CN114521647B - 具有辅助提高抗氧化能力的海带益生菌发酵物及其制备方法 - Google Patents
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Abstract
本发明涉及具有辅助提高抗氧化能力的海带益生菌发酵物及其制备方法,属于保健食品技术领域。本发明所述海带益生菌发酵物的主要原料包括海带,罗伊氏乳杆菌,戊糖片球菌,嗜酸乳杆菌和葡萄糖。将海带原料磨粉后,通过不同极性溶剂提取及蛋白酶酶解,获得海带酶解活性物质及海带全活性物质;将已活化的罗伊氏乳杆菌,戊糖片球菌,嗜酸乳杆菌接种于海带酶解活性物质及海带全活性物质液体培养基中发酵,制备具有辅助提高抗氧化能力的海带益生菌发酵物。经动物实验验证,海带益生菌发酵物能够显著提高D‑半乳糖诱导的小鼠血清总抗氧化能力、超氧化物歧化酶活力,降低脂质氧化产物丙二醛的水平,显著修复肝脏组织损伤,起到延缓衰老的作用。
Description
技术领域
本发明属于保健食品技术领域,具体涉及具有辅助提高抗氧化能力的海带益生菌发酵物及其制备方法。
背景技术
衰老是一个由遗传和环境共同介导的复杂生命过程,由多种因素如氧化应激、细胞凋亡和DNA损伤等引起。在大多数情况下,与衰老有关的疾病和程序性细胞死亡主要由细胞氧化应激引起。
衰老是不可逆转的生命过程,伴随着组织器官功能退化,学习记忆能力降低,氧化应激反应增强,免疫能力下降等多种生理过程。人体在不可避免地产生自由基的同时,也在产生着抵抗自由基的抗氧化物质,以抵消自由基对人体细胞的氧化攻击。越来越多的证据表明,可通过膳食干预,通过补充抗氧化物质,提高机体抗氧化能力,清除衰老过程产生的过量自由基,减少其对细胞和组织的破坏,通过调控衰老过程中重要基因的表达水平及相互作用来延缓衰老进程。
海带是一种低热量大型食用褐藻,含有丰富的矿物质,褐藻淀粉,褐藻胶,蛋白质,甘露醇,膳食纤维等营养成分。目前,我国海带产量居世界第一,海带资源的功能性开发和利用成为当前研究的热点。已有研究表明,海带具有抗氧化、降血脂、降血糖、调节免疫和抗肿瘤等多种生物活性与保健功能。
益生菌是一类对人体有益的微生物,罗伊氏乳杆菌、戊糖片球菌、嗜酸乳杆菌是一类已经应用于生产的益生菌,在发酵过程中能利用发酵体系的营养成分,生成大量的有机酸及各种氨基酸等代谢物,提高活性物质消化率、增强发酵体系的抗氧化能力等。
本发明公开了具有辅助提高抗氧化能力的海带益生菌发酵制品及其制备方法,该方法以海带为原料,经过不同极性溶剂梯度提取分级,再通过益生菌发酵后,获得具有辅助提高抗氧化能力的海带益生菌发酵复合物。经动物实验验证,海带益生菌发酵复合物能够显著提高D-半乳糖诱导的衰老小鼠的血清总抗氧化能力、血清超氧化物歧化酶水平及降低血清中丙二醛的含量,能够显著改善肝脏组织损伤。本发明开辟了一条海带高值化综合开发利用的新途径,获得具有辅助提高抗氧化能力的海带益生菌发酵制品,促进了海带资源的开发利用,提升海带的附加值。
发明内容
本发明的目的在于提供具有辅助提高抗氧化能力的海带益生菌发酵物及其制备方法,海带经益生菌发酵,所得发酵物具有显著延缓衰老,缓解肝脏损伤等功效。
为了实现上述目的,本发明采用如下技术方案:
具有辅助提高抗氧化能力的海带益生菌发酵物的制备方法,其特征在于:将海带原料超微粉碎后,通过不同极性溶剂提取及复合蛋白酶酶解,获得海带酶解活性成分及海带全活性提取物,然后将益生菌菌株接种于含有海带酶解活性成分及海带全活性提取物的液体培养基中发酵,制得具有辅助提高抗氧化能力的海带益生菌发酵物。所述不同极性溶剂包括95vol%乙醇、55vol%乙醇和水。所述益生菌菌株包括:罗伊氏乳杆菌(Lactobacillus reuteri)、戊糖片球菌(Pediococcus pentosaceus)、嗜酸乳杆菌(Lactobacillus acidophilus)中的一种或几种。
上述具有辅助提高抗氧化能力的海带益生菌发酵物的制备方法,具体包括如下步骤:
(1)海带酶解活性物质及海带全活性物质的制备:
