CN114081184B - 一种具有预防抑郁和焦虑行为形成的益生菌剂及其应用 - Google Patents
一种具有预防抑郁和焦虑行为形成的益生菌剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有预防抑郁和焦虑行为形成的益生菌剂及其应用,本发明提供的植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033这3株菌均具有耐酸耐胆盐的特性,可在人体肠道环境中定值繁殖生长,并且这3株菌还可以产生神经递质或其前体物质,例如色氨酸和5‑羟色胺,这些物质能使人产生愉悦情绪,并且低聚半乳糖和低聚异麦芽糖促进有益菌的增殖,使其产生使人愉悦的神经递质,同时益生菌组合物和功能性物质又都可以调节肠道健康,相辅相成,从而达到调节人体情绪的目的,进一步提高了改善抑郁、焦虑症状的作用效果。
Description
技术领域
本发明涉及微生态制剂技术领域,具体涉及一种具有预防抑郁和焦虑行为形成的益生菌剂及其应用。
背景技术
抑郁症是一种发病率和死亡率高的全球性情绪障碍疾病,目前对其致病机理尚不清楚,据世界卫生组织预测,到2030年抑郁症将高居全球疾病负担第一位,抑郁症一线治疗药物以纠正中枢单胺神经递质失调的药物为主,如5-羟色胺再摄取抑制药和5-羟色胺/去甲肾上腺素双重再摄取抑制药等,但这些药物存在反应率低和副作用大等缺点,首发抑郁症患者对这些传统抗抑郁药的响应率只有40-60%,因此,探寻抑郁症新型防治策略是迫在眉睫的任务。
随着越来越多的证据表明肠道微生物在抑郁症和焦虑症发生、发展中扮演了极其重要的角色。譬如,用于诱导啮齿类动物抑郁和焦虑症状形成的慢性应激能够改变肠道菌群丰度,受影响的菌种包括异杆菌、双歧杆菌、土杆菌、梭状杆菌、颤螺菌等,长期摄入植物乳杆菌、长双歧杆菌等益生菌具有显著抑制抑郁和焦虑样行为形成的作用,同时不表现出明显的副作用。此外,婴儿型双歧杆菌也具有显著的抗抑郁特性,神经炎症反应在慢性应激诱导抑郁症和焦虑症形成中具有十分重要的作用,由肠道微生物菌群破坏引起的肠道屏障功能受损是慢性应激导致神经炎症发生的关键病理事件,因此,改善肠道微生物菌群丰度(增加有益菌数量、减少有害菌数量),对缓解由应激引起的神经炎症反应和随之而来的抑郁焦虑等情绪变化具有积极的理论和现实意义。
鉴于现代社会竞争剧烈,在社会和环境双重因素作用下,人群精神健康水平普遍处于亚健康状态,肠道菌群长期紊乱,神经炎症低度长期活跃都是构成情绪障碍疾病形成的关键因素,因此,开发能够改善肠道菌群环境的益生菌对改善人群整体精神健康水平具有重要的社会意义和商业价值。
发明内容
针对现有技术的不足,本发明的目的在于提供一种具有预防抑郁和焦虑行为形成的益生菌剂及其应用,通过改善肠道菌群环境来预防和改善抑郁症状。
为了实现上述目的,本发明采取如下技术方案:
一种具有预防抑郁和焦虑行为形成的益生菌剂,所述益生菌剂由益生菌组合物和功能性物质组成,所述益生菌组合物为植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033。
优选的,所述益生菌组合物中的总活菌数≥2.0×1011CFU/g。
优选的,所述益生菌组合物中植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033的质量比为1-2:1-3:1-2。
