CN106579464A - 一种治疗肝硬化的营养组合物及其制备方法 - Google Patents
一种治疗肝硬化的营养组合物及其制备方法 Download PDFInfo
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Abstract
本发明属于功能营养食品领域,具体涉及一种治疗肝硬化的营养组合物及其制备方法。本发明根据我国肝硬化人群的营养代谢特点及所需营养素,科学复配用于肝硬化治疗的营养组合物,通过合理的营养干预,改善肝病患者的营养代谢状态、促进肝细胞再生、促进肝功能的恢复,能有效改善肝硬化患者的生化指标和Child‑Pugh评分,提高生活质量。
Description
技术领域:
本发明属于功能营养食品领域,具体涉及一种治疗肝硬化的营养组合物及其制备方法。
背景技术:
肝硬化是临床常见的慢性进行性肝病,由一种或多种病因长期或反复作用形成的弥漫性肝损害。引起肝硬化的病因很多,可分为病毒性肝炎肝硬化、酒精性肝硬化、代谢性肝硬化、胆汁淤积性肝硬化、肝静脉回流受阻性肝硬化、自身免疫性肝硬化、毒物或药物性肝硬化、营养不良性肝硬化、隐源性肝硬化等。
肝硬化患者由于能量摄入与能量消耗平衡的破坏,几乎所有患者都存在营养不良和代谢障碍。肝硬化患者的营养不良主要表现为对葡萄糖的利用减低,而对脂肪和蛋白质的消耗增多;肝硬化患者主要表现为白天代谢低夜间代谢高。尽管可以通过服用抗病毒药物控制病毒性肝硬化的进展,通过戒酒缓解酒精性肝硬化,但目前改变肝硬化无特效药,且不宜滥用药物,否则将加重肝脏负担而适得其反。
睡前加餐是一种缩短空腹期并能提高基质利用率,改善肝硬化患者营养状态的干预措施。作者通过前期临床研究和荟萃分析,总结LES(late evening snack,睡前加餐)对基质利用和营养状态的调节作用,睡前小餐均能减少脂质氧化并能改善氮平衡。日间服用等热量、等氮量的小餐不具有LES的代谢或临床益处。
我国肝硬化人群以病毒性肝病为主要人群,其能量和营养素的代谢特点是:蛋白质代谢处于负平衡状态,氨基酸代谢发生明显异常(支链氨基酸/芳香族氨基酸下降);脂溶性维生素、B族维生素、叶酸、钙、镁、锌、硒摄入量严重缺乏;同时肠黏膜屏障功能降低,营养不足和营养失衡使得肠绒毛的形态和功能障碍。针对肝硬化患者的营养不良,单纯的食品添加剂和/或现有的成品食物作为睡前加餐食品不能达到完全合理化的营养搭配,进而有必要为肝硬化患者提供一款适合其睡前加餐的营养餐包。
本发明依据我国肝硬化人群的营养代谢特点及所需营养素需求进行设计:多种营养成分的有机复合、协同作用,达到辅助肝硬化患者治疗的功效。
发明内容:
为了解决上述技术问题,本发明提供一种治疗肝硬化的营养组合物,所述营养组合物重量份数组成如下:
浓缩乳清蛋白5-50份,大豆分离蛋白5-50份,玉米低聚肽1-5份,磷脂2-5份,麦芽糊精5-50份,菊粉1-20份,牛樟芝提取物1-3份,牛磺酸1-5份,缬氨酸1-4份,亮氨酸1-6份,异亮氨酸1-4份,精氨酸1-4份,还原型谷胱甘肽1-4份,复合矿物质0.01-0.2份,复合维生素0.05-0.1份;
所述复合矿物质重量份数组成如下:碳酸钙10份、乳酸镁8份、乳酸锌1份;
所述复合维生素重量份数组成如下:维生素A 1份、维生素D 0.002份、维生素E 10份、维生素B1 2份、维生素B2 2份、维生素B6 2份、维生素B12 2份、维生素C 100份、烟酸20份、叶酸1份、泛酸10份;
所述玉米低聚肽的制备方法如下:
(1)向玉米蛋白粉中加入重量体积比10-20倍的水,混合均匀,调整pH8-10,加热至50-80℃保温搅拌20-60min;将料液经过离心分离操作后弃去清液,留用渣料;重复上述步骤1-3次得渣料;