A.取海带原料置于50 ℃烘箱中烘干,然后超微粉碎后过40目筛备用;
B.将步骤A得到的海带粉,先以95vol%乙醇为溶剂超声辅助提取1 h,功率300 W,温度45 ℃,料液比1:10;经过滤,得海带95vol%乙醇提取液和剩余物1,剩余物1置于50 ℃烘箱中干燥;海带95vol%乙醇提取液经60 ℃减压浓缩、冷冻干燥得海带95vol%乙醇提取物;
C.取步骤B烘干的剩余物1,以55vol%乙醇为溶剂超声辅助提取1 h,功率300 W,温度45 ℃,料液比1:10;经过滤,得海带55vol%乙醇提取液和剩余物2,剩余物2置于50 ℃烘箱中干燥;海带55vol%乙醇提取液经60 ℃减压浓缩、冷冻干燥得海带55vol%乙醇提取物;
D.将步骤C烘干的剩余物2于100 °C水浴提取2 h,料液比1:40,经过滤,得海带水提取液和剩余物3,剩余物3置于50 ℃烘箱中干燥;海带水提取液经60 ℃减压浓缩、冷冻干燥得海带水提取物;
E.再将步骤D烘干的剩余物3用浓度为1%复合蛋白酶溶液(Solarbio、C8800)(w/v)53 °C水浴酶解4 h,pH 7.5,料液比1:40,过滤得海带酶解液,经60 ℃减压浓缩、冷冻干燥得海带酶解活性成分;
F.将上述制备得到的海带95vol%乙醇提取物、55vol%乙醇提取物、水提取物以及酶解活性成分混合,即组成海带全活性提取物;
(2)海带益生菌发酵物的制备
1)分别将步骤E所得海带酶解活性成分和步骤F所得海带全活性提取物配制成质量浓度为20 mg/mL的海带活性成分液体培养基,并加入2%葡萄糖(w/w),调节pH值为6.8,经80 °C巴氏杀菌20 min后,冷却得海带酶解活性成分发酵培养基和海带全活性物质发酵培养基;
2)将罗伊氏乳杆菌(Lactobacillus reuteri)、戊糖片球菌(Pediococcus pentosaceus)、嗜酸乳杆菌(Lactobacillus acidophilus)分别转接于MRS培养基连续活化两代后备用;
3)将活化后的罗伊氏乳杆菌、戊糖片球菌、嗜酸乳杆菌混合菌液接种于含有海带酶解活性成分的液体发酵培养基和含有海带全活性提取物的液体发酵培养基中,混合菌液中菌株比例为1:1:1,各菌液浓度109 CFU/mL,接种量5%~10%(v/v),密闭后置于37 °C恒温培养箱中静止发酵48 h,得海带益生菌发酵液。
上述方法制备所得的海带益生菌发酵物。
具有辅助提高抗氧化能力的海带益生菌发酵制品,所述发酵制品中含有上述海带益生菌发酵物。海带益生菌发酵物经过调配,可用于制成海带益生菌发酵饮料,海带益生菌发酵口服液,也可经过冻干制成海带益生菌发酵固体饮料。
对本发明的制备方法和原理作详细说明:
海带:海带又称昆布、江白菜、纶布、海昆布。含有丰富的碳水化合物,矿物质,较少的蛋白质和脂肪,具有较强的抗氧化功能,降血脂,免疫调节等功效。
罗伊氏乳杆菌(Lactobacillus reuteri):罗伊氏乳杆菌对肠黏膜具有很强的黏附能力,可改善肠道菌群分布,改善人体机能,提高免疫力从而促进人体健康。
戊糖片球菌(Pediococcus pentosaceus):戊糖片球菌,最适生长温度35 ℃,最高生长温度42~45 ℃。分布于麦芽汁及发酵植物材料中,常应用于食品发酵中。
嗜酸乳杆菌(Lactobacillus acidophilus):已有研究表明,嗜酸乳杆菌可以顺利通过胃肠高酸、高胆汁盐环境到达肠内,可作为食补因子在人体肠道中正常繁殖,形成优势菌,充分发挥促进机体健康特性,起到整肠作用。
复合益生菌对海带营养活性成分进行发酵,一方面益生菌利用海带营养活性物质进行增殖,另一方面,海带活性物质在益生菌的发酵作用下,更容易被人体消化吸收。发酵液中富含益生菌和海带活性物质,动物实验表明其具有显著的抗衰老作用。
本发明的有益之处:
1.本发明采用天然食品材料为原料,能够直接提高抗氧化能力、延缓衰老。
2.本发明采用的发酵益生菌,安全可靠,适于工业化生产。
3.本发明为具有辅助提高抗氧化能力的益生菌发酵海带制备方法,该方法制备的产品在提高D-半乳糖亚急性衰老小鼠的抗氧化能力方面具有显著效果,拥有较好的开发前景。
