缩、透析(4℃,48h)和冻干后,将馏分(1mg/mL)应用于SephadexG-200柱上进行纯化。
一种如上述所述的柚子多糖提取物作为饲料添加剂的应用。将上述的柚子多糖提取物作为饲料的添加剂应用到饲料中能够提高饲料的营养价值。在本实施例中主要研究了该柚子多糖提取物作为石斑鱼饲料的添加剂的应用。当然该柚子多糖提取物还可以应用到其他饲料或食物中。
本发明还公开了一种石斑鱼饲料,包括上述的柚子多糖提取物和基础饲料,其中柚子多糖提取物占基础饲料质量的0.03-0.06%。实验研究表明根据生长性能,其适宜添加量是基础饲料质量的0.03-0.06%,其能显著增强石斑鱼免疫力,提高抗氧化能力和具有保肝护肝的作用。添加量过高会影响石斑鱼的生长性能和肝脏结构以及免疫力,添加量过低则达不到效果具体实验验证见下文。
所述的石斑鱼基础饲料,其成分组成为:粗蛋白44.52-44.83%、粗脂肪11.58-11.91%、粗灰分11.28-11.89%、水分6.36-6.71%。
具体的实施例1
公开了一种石斑鱼饲料,所述石斑鱼饲料包括以下按重量份数计的原料:
实施例2
给出了另一具体的饲料配比:所述石斑鱼饲料包括以下按重量份数计的原料:
实施例3
给出了另一具体的饲料配比,所述石斑鱼饲料包括以下按重量份数计的原料:
本发明的另一实施例还公开了一种上述石斑鱼饲料的制备方法,包括如下步骤:
步骤1:取鱼粉、豆粕、面粉并混合均匀;
步骤2:往步骤1中的混合物中加入充分混匀的复合维生素矿物盐、氯化胆碱、维生素C、磷酸二氢钙,并搅拌均匀;
步骤3:往步骤S2中获得的混合物中加入鱼油、豆油和卵磷脂,搅拌均匀以形成混合物物;
步骤4:准备步骤3中混合物质量总和25-35%的水,将柚子多糖提取物与啤酒酵母混合均匀后充分溶解在水中充分搅拌以形成混合溶液,其中水的温度控制在70-80℃之间;
步骤5:步骤4中的混合溶液添加到步骤3中的混合物中的混合溶液中并充分混合,制成颗粒料后晾干。
本发明实施例公开的石斑鱼的饲料由于添加了柚子多糖提取物,不仅可以提高养殖鱼类的生长性能、饲料效率和免疫力以及改善肝脏结构、提高抗氧化能力,而且无毒副作用、环境友好。柚子多糖提取物作为一种功能性绿色添加剂,其在饲料中添加所取得的效果正是在目前水产养殖发展过程中所需要的。
添加柚子多糖提取物的饲料对石斑鱼的影响实验验证
本实验为了探究饲料中添加柚子多糖提取物(YZ-0.5)对石斑鱼生长性能的影响,在基础饲料中添加柚子多糖提取物(YZ-0.5)饲喂石斑鱼,在实验中采用的石斑鱼为虎龙杂交石斑鱼,当然也可以采用珍珠龙胆杂交石斑鱼或者其他石斑鱼,设置4个梯度并形成四组YZ- 0.5-150、YZ-0.5-300、YZ-0.5-600和YZ-0.5-1200,具体的每组的饲料成分如下表1,在这四组中柚子多糖提取物(YZ-0.5)的添加量分别为150、300、600、1200mg/kg,试验养殖期为8周,并同时设置对照组YZ-0.5-0,其中柚子多糖提取物(YZ-0.5)的添加量为0。
表1试验饲料组成及营养水平(克/千克)
添加柚子多糖提取物(YZ-0.5)的石斑鱼饲料对石斑鱼生长性能的影响
为了探究添加不同浓度的添加柚子多糖提取物(YZ-0.5)的石斑鱼饲料对石斑鱼生长性能的影响,对喂养8周后的石斑鱼进行了研究,具体的对石斑鱼生长性能,饲料利用和形态学参数的影响分别如表2和表3所示。
表2饲料中添加柚子多糖提取物(YZ-0.5)对石斑鱼生长性能和饲料利用的影响
IBW:初始体重;FBW:终末体重;WG:增重率;FE:饲料效率。