优选的,所述功能性物质由沙棘提取物、低聚异麦芽糖和低聚半乳糖组成。
优选的,所述沙棘提取物、低聚异麦芽糖和低聚半乳糖的质量比为10:5-8:2-6。
优选的,所述益生菌剂中益生菌组合物的含量为5-20%。
本发明还提供上述益生菌剂在食品、保健品或药品中的应用。
与现有技术相比,本发明具有如下有益效果:
(1)乳杆菌和双歧杆菌均属于常见的益生菌,不同的是本发明提供的植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033这3株菌均具有耐酸耐胆盐的特性,于胃酸模拟环境下(pH为2-3),这3株菌的存活率为96%,于肠道模拟环境下(pH为7),这3株菌的存活率高达99%,可在人体肠道环境中定值繁殖生长,并且这3株菌还可以产生神经递质或其前体物质,例如色氨酸和5-羟色胺,这些物质能使人产生愉悦情绪,此外,副干酪乳杆菌LPC45还能产生维生素VK,维生素VK可以安神,让人保持心情平和,有效防止焦虑抑郁症状的发生。
(2)本发明提供的低聚半乳糖可作为底物被植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033利用,可促进有益菌的增殖,同时能够降低肠道pH,抑制有害菌的生长代谢;低聚异麦芽糖含有α-1,6糖苷键,因人体缺少水解α-1,6糖苷键的酶,在胃和小肠内基本不被消化,能被大肠内的有益菌尤其是短双歧杆菌BB033所利用,而大肠杆菌、梭状杆菌等有害菌不能利用,因而具有改善肠道微生态环境的作用;沙棘提取物具有改善抑郁症病人机体多巴胺水平和调节大脑五羟色胺水平的作用,能够调节人体情绪,与益生菌产生的神经递质协同作用,从而改善抑郁得到症状。
(3)本发明提供的益生菌组合物和功能性物质之间具有协同作用,低聚半乳糖和低聚异麦芽糖促进有益菌的增殖,使其产生使人愉悦的神经递质,同时益生菌组合物和功能性物质又都可以调节肠道健康,相辅相成,从而达到调节人体情绪的目的,进一步提高了改善抑郁、焦虑症状的作用效果,本发明的组合物具有温和、无副作用的特点,可有效改善抑郁、焦虑患者的症状。
具体实施方式
以下通过具体较佳实施例对本发明作进一步详细说明,但本发明并不仅限于以下的实施例。
需要说明的是,无特殊说明外,本发明中涉及到的化学试剂均通过商业渠道购买。
植物乳杆菌Lp3a选自江苏新申奥生物科技有限公司,生物保藏信息:CGMCCNo.17054,保藏时间2019.01.02;
副干酪乳杆菌LPC45选自江苏新申奥生物科技有限公司,生物保藏信息:CGMCCNo.22772,保藏时间2021.06.24;
短双歧杆菌BB033选自江苏新申奥生物科技有限公司,生物保藏信息:CGMCCNo.22777,保藏时间2021.06.24。
植物乳杆菌Lp3a冻干菌粉的制备:
(1)液体深层发酵培养
将植物乳杆菌Lp3a保藏菌种以2%接种量接种至用250mL三角瓶装着的150mL种子培养基中,于37℃,5%的CO2培养箱中静置培养,24h后,以3%的接种量接种至用1L三角瓶装着的800mL种子培养基中,于37℃,5%的CO2培养箱中静置培养,培养约7h后,以3%的接种量接种至200mL发酵罐中,内装160mL发酵培养基;
发酵培养条件为:温度37℃,转速50rpm,压力0.05MPa,通风量1.5L/h,发酵过程中pH值用30%的氢氧化钾溶液控制在6.2,待发酵液OD600值和pH值趋于稳定后开始放罐,培养时间为8h,放罐时罐温降至20℃,植物乳杆菌Lp3a发酵液放罐活菌数为1011cfu/mL;