附图说明
图1为海带益生菌发酵液对D-半乳糖亚急性衰老小鼠血清总抗氧化能力(T-AOC)的影响。
图2为海带益生菌发酵液对D-半乳糖亚急性衰老小鼠血清超氧化物歧化酶能力(SOD)的影响。
图3为海带益生菌发酵液对D-半乳糖亚急性衰老小鼠血清丙二醛(MDA)含量的影响。
图4为海带益生菌发酵液对D-半乳糖亚急性衰老小鼠肝脏组织形态(400×)的影响。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1
具有辅助提高抗氧化能力的海带益生菌发酵制品的制备方法,包括以下步骤:
(1)海带酶解活性物质及海带全活性物质的制备:
A.取海带原料置于50 ℃烘箱烘干,超微粉碎后过40目筛备用;
B.将步骤A得到的海带粉,先以95vol%乙醇为溶剂超声辅助提取1 h(功率300 W,温度45 ℃,料液比1:10),经过滤,得海带95vol%乙醇提取液和剩余物1,剩余物1置于50 ℃烘箱中烘干;海带95vol%乙醇提取液,经60 ℃减压浓缩、冷冻干燥得海带95vol%乙醇提取物;
C.取步骤B烘干的剩余物1,以55vol%乙醇为溶剂超声辅助提取1 h(功率300 W,温度45 ℃,料液比1:10),经过滤,得海带55vol%乙醇提取液和剩余物2,剩余物2置于50 ℃烘箱中烘干;海带55vol%乙醇提取液,经60 ℃减压浓缩、冷冻干燥得海带55vol%乙醇提取物;
D.将步骤C烘干的剩余物2于100 °C水浴提取2 h(料液比1:40),经过滤,得海带水提取液和剩余物3,剩余物3置于50 ℃烘箱中烘干;海带水提取液,经60 ℃减压浓缩、冷冻干燥得海带水提取物;
E.再将步骤D烘干的剩余物3用浓度为1%的复合蛋白酶溶液(Solarbio、C8800)(w/v)53°C水浴酶解4 h(pH 7.5,料液比1:40),过滤得海带酶解液,经60 ℃减压浓缩、冷冻干燥得海带酶解活性成分。
F.将制备得到的海带95vol%乙醇提取物、55vol%乙醇提取物、水提取物以及酶解活性成分混合,即组成海带全活性提取物。
(2)海带益生菌发酵物的制备
1)分别将步骤E所得海带酶解活性成分和步骤F所得海带全活性提取物配制成质量浓度为20 mg/mL的海带活性成分液体培养基,并加入2%葡萄糖(w/w),调节pH值为6.8,经80 °C巴氏杀菌20 min后,冷却得海带酶解活性成分液体发酵培养基和海带全活性物质液体发酵培养基;
2)将罗伊氏乳杆菌(Lactobacillus reuteri)、戊糖片球菌(Pediococcus pentosaceus)、嗜酸乳杆菌(Lactobacillus acidophilus)分别转接于MRS培养基连续活化两代后备用;
3)将活化后的罗伊氏乳杆菌、戊糖片球菌、嗜酸乳杆菌混合菌液分别接种于含有海带酶解活性成分的液体发酵培养基和含有海带全活性物质的液体发酵培养基中,混合菌液中菌株比例为1:1:1,各菌液浓度为109 CFU/mL;接种量5%~10%(v/v),密闭后置于37 °C恒温培养箱静止发酵48 h,得海带酶解活性成分益生菌发酵物和海带全活性提取物益生菌发酵物;
(3)海带益生菌发酵制品的制备
将上述海带益生菌发酵液经过调配,可用于制成海带益生菌发酵饮料,海带益生菌发酵口服液,也可经过冻干制成海带益生菌发酵固体饮料。
实施例2 动物实验
实验方法:
以SPF级雄性ICR小鼠为研究对象,通过连续每天腹腔注射D-半乳糖150 mg/kg∙d构建衰老动物模型,然后进行为期8周的海带酶解活性成分益生菌发酵物和海带全活性物质益生菌发酵物干预喂养试验。实验设4组,每组12只,按200 mg/(kg∙bw∙d)剂量灌胃活性物质,自由进食和饮水,控制黑暗和光照12 h交替进行,室温24±1 °C:正常对照组(NC):灌胃生理盐水,腹腔注射生理盐水;衰老对照组(MC):灌胃生理盐水;腹腔注射D-半乳糖;海带酶解活性成分益生菌发酵组(KMF):用海带酶解活性成分益生菌发酵物灌胃的实验组,腹腔注射D-半乳糖;海带全活性物质益生菌发酵组(KXF):用海带全活性物质益生菌发酵物灌胃的实验组,腹腔注射D-半乳糖。给予受试样品,56天后,麻醉已禁食12 h的ICR小鼠,在洁净工作台中,先通过眼球取血,将血液收集在采血管中,再解剖获得小鼠肝脏组织,通过生化试剂盒分析血清中总抗氧化酶(T-AOC)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平,通过HE染色分析小鼠肝脏组织形态结构。