表3饲料中添加柚子多糖提取物(YZ-0.5)对石斑鱼形态学参数的影响
HSI:肝体比;VSI:脏体比;CF肥满度。
研究表明与对照组相比,添加相对较低剂量(分别为150mg/kg、300mg/kg和600mg/kg)YZ-0.5的饲料可提高石斑鱼的生长性能,而添加较高剂量(1200mg/kg)YZ-0.5的饲料会使石斑鱼的生长性能下降。此外,150mg/kg、300mg/kg和600mg/kg组YZ-0.5对改善石斑鱼饲料效率有显著的效果。在本研究中发现,YZ-0.5-300、YZ-0.5-600和YZ-0.5-1200 组中的肝体比(HSI)显著的低于YZ-0.5-0组(P<0.05)。YZ-0.5-0组脏体比(VSI)和肥满度 (CF)显著的高于其他实验组的。这说明饲喂添加YZ-0.5饲料对鱼的形态学参数(如HSI、 VSI)有显著影响,可以改善鱼类的脂质沉积。
添加柚子多糖提取物(YZ-0.5)的石斑鱼饲料对石斑鱼肝脏形态结构和组织化学的影响
为了探究添加柚子多糖提取物(YZ-0.5)的石斑鱼饲料对石斑鱼肝脏形态结构和组织化学的影响。在各组石斑鱼养殖8周后,取肝脏组织做HE染色和油红O染色(图1,图2)。肝脏是代谢过程的关键器官,对营养物质的消化、代谢和储存起着至关重要的作用,肝脏的正常功能会受到各种因素的影响。肝脏脂质积累被认为是一种浪费,因为它转移了生长中的能量,导致产量下降,饲料效率低下,生长不良。这种病理状态对养殖鱼类的健康有害。研究发现高脂饲料喂养的石斑鱼肝细胞肿胀,大量肝细胞空泡化,细胞核偏移。饲料中加入银杏叶提取物(GBE)后可逆转鱼类肝脏这种病理损伤。在另一项研究中,观察到在饲喂氧化鱼油的鱼体内出现了肝细胞空泡化和细胞核偏移等异常现象。我们的结果得到了组织病理学检测的支持,饲喂YZ-0.5-150、YZ-0.5-300和YZ-0.5-600组饲料的鱼肝细胞形态正常,细胞核呈卵圆形,核仁突出。而在YZ-0.5-0和YZ-0.5-1200组肝细胞出现大量的空泡化,细胞核肥大,部分核仁解体和肝细胞结构损伤。肝脏油红O染色结果的脂滴含量进一步证明了以上结果。与YZ-0.5-0组相比,饲喂添加YZ-0.5剂量达到600mg/kg的饲料的鱼,肝脏脂滴含量显著的减少,但当添加量更高(达到1200mg/kg)时,肝脏中的脂滴含量又增加了。表明饲料中添加YZ-0.5可降低肝脏中脂滴的含量,显著改善杂交石斑鱼肝脏结构。
添加柚子多糖提取物(YZ-0.5)的石斑鱼饲料对石斑鱼肝脏脂代谢相关基因的mRNA表达的影响
图3表示了饲料中添加不同含量的柚子多糖提取物(YZ-0.5)对石斑鱼肝脏脂代谢相关基因的mRNA表达的影响。由图可知当饲料中YZ-0.5的添加量增加到600mg/kg时,脂蛋白脂肪酶(LPL)的mRNA表达量显著的升高(P<0.05)。与YZ-0.5-0组相比,脂蛋白脂肪酶(LPL)的mRNA表达量在YZ-0.5添加组显著的上调(P<0.05)。与YZ-0.5-0组组相比,过氧化氢酶体增殖物激活受体α(PPARα)的mRNA表达量在YZ-0.5-300,YZ-0.5-600和YZ -0.5-1200组显著的上调(P<0.05)。
柚子多糖提取物(YZ-0.5)对石斑鱼免疫功能的提升机制探究
为了进一步研究柚子多糖提取物(YZ-0.5)对石斑鱼免疫功能的提升机制,分离培养了石斑鱼原代肝细胞,建立肝细胞脂肪变性模型,然后再用不同浓度的柚子多糖提取物(YZ-0. 5)进行干预,深入探讨YZ-0.5降低石斑鱼脂肪沉积的代谢机制。