(2)向上述渣料中加入8-15倍的水(m:v)调浆,调节pH8-10,温度50-70℃,按8000-10000U/g玉米蛋白粉添加碱性蛋白酶,酶解2-3h;
(3)降温至30-50℃后,调节发酵液pH至5-7,按10000-15000U/g玉米蛋白粉添加中性蛋白酶,酶解5-6h;
(4)调节pH6-7,温度40-60℃,按400-1000U/g玉米蛋白粉添加风味蛋白酶,酶解3-5h,酶解结束后,加热至85℃-95℃,灭酶15-20分钟;
(5)离心浓缩后,喷雾干燥;
所述牛樟芝提取物的制备方法如下:
牛樟芝子实体粉碎,过40-50目筛后,添加牛樟芝子实体3-6倍重量无水乙醇浸渍提取,控制温度30-45℃,2-4小时后调整温度为55-60℃1-2小时,离心,乙醇提取液浓缩、干燥得到乙醇提取物;乙醇提取后的牛樟芝子实体残渣中添加2-4倍75-85℃热水,浸泡1-2h;之后进行超声浸提,超声条件为200-300W,频率30-40KHz、40-50℃、1-2h;之后进行高压脉冲电场浸提,脉冲条件为电场强度25-35kV/cm,脉冲频率200-1000Hz,0.5-1h,连续超声高压脉冲浸提2-3次,离心后将提取液真空浓缩后喷雾干燥,得到热水提取物;将上述乙醇提取物和热水提取物合并粉碎,过60目筛,即得牛樟芝子实体提取物;剩余的牛樟芝子实体残渣保存待用;
所述还原型谷胱甘肽的制备方法如下:
(1)向上述牛樟芝子实体残渣中按20-40倍(m:v)加水,并于121℃灭菌20min;
(2)配置500g/L的葡萄糖母液,并于115℃灭菌20min后加入到步骤(1)所得溶液,使葡萄糖终浓度达到100g/L,得发酵基质;
(3)按5-10%的接种量,向步骤(2)所得发酵基质中接入酿酒酵母种子液进行发酵,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,继续发酵18-24h;
(4)发酵结束后离心分离上清,经浓缩后,冷冻干燥得还原型谷胱甘肽;
所述的种子液培养条件:
取酿酒酵母斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
所述酿酒酵母菌株具体为酿酒酵母(Saccharomyces cerevisiae)tlj2016,该菌株已于2016年7月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.12789,保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101;
所述酿酒酵母tlj2016是一株经过诱变后获得的菌株,tlj2016其葡萄糖耐受能力以及L-半胱氨酸耐受能力均得到提高,一方面在高浓度葡萄糖培养下能够提高细胞密度,另一方面L-半胱氨酸耐受能力的提高将有利于GSH在胞内大量合成,从而提高菌株大规模生产GSH的能力;
由于所述酿酒酵母tlj2016是一株可在外源添加L-半胱氨酸条件下大量合成半胱氨酸的酵母菌,将经过初步浸提之后的牛樟芝子实体残渣作为发酵基质,充分利用其未被提取的营养成分,使用酿酒酵母tlj2016在添加L-半胱氨酸的条件下发酵生产还原型谷胱甘肽,一方面,还原型谷胱甘肽对治疗肝功能异常脂肪肝及对促进肝脏脂肪代谢、降低血脂、修复损伤的肝细胞有明显的功效,另一方面,采用牛樟芝子实体残渣作为发酵基质,实现废物利用,降低了经济成本,提高原料利用率;
其余原料均为市售产品;
所述治疗肝硬化的营养组合物的制备方法为:按本领域常规方法加工为片剂、粉剂、颗粒剂、口服液等,作为睡前加餐于每日20:00-21:00直接服用,每日用量为40~120g。