植物乳杆菌Lp3a的种子培养基为:葡萄糖8g/L、蔗糖60g/L、酵母提取物15g/L、乙酸钠8g/L、柠檬酸氢二铵4g/L、七水硫酸镁0.6g/L、一水硫酸锰0.3g/L,pH=6.2;
植物乳杆菌Lp3a的发酵培养基为:葡萄糖10g/L、蔗糖80g/L、酵母提取物40g/L、乙酸钠5g/L、柠檬酸氢二铵2g/L、七水硫酸镁0.58g/L、一水硫酸锰0.25g/L,pH=6.2;
(2)冻干菌剂的制备:待植物乳杆菌Lp3a发酵液温度降至20℃左右时,离心收集菌泥,离心菌体管道降温冷却,离心结束后收得菌泥,然后加入脱脂奶粉1.92kg,乳糖1.12kg,海藻糖0.8kg,抗结块剂0.16kg作为冻干保护剂,用10L无菌水溶解混匀,然后,混合物均匀铺于托盘上,每个托盘装1L,将托盘置于隔板上,在物料中放置温度探头,再进行冻干,收集菌粉,置于-20℃冰箱保存。
副干酪乳杆菌LPC45冻干菌粉的制备:
(1)液体深层发酵培养
将副干酪乳杆菌LPC45保藏菌种以2%接种量接种至用250mL三角瓶装着的150mL种子培养基中,于37℃,5%的CO2培养箱中静置培养,24h后,以3%的接种量接种至用1L三角瓶装着的800mL种子培养基中,于37℃,5%的CO2培养箱中静置培养,培养约7h后,以3%的接种量接种至200mL发酵罐中,内装160mL发酵培养基;
发酵培养条件为:温度37℃,转速50rpm,压力0.05MPa,通风量1.5L/h,发酵过程中pH值用30%的氢氧化钾溶液控制在6.4,待发酵液OD600值和pH值趋于稳定后开始放罐,培养时间为8h,放罐时罐温降至20℃,副干酪乳杆菌LPC45发酵液放罐活菌数为1011cfu/mL;
副干酪乳杆菌LPC45的种子培养基为:葡萄糖8g/L、蔗糖60g/L、酵母提取物15g/L、乙酸钠8g/L、柠檬酸氢二铵4g/L、七水硫酸镁0.6g/L、一水硫酸锰0.3g/L,pH=6.4;
副干酪乳杆菌LPC45的发酵培养基为:葡萄糖10g/L、蔗糖80g/L、酵母提取物40g/L、乙酸钠5g/L、柠檬酸氢二铵2g/L、七水硫酸镁0.58g/L、一水硫酸锰0.25g/L,pH=6.4;
(2)冻干菌剂的制备:待副干酪乳杆菌LPC45发酵液温度降至20℃左右时,离心收集菌泥,离心菌体管道降温冷却,离心结束后收得菌泥,然后加入脱脂奶粉1.92kg,乳糖1.12kg,海藻糖0.8kg,抗结块剂0.16kg作为冻干保护剂,用10L无菌水溶解混匀,然后,混合物均匀铺于托盘上,每个托盘装1L,将托盘置于隔板上,在物料中放置温度探头,再进行冻干,收集菌粉,置于-20℃冰箱保存。
短双歧杆菌BB033冻干菌粉的制备:
(1)液体深层发酵培养
将短双歧杆菌BB033保藏菌种以2%接种量接种至用250mL三角瓶装着的150mL种子培养基中,于37℃,5%的CO2培养箱中静置培养,24h后,以3%的接种量接种至用1L三角瓶装着的800mL种子培养基中,于37℃,5%的CO2培养箱中静置培养,培养约7h后,以3%的接种量接种至200mL发酵罐中,内装160mL发酵培养基;
发酵培养条件为:温度37℃,转速50rpm,压力0.05MPa,通风量1.5L/h,发酵过程中pH值用30%的氢氧化钾溶液控制在6.5,待发酵液OD600值和pH值趋于稳定后开始放罐,培养时间为8h,放罐时罐温降至20℃,短双歧杆菌BB033发酵液放罐活菌数为1011cfu/mL;