用CCK-8法检测了不同浓度柚子多糖提取物(YZ-0.5)对细胞增殖的影响,如图4所示,结果表明75-600μg/ml的YZ-0.5孵育肝细胞后,细胞的存活率高于95%,说明75-600μg/ml的YZ-0.5对肝细胞没有毒(图4A,B,C)。为了证实YZ-0.5可以减轻LE诱导的细胞活力下降,我们用2ml/L 20%LE预处理肝细胞48h,然后用不同浓度的YZ-0.5再孵育肝细胞24h;用CCK-8分析细胞存活率(图4D)。结果显示模型组肝细胞的存活率为76.37±1.9 2%,与对照组相比显著的下降(P<0.05)。与模型组相比,自然恢复组的细胞存活率升高了(P <0.05),而150μg/ml YZ-0.5组细胞存活率显著的高于自然恢复组(P<0.05)。
图5为YZ-0.5对20%LE诱导的肝细胞形态变化的影响,石斑鱼肝细胞形态通过HE染色进行分析。对照组肝细胞排列整齐,细胞边缘清晰,细胞核正常。模型组细胞形态结构损伤,部分细胞核裂解,有大量空泡形成。自然恢复组,细胞形态损伤和细胞核肥大有所恢复,但细胞状态不是很好。当加入低、中和高三种浓度的YZ-0.5处理肝细胞后,观察到不同的细胞形态。在75和150μg/ml YZ-0.5组,细胞形态损伤显著的降低。然而,在300μg/ml YZ-0.5组,细胞有肿胀现象。
细胞用V-APC/7AAD进行双染,然后用流式细胞仪检测细胞的凋亡率(图5)。结果表明,模型组中细胞的凋亡率(Q2+Q4:晚期凋亡及坏死细胞和早期凋亡细胞)为19.62±0.55%,显著高于对照组中的凋亡率(5.70±0.14%)(P<0.05)。自然恢复组与模型组相比凋亡率显著上升(P<0.05)。然而,随着不同浓度YZ-0.5(75,150和300μg/mL)的加入,与自然恢复组相比,凋亡率显著的下降(P<0.05)。YZ-0.5浓度为150μg/mL组,凋亡率最低,为5.35±0.41%。
为了检测柚子多糖提取物(YZ-0.5)对脂质积累分子代谢的影响,脂肪生成和脂质分解相关基因的表达如图6所示。模型组葡萄糖6-磷酸脱氢酶(G6PD)和苹果酸酶1(ME1)的mRNA表达水平较对照组明显上调(P<0.05)。自然恢复组比模型组低(P<0.05)。然而,YZ -0.5(75μg/ml、150μg/ml和300μg/ml)组与自然恢复组相比葡萄糖6-磷酸脱氢酶(G6 PD)和苹果酸酶1(ME1)显著下调(P<0.05)。与对照组相比,模型组脂肪分解相关基因过氧化氢酶体增殖物激活受体α(PPARα)显著的下降(P<0.05)。自然恢复组过氧化氢酶体增殖物激活受体α(PPARα)基因表达量与模型组相比显著的升高(P<0.05)。与自然恢复组相比,150和300μg/ml YZ-0.5组PPARα基因表达水平显著的上调(P<0.05)。
图7为YZ-0.5对20%LE诱导的肝细胞脂代谢相关基因表达的影响;本研究根据体内和体外实验研究表明柚子多糖提取物(YZ-0.5)通过下调脂肪合成基因和上调脂肪分解基因可抑制石斑鱼脂肪沉积。此外,对鱼摄入高脂饲料后引发的健康问题可通过YZ-0.5对肝脏的保护作用和增强免疫功能进行改善。这些结果可能有助于研究人员更好地了解脂质代谢的复杂调控机制,为膳食干预抑制脂质过度积累、改善鱼类健康提供有益信息。
实验证实,柚子多糖提取物(YZ-0.5)对石斑鱼具有特别的改善肝脏免疫功能,提高抗氧化能力和保肝护肝的作用,且石斑鱼是目前养殖规模较大的一个品种具有较好的市场前景。