有益效果:
1、本发明根据我国肝硬化人群的营养代谢特点及所需营养素,科学复配用于肝硬化治疗的营养组合物,通过合理的营养干预,改善肝病患者的营养代谢状态、促进肝细胞再生、促进肝功能的恢复,能有效改善肝硬化患者的生化指标和Child-Pugh评分,提高生活质量;
2、本发明所添加的谷胱甘肽是利用牛樟芝提取物剩余残渣作为发酵基质进行获取的,实现废物利用,降低了经济成本,提高原料利用率;
3、本发明制备谷胱甘肽采用经过诱变后获得的菌株tlj2016其葡萄糖耐受能力以及L-半胱氨酸耐受能力均得到提高,一方面在高浓度葡萄糖培养下能够提高细胞密度,另一方面L-半胱氨酸耐受能力的提高将有利于GSH(还原型谷胱甘肽)在胞内大量合成,从而提高菌株大规模生产GSH的能力。
具体实施方式:
实施例1:一种治疗肝硬化的营养组合物
一种治疗肝硬化的营养组合物,所述营养组合物重量份数组成如下:
浓缩乳清蛋白5份,大豆分离蛋白50份,玉米低聚肽1份,磷脂5份,麦芽糊精5份,菊粉20份,牛樟芝提取物1份,牛磺酸5份,缬氨酸1份,亮氨酸6份,异亮氨酸1份,精氨酸4份,还原型谷胱甘肽1份,复合矿物质0.2份,复合维生素0.05份;
按照上述配方比例称取各组分,粉碎,过80目筛,然后混合10min,得粉剂;
所述复合矿物质重量份数组成如下:碳酸钙10份、乳酸镁8份、乳酸锌1份;
所述复合维生素重量份数组成如下:维生素A 1份、维生素D 0.002份、维生素E 10份、维生素B1 2份、维生素B2 2份、维生素B6 2份、维生素B12 2份、维生素C 100份、烟酸20份、叶酸1份、泛酸10份;
所述玉米低聚肽的制备方法如下:
(1)向玉米蛋白粉中加入重量体积比10倍的水,混合均匀,调整pH8,加热至50℃保温搅拌20min;将料液经过离心分离操作后弃去清液,留用渣料;重复上述步骤1次得渣料;
(2)向上述渣料中加入8倍的水(m:v)调浆,调节pH8,温度50℃,按8000U/g玉米蛋白粉添加碱性蛋白酶,酶解2h;
(3)降温至30℃后,调节发酵液pH至5,按10000U/g玉米蛋白粉添加中性蛋白酶,酶解5h;
(4)调节pH6,温度40℃,按400U/g玉米蛋白粉添加风味蛋白酶,酶解3h,酶解结束后,加热至85℃,灭酶15分钟;
(5)离心浓缩后,喷雾干燥;
所述牛樟芝提取物的制备方法如下:
牛樟芝子实体粉碎,过40目筛后,添加牛樟芝子实体3倍重量无水乙醇浸渍提取,控制温度30℃,2小时后调整温度为55℃保持1小时,离心,乙醇提取液浓缩、干燥得到乙醇提取物;乙醇提取后的牛樟芝子实体残渣中添加2倍75℃热水,浸泡1h;之后进行超声浸提,超声条件为200W,频率30KHz、40℃、1h;之后进行高压脉冲电场浸提,脉冲条件为电场强度25kV/cm,脉冲频率200Hz,0.5h,连续超声高压脉冲浸提2次,离心后将提取液真空浓缩后喷雾干燥,得到热水提取物;将上述乙醇提取物和热水提取物合并粉碎,过60目筛,即得牛樟芝子实体提取物;剩余的牛樟芝子实体残渣保存待用;
所述还原型谷胱甘肽的制备方法如下:
(1)向上述牛樟芝子实体残渣中按20倍(m:v)加水,并于121℃灭菌20min;
(2)配置500g/L的葡萄糖母液,并于115℃灭菌20min后加入到步骤(1)所得溶液,使葡萄糖终浓度达到100g/L,得发酵基质;
(3)按5%的接种量,向步骤(2)所得发酵基质中接入酿酒酵母种子液进行发酵,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,继续发酵18h;发酵结束后,测定发酵液中GSH的含量为2108mg/L;
(4)发酵结束后离心分离上清,经浓缩后,冷冻干燥得还原型谷胱甘肽;