实验结果如图所示,经海带发酵活性物质干预的KMF组和KXF组衰老小鼠的血液指标与空白组与模型组相比均有显著变化,海带酶解活性物质益生菌发酵物和海带全活性物质益生菌发酵物均能够显著提高小鼠血清中T-AOC(图1)和SOD活力(图2),降低MDA含量(图3),增强小鼠抗衰老能力。从肝脏组织切片结果(图4)可以看出,与MC组相比,KMF组和KXF组肝脏组织细胞呈有序化发展,排列更加整齐,细胞核深染减少,表明海带益生菌发酵物对D-半乳糖所致的肝损伤具有保护作用。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (3)
1.具有辅助提高抗氧化能力的海带益生菌发酵物的制备方法,其特征在于:所述制备方法具体包括如下步骤:
(1)海带酶解活性物质及海带全活性物质的制备:
A. 取海带原料置于50℃烘箱烘干,然后超微粉碎后过40目筛备用;
B. 将步骤A得到的海带粉,先以95vol%乙醇为溶剂超声辅助提取1h,超声功率300W,温度45℃,料液比1:10;经过滤,得海带95vol%乙醇提取液和剩余物1,剩余物1置于50℃烘箱中干燥;海带95vol%乙醇提取液经60℃减压浓缩、冷冻干燥得海带95vol%乙醇提取物;
C. 取步骤B烘干的剩余物1,以55vol%乙醇为溶剂超声辅助提取1h,超声功率300W,温度45℃,料液比1:10;经过滤,得海带55vol%乙醇提取液和剩余物2,剩余物2置于50℃烘箱中干燥;海带55vol%乙醇提取液经60℃减压浓缩、冷冻干燥得海带55vol%乙醇提取物;
D. 将步骤C烘干的剩余物2于100℃水浴提取2 h,料液比1:40,经过滤,得海带水提取液和剩余物3,剩余物3置于50℃烘箱中干燥;海带水提取液经60℃减压浓缩、冷冻干燥得海带水提取物;
E. 再将步骤D烘干的剩余物3用浓度为1% w/v复合蛋白酶溶液53℃水浴酶解4h,pH7.5,料液比1:40,过滤得海带酶解液,经60℃减压浓缩、冷冻干燥得海带酶解活性成分;
F.将上述制备得到的海带95vol%乙醇提取物、55vol%乙醇提取物、水提取物以及酶解活性成分混合,即组成海带全活性提取物;
(2)海带益生菌发酵物的制备:
1)分别将步骤E所得海带酶解活性成分和步骤F所得海带全活性提取物配制成浓度为20mg/mL的海带活性成分液体培养基,并加入2% w/w葡萄糖,调节pH值为6.8,经80℃巴氏杀菌20min后,冷却得含有海带酶解活性成分的液体发酵培养基和含有海带全活性提取物的液体发酵培养基;
2)将罗伊氏乳杆菌(Lactobacillus reuteri)、戊糖片球菌(Pediococcus pentosaceus)、嗜酸乳杆菌(Lactobacillus acidophilus)分别转接于MRS培养基连续活化两代后备用;
3)将活化后的罗伊氏乳杆菌、戊糖片球菌、嗜酸乳杆菌混合菌液分别接种于含有海带酶解活性成分的液体发酵培养基和含有海带全活性提取物的液体发酵培养基中,混合菌液中菌株比例为1:1:1,各菌液浓度109CFU/mL,接种量5%~10% v/v,密闭后置于37℃恒温培养箱静止发酵48h,得海带酶解活性成分益生菌发酵物和海带全活性提取物益生菌发酵物。
2.如权利要求1所述方法制备所得的海带益生菌发酵物。
3.具有辅助提高抗氧化能力的海带益生菌发酵制品,其特征在于:所述发酵制品中含有权利要求2所述的海带益生菌发酵物。
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