以上内容是结合具体实施方式对本发明作进一步详细说明,不能认定本发明具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明的构思的前提下,还可以做出若干简单的推演或替换,都应当视为属于本发明所提交的权利要求书确定的保护范围。
Claims (10)
1.一种柚子多糖提取物,其特征在于,其糖链的主链为→2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→4)-α-D-GalAp-(1,支链为→5)-α-L-Araf-(1→5)-α-L-Araf-(1→3,6)-β-D-Galp-(1→6)-β-D-Galp-(1→。
3.根据权利要求1所述的柚子多糖提取物,其特征在于,柚子多糖提取物的糖链的主链中1,5-linked Araf的含量为22.10-22.90%;1,4-linked Glcp的含量为14.35-14.50%;1,4-linked Galp的含量分别为17.10-17.90%。
4.根据权利要求1所述的柚子多糖提取物,其特征在于,柚子多糖提取物的糖链的主链中1,5-linked Araf的含量为22.40%;1,4-linked Glcp的含量为14.42%;1,4-link edGalp的含量分别为17.68%。
5.一种从柚子中提取如权利要求1-4任一项所述的柚子多糖提取物的提取方法,其特征在于,包括如下步骤:
S1:柚子幼果干燥后制成粉末,按照料液比为1:30加入去离子水,用果胶酶和纤维素酶(2%w/v,pH6.0,50℃)发酵1.5-3h;
S2:对步骤S1获取的产物灭酶后,通过超声波破壁萃取;
S3:超声破壁萃取后,对萃取液进行离心和浓缩,采用三氯乙酸脱蛋白,用四倍体积的乙醇在4℃条件下沉淀过夜分层,取沉淀;
S4:将下层的沉淀溶于去离子水中,并注入DEAE-Sepharose快速流动色谱柱中,加入超纯水和不同浓度的梯度NaCI溶液(0.2,0.5,2mol/L,pH7.0),在5mL/min下洗脱柱;收集成分后浓缩、透析(4℃,48h)和冻干后,将馏分(1mg/mL)应用于SephadexG-200柱上进行纯化。
6.一种如权利要求1至4任一项所述的柚子多糖提取物作为饲料添加剂的应用。
7.一种石斑鱼饲料,其特征在于,包括如权利要求1至4任一项所述的柚子多糖提取物和基础饲料,其中柚子多糖提取物占基础饲料质量的0.03-0.06%。
8.根据权利要求7所述的石斑鱼饲料,其特征在于,所述的基础饲料,其成分组成为:粗蛋白44.52-44.83%、粗脂肪11.58-11.91%、粗灰分11.28-11.89%、水分6.36-6.71%。
10.一种如权利要求7至9任一项所述的石斑鱼饲料的制备方法,其特征在于,包括如下步骤:
步骤1:取鱼粉、豆粕、面粉并混合均匀;
步骤2:往步骤1中的混合物中加入充分混匀的复合维生素矿物盐、氯化胆碱、维生素C、磷酸二氢钙,并搅拌均匀;
步骤3:往步骤S2中获得的混合物中加入鱼油、豆油和卵磷脂,搅拌均匀以形成混合物物;
步骤4:准备步骤3中混合物质量总和25-35%的水,将柚子多糖提取物与啤酒酵母混合均匀后充分溶解在水中充分搅拌以形成混合溶液,其中水的温度控制在70-80℃之间;
步骤5:步骤4中的混合溶液添加到步骤3中的混合物中的混合溶液中并充分混合,制成颗粒料后晾干。
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