所述的种子液培养条件:
取酿酒酵母斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
所述酿酒酵母菌株具体为酿酒酵母(Saccharomyces cerevisiae)tlj2016,保藏编号为CGMCC No.12789;
其余原料均为市售。
实施例2:
一种治疗肝硬化的营养组合物,所述营养组合物重量份数组成如下:
浓缩乳清蛋白50份,大豆分离蛋白5份,玉米低聚肽5份,磷脂2份,麦芽糊精50份,菊粉1份,牛樟芝提取物3份,牛磺酸1份,缬氨酸4份,亮氨酸1份,异亮氨酸4份,精氨酸1份,还原型谷胱甘肽4份,复合矿物质0.01份,复合维生素0.1份;
按照上述配方比例称取各组分,粉碎,过80目筛,然后混合10min,得粉剂;
所述复合矿物质重量份数组成如下:碳酸钙10份、乳酸镁8份、乳酸锌1份;
所述复合维生素重量份数组成如下:维生素A 1份、维生素D 0.002份、维生素E 10份、维生素B1 2份、维生素B2 2份、维生素B6 2份、维生素B12 2份、维生素C 100份、烟酸20份、叶酸1份、泛酸10份;
所述玉米低聚肽的制备方法如下:
(1)向玉米蛋白粉中加入重量体积比20倍的水,混合均匀,调整pH10,加热至80℃保温搅拌60min;将料液经过离心分离操作后弃去清液,留用渣料;重复上述步骤3次得渣料;
(2)向上述渣料中加入15倍的水(m:v)调浆,调节pH10,温度70℃,按10000U/g玉米蛋白粉添加碱性蛋白酶,酶解3h;
(3)降温至50℃后,调节发酵液pH至7,按15000U/g玉米蛋白粉添加中性蛋白酶,酶解6h;
(4)调节pH7,温度60℃,按1000U/g玉米蛋白粉添加风味蛋白酶,酶解5h,酶解结束后,加热至95℃,灭酶20分钟;
(5)离心浓缩后,喷雾干燥;
所述牛樟芝提取物的制备方法如下:
牛樟芝子实体粉碎,过50目筛后,添加牛樟芝子实体6倍重量无水乙醇浸渍提取,控制温度45℃,4小时后调整温度为60℃保持2小时,离心,乙醇提取液浓缩、干燥得到乙醇提取物;乙醇提取后的牛樟芝子实体残渣中添加4倍85℃热水,浸泡2h;之后进行超声浸提,超声条件为300W,频率40KHz、50℃、2h;之后进行高压脉冲电场浸提,脉冲条件为电场强度35kV/cm,脉冲频率1000Hz,1h,连续超声高压脉冲浸提3次,离心后将提取液真空浓缩后喷雾干燥,得到热水提取物;将上述乙醇提取物和热水提取物合并粉碎,过60目筛,即得牛樟芝子实体提取物;剩余的牛樟芝子实体残渣保存待用;
所述还原型谷胱甘肽的制备方法如下:
(1)向上述牛樟芝子实体残渣中按40倍(m:v)加水,并于121℃灭菌20min;
(2)配置500g/L的葡萄糖母液,并于115℃灭菌20min后加入到步骤(1)所得溶液,使葡萄糖终浓度达到100g/L,得发酵基质;
(3)按10%的接种量,向步骤(2)所得发酵基质中接入酿酒酵母种子液进行发酵,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,继续发酵24h,发酵结束后,测定发酵液中GSH的含量为2304mg/L;
(4)发酵结束后离心分离上清,经浓缩后,冷冻干燥得还原型谷胱甘肽;
所述的种子液培养条件:
取酿酒酵母斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
所述酿酒酵母菌株具体为酿酒酵母(Saccharomyces cerevisiae)tlj2016,保藏编号为CGMCC No.12789;
其余原料均为市售。
实施例3
一种治疗肝硬化的营养组合物,所述营养组合物重量份数组成如下:
浓缩乳清蛋白30份,大豆分离蛋白30份,玉米低聚肽3份,磷脂3份,麦芽糊精30份,菊粉10份,牛樟芝提取物2份,牛磺酸3份,缬氨酸3份,亮氨酸4份,异亮氨酸2份,精氨酸2份,还原型谷胱甘肽2份,复合矿物质0.1份,复合维生素0.08份;
按照上述配方比例称取各组分,粉碎,过80目筛,然后混合10min,得粉剂;
所述复合矿物质重量份数组成如下:碳酸钙10份、乳酸镁8份、乳酸锌1份;
所述复合维生素重量份数组成如下:维生素A 1份、维生素D 0.002份、维生素E 10份、维生素B1 2份、维生素B2 2份、维生素B6 2份、维生素B12 2份、维生素C 100份、烟酸20份、叶酸1份、泛酸10份;
所述玉米低聚肽的制备方法如下:
(1)向玉米蛋白粉中加入重量体积比15倍的水,混合均匀,调整pH9,加热至65℃保温搅拌40min;将料液经过离心分离操作后弃去清液,留用渣料;重复上述步骤2次得渣料;
(2)向上述渣料中加入10倍的水(m:v)调浆,调节pH9,温度60℃,按9000U/g玉米蛋白粉添加碱性蛋白酶,酶解2.5h;
(3)降温至40℃后,调节发酵液pH至6,按12500U/g玉米蛋白粉添加中性蛋白酶,酶解5h;
(4)调节pH7,温度50℃,按700U/g玉米蛋白粉添加风味蛋白酶,酶解4h,酶解结束后,加热至90℃,灭酶20分钟;
(5)离心浓缩后,喷雾干燥;
所述牛樟芝提取物的制备方法如下:
牛樟芝子实体粉碎,过45目筛后,添加牛樟芝子实体5倍重量无水乙醇浸渍提取,控制温度40℃,3小时后调整温度为60℃保持2小时,离心,乙醇提取液浓缩、干燥得到乙醇提取物;乙醇提取后的牛樟芝子实体残渣中添加3倍80℃热水,浸泡2h;之后进行超声浸提,超声条件为250W,频率35KHz、45℃、1.5h;之后进行高压脉冲电场浸提,脉冲条件为电场强度30kV/cm,脉冲频率800Hz,1h,连续超声高压脉冲浸提3次,离心后将提取液真空浓缩后喷雾干燥,得到热水提取物;将上述乙醇提取物和热水提取物合并粉碎,过60目筛,即得牛樟芝子实体提取物;剩余的牛樟芝子实体残渣保存待用;
所述还原型谷胱甘肽的制备方法如下:
(1)向上述牛樟芝子实体残渣中按30倍(m:v)加水,并于121℃灭菌20min;
(2)配置500g/L的葡萄糖母液,并于115℃灭菌20min后加入到步骤(1)所得溶液,使葡萄糖终浓度达到100g/L,得发酵基质;
(3)按8%的接种量,向步骤(2)所得发酵基质中接入酿酒酵母种子液进行发酵,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,继续发酵20h,发酵结束后,测定发酵液中GSH的含量为2641mg/L;
(4)发酵结束后离心分离上清,经浓缩后,冷冻干燥得还原型谷胱甘肽;
所述的种子液培养条件:
取酿酒酵母斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
所述酿酒酵母菌株具体为酿酒酵母(Saccharomyces cerevisiae)tlj2016,保藏编号为CGMCC No.12789;
其余原料均为市售。
实施例4效果实验
(1)将100例年龄18-65岁肝硬化患者随机分A、B组
A组Child-Pugh评分为B级36例,C级14例;B组Child-Pugh评分为B级35例,C级15例;
排除标准:①合并甲肝、丙肝、戊肝、艾滋、梅毒感染及免疫性疾病者;②无法经肠营养者;③合并严重感染、消化道出血、未控制的高血压及糖尿病、肾功能不全、甲状腺功能亢进或减退等疾病者;
A组50例:单一碳水化合物的睡前加餐;
B组50例:睡前加餐包;
A组单一碳水化合物的睡前加餐总热卡与B组中餐包总热卡相等,实现形式为提供统一的面包来完成;
B组睡前加餐包为本发明实施例3制备的产品120g;
睡前加餐均于每日20:00-21:00摄入,同时肝硬化患者均接受常规治疗,包括原发病治疗、保肝药物使用等;
实验周期2个月。
(2)评价指标:
生化指标:血液生化指标包括白蛋白、前白蛋白、总胆红素、肝酶、碱性磷酸酶、凝血指标等,见表1;
Child-Pugh评分:见表2;
实验结果表明,两组肝功能均有改善,B组优于A组,未发现明显不良反应。
表1
表2 Child-Pugh评分
实施例5高糖条件下tlj2016发酵产GSH能力实验
(1)摇瓶培养
取tlj2016斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
(2)5L发酵罐培养
将种子液按10%接种量,接入装有3L发酵培养基的发酵罐中,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,总发酵时间为50h;
发酵培养基(g/L):(NH4)2SO4 10、葡萄糖100、K2HPO4·3H2O 8、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
发酵结束后,测定发酵液中GSH的含量为3308mg/L。
实施例6 L-半胱氨酸耐受力实验
将出发菌株以及tlj2016斜面菌种各一环,分别接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养进行培养,在培养至12h时,向摇瓶中加入不同终浓度的L-半胱氨酸,再培养10h,测定细胞干重,结果表3、4;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖20、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
表3:出发菌株L-半胱氨酸耐受力
L-半胱氨酸浓度mmol/L | 0 | 5 | 10 | 15 | 20 | 40 |
出发菌株干重g/L | 22.6 | 15.7 | 10.2 | 4.3 | 2.2 | 0.8 |
GSH浓度(mg/L) | 35.6 | 46.7 | 43.2 | 40.7 | 37.9 | 25.3 |
表4 tlj2016L-半胱氨酸耐受力
L-半胱氨酸浓度mmol/L | 0 | 5 | 10 | 15 | 20 | 40 |
tlj2016干重g/L | 25.7 | 28.5 | 23.6 | 21.2 | 20.6 | 18.7 |
GSH浓度(mg/L) | 73.2 | 98.3 | 113.5 | 121.7 | 127.5 | 135.8 |
从表3、4的结果可以看出,对于出发菌株,培养基中添加L-半胱氨酸,细胞停止生长,并且开始自溶,导致GSH增长率随着L-半胱氨酸浓度的升高而降低;而低浓度L-半胱氨酸下,tlj2016仍能够缓慢生长,随着L-半胱氨酸浓度的提高,tlj2016菌株的细胞干重缓慢下降,而GSH浓度持续增长,这一结果将有利于GSH生产过程中通过添加前体氨基酸-L-半胱氨酸提促进GSH的生产。
Claims (8)
1.一种治疗肝硬化的营养组合物,其特征在于,所述营养组合物重量份数组成如下:浓缩乳清蛋白5-50份,大豆分离蛋白5-50份,玉米低聚肽1-5份,磷脂2-5份,麦芽糊精5-50份,菊粉1-20份,牛樟芝提取物1-3份,牛磺酸1-5份,缬氨酸1-4份,亮氨酸1-6份,异亮氨酸1-4份,精氨酸1-4份,还原型谷胱甘肽1-4份,复合矿物质0.01-0.2份,复合维生素0.05-0.1份;
所述牛樟芝提取物的制备方法如下:
牛樟芝子实体粉碎,过40-50目筛后,添加牛樟芝子实体3-6倍重量无水乙醇浸渍提取,控制温度30-45℃,2-4小时后调整温度为55-60℃1-2小时,离心,乙醇提取液浓缩、干燥得到乙醇提取物;乙醇提取后的牛樟芝子实体残渣中添加2-4倍75-85℃热水,浸泡1-2h;之后进行超声浸提,超声条件为200-300W,频率30-40KHz、40-50℃、1-2h;之后进行高压脉冲电场浸提,脉冲条件为电场强度25-35kV/cm,脉冲频率200-1000Hz,0.5-1h,连续提取超声高压脉冲浸提2-3次,离心后将提取液真空浓缩、喷雾干燥,得到热水提取物;将上述乙醇提取物和热水提取物合并粉碎,过60目筛,即得牛樟芝子实体提取物;剩余的牛樟芝子实体残渣保存待用;
所述还原型谷胱甘肽的制备方法如下:
(1)向上述牛樟芝子实体残渣中按20-40倍加水,并于121℃灭菌20min;
(2)向步骤(1)所得溶液,加入葡萄糖终浓度达到100g/L,得发酵基质;
(3)按5-10%的接种量,向步骤(2)所得发酵基质中接入酿酒酵母种子液进行发酵,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,继续发酵18-24h;
(4)发酵结束后离心分离上清,经浓缩后,冷冻干燥得还原型谷胱甘肽。
2.如权利要求1所述的一种治疗肝硬化的营养组合物,其特征在于,所述的种子液培养条件:
取酿酒酵母斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基按g/L计:(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0。
3.如权利要求2所述的一种治疗肝硬化的营养组合物,其特征在于,所述酿酒酵母菌株具体为酿酒酵母(Saccharomyces cerevisiae)tlj2016,保藏编号为CGMCC No.12789。
4.如权利要求1所述的一种治疗肝硬化的营养组合物,其特征在于,所述复合矿物质重量份数组成如下:碳酸钙10份、乳酸镁8份、乳酸锌1份。
5.如权利要求1所述的一种治疗肝硬化的营养组合物,其特征在于,所述复合维生素重量份数组成如下:维生素A 1份、维生素D 0.002份、维生素E 10份、维生素B1 2份、维生素B22份、维生素B6 2份、维生素B12 2份、维生素C 100份、烟酸20份、叶酸1份、泛酸10份。
6.如权利要求1所述的一种治疗肝硬化的营养组合物,其特征在于,所述玉米低聚肽的制备方法如下:
(1)向玉米蛋白粉中加入重量体积比10-20倍的水,混合均匀,调整pH8-10,加热至50-80℃保温搅拌20-60min;将料液经过离心分离操作后弃去清液,留用渣料;重复上述步骤1-3次得渣料;
(2)向上述渣料中加入8-15倍的水调浆,调节pH8-10,温度50-70℃,按8000-10000U/g玉米蛋白粉添加碱性蛋白酶,酶解2-3h;
(3)降温至30-50℃后,调节发酵液pH至5-7,按10000-15000U/g玉米蛋白粉添加中性蛋白酶,酶解5-6h;
(4)调节pH6-7,温度40-60℃,按400-1000U/g玉米蛋白粉添加风味蛋白酶,酶解3-5h,酶解结束后,加热至85℃-95℃,灭酶15-20分钟;
(5)离心浓缩后,喷雾干燥。
7.如权利要求1-6任意一项治疗肝硬化的营养组合物,其特征在于,服用方法如下:作为睡前加餐于每日20:00-21:00直接服用,每日用量为40~120g。
8.一种治疗肝硬化的营养组合物的制备方法,其特征在于,具体如下:
(1)所述营养组合物重量份数组成如下:浓缩乳清蛋白5-50份,大豆分离蛋白5-50份,玉米低聚肽1-5份,磷脂2-5份,麦芽糊精5-50份,菊粉1-20份,牛樟芝提取物1-3份,牛磺酸1-5份,缬氨酸1-4份,亮氨酸1-6份,异亮氨酸1-4份,精氨酸1-4份,还原型谷胱甘肽1-4份,复合矿物质0.01-0.2份,复合维生素0.05-0.1份;
按上述原料组成加工为片剂、粉剂、颗粒剂、口服液;
(2)所述复合矿物质重量份数组成如下:碳酸钙10份、乳酸镁8份、乳酸锌1份;
(3)所述复合维生素重量份数组成如下:维生素A 1份、维生素D 0.002份、维生素E 10份、维生素B1 2份、维生素B2 2份、维生素B6 2份、维生素B12 2份、维生素C 100份、烟酸20份、叶酸1份、泛酸10份;
(4)所述玉米低聚肽的制备方法如下:
①向玉米蛋白粉中加入重量体积比10-20倍的水,混合均匀,调整pH8-10,加热至50-80℃保温搅拌20-60min;将料液经过离心分离操作后弃去清液,留用渣料;重复上述步骤1-3次得渣料;
②向上述渣料中加入8-15倍的水调浆,调节pH8-10,温度50-70℃,按8000-10000U/g玉米蛋白粉添加碱性蛋白酶,酶解2-3h;
③降温至30-50℃后,调节发酵液pH至5-7,按10000-15000U/g玉米蛋白粉添加中性蛋白酶,酶解5-6h;
④调节pH6-7,温度40-60℃,按400-1000U/g玉米蛋白粉添加风味蛋白酶,酶解3-5h,酶解结束后,加热至85℃-95℃,灭酶15-20分钟;
⑤离心浓缩后,喷雾干燥;
(5)所述牛樟芝提取物的制备方法如下:
牛樟芝子实体粉碎,过40-50目筛后,添加牛樟芝子实体3-6倍重量无水乙醇浸渍提取,控制温度30-45℃,2-4小时后调整温度为55-60℃1-2小时,离心,乙醇提取液浓缩、干燥得到乙醇提取物;乙醇提取后的牛樟芝子实体残渣中添加2-4倍75-85℃热水,浸泡1-2h;之后进行超声浸提,超声条件为200-300W,频率30-40KHz、40-50℃、1-2h;之后进行高压脉冲电场浸提,脉冲条件为电场强度25-35kV/cm,脉冲频率200-1000Hz,0.5-1h,连续超声高压脉冲浸提2-3次,离心后将提取液真空浓缩后喷雾干燥,得到热水提取物;将上述乙醇提取物和热水提取物合并粉碎,过60目筛,即得牛樟芝子实体提取物;剩余的牛樟芝子实体残渣保存待用;
(6)所述还原型谷胱甘肽的制备方法如下:
①向上述牛樟芝子实体残渣中按20-40倍加水,并于121℃灭菌20min;
②向步骤①所得溶液,加入葡萄糖,终浓度达到100g/L,得发酵基质;
③按5-10%的接种量,向步骤②所得发酵基质中接入酿酒酵母种子液进行发酵,30℃,通气量6L/min,罐压0.03MPa,500rpm,恒pH6.0条件下进行发酵培养,发酵至30h时,一次性添加终浓度为25mmol/L的L-半胱氨酸,继续发酵18-24h;
④发酵结束后离心分离上清,经浓缩后,冷冻干燥得还原型谷胱甘肽;
所述的种子液培养条件:
取酿酒酵母斜面菌种一环,接入装有30mL摇瓶培养基的250mL摇瓶中150rpm,30℃培养30h得种子液;
摇瓶培养基(g/L):(NH4)2SO4 6、葡萄糖35、K2HPO4·3H2O 3、KH2PO4 0.5、酵母粉11、MnSO4 0.1、KCL 0.1、FeSO4 0.1、MgSO4·7H2O 0.1,pH6.0;
所述酿酒酵母菌株具体为酿酒酵母(Saccharomyces cerevisiae)tlj2016;
其余原料均为市售。
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