短双歧杆菌BB033的种子培养基为:葡萄糖8g/L、蔗糖60g/L、酵母提取物15g/L、乙酸钠8g/L、柠檬酸氢二铵4g/L、七水硫酸镁0.6g/L、一水硫酸锰0.3g/L、0.5gL-半胱氨酸盐酸盐,pH=6.5;
短双歧杆菌BB033的发酵培养基为:葡萄糖10g/L、蔗糖80g/L、酵母提取物40g/L、乙酸钠5g/L、柠檬酸氢二铵2g/L、七水硫酸镁0.58g/L、一水硫酸锰0.25g/L、0.5gL-半胱氨酸盐酸盐,pH=6.5;
(2)冻干菌剂的制备:待短双歧杆菌BB033发酵液温度降至20℃左右时,离心收集菌泥,离心菌体管道降温冷却,离心结束后收得菌泥,然后加入脱脂奶粉1.92kg,乳糖1.12kg,海藻糖0.8kg,抗结块剂0.16kg作为冻干保护剂,用10L无菌水溶解混匀,然后,混合物均匀铺于托盘上,每个托盘装1L,将托盘置于隔板上,在物料中放置温度探头,再进行冻干,收集菌粉,置于-20℃冰箱保存。
实施例1
一种具有预防抑郁和焦虑行为形成的益生菌剂的制备方法,包括如下步骤:
将益生菌组合物和功能性物质混合均匀,即得到益生菌剂,其中益生菌组合物为0.25g植物乳杆菌Lp3a冻干菌粉、0.25g副干酪乳杆菌LPC45冻干菌粉和0.25g短双歧杆菌BB033冻干菌粉的混合菌粉,功能性物质为4.5g沙棘提取物、2.7g低聚异麦芽糖和1.8g低聚半乳糖的混合物。
实施例2
一种具有预防抑郁和焦虑行为形成的益生菌剂的制备方法,包括如下步骤:
将益生菌组合物和功能性物质混合均匀,即得到益生菌剂,其中益生菌组合物为0.2g植物乳杆菌Lp3a冻干菌粉、0.3g副干酪乳杆菌LPC45冻干菌粉和0.2g短双歧杆菌BB033冻干菌粉的混合菌粉,功能性物质为4.5g沙棘提取物、2.7g低聚异麦芽糖和1.8g低聚半乳糖的混合物。
实施例3
一种具有预防抑郁和焦虑行为形成的益生菌剂的制备方法,包括如下步骤:
将益生菌组合物和功能性物质混合均匀,即得到益生菌剂,其中益生菌组合物为0.4g植物乳杆菌Lp3a冻干菌粉、0.6g副干酪乳杆菌LPC45冻干菌粉和0.4g短双歧杆菌BB033冻干菌粉的混合菌粉,功能性物质为4g沙棘提取物、2g低聚异麦芽糖和2g低聚半乳糖的混合物。
实施例4
一种具有预防抑郁和焦虑行为形成的益生菌剂的制备方法,包括如下步骤:
将益生菌组合物和功能性物质混合均匀,即得到益生菌剂,其中益生菌组合物为0.5g植物乳杆菌Lp3a冻干菌粉、0.5g副干酪乳杆菌LPC45冻干菌粉和0.5g短双歧杆菌BB033冻干菌粉的混合菌粉,功能性物质为4g沙棘提取物、2.4g低聚异麦芽糖和1.6g低聚半乳糖的混合物。
对比例1
一种益生菌剂的制备方法,包括如下步骤:
将0.4g植物乳杆菌Lp3a冻干菌粉、0.6g副干酪乳杆菌LPC45冻干菌粉和0.4g短双歧杆菌BB033冻干菌粉混合均匀,即得到益生菌剂。
对比例2
一种益生菌剂的制备方法,包括如下步骤:
将益生菌组合物和功能性物质混合均匀,即得到益生菌剂,其中益生菌组合物为0.6g副干酪乳杆菌LPC45冻干菌粉和0.4g短双歧杆菌BB033冻干菌粉的混合菌粉,功能性物质为4g沙棘提取物、2g低聚异麦芽糖和2g低聚半乳糖的混合物。
对比例3
一种益生菌剂的制备方法,包括如下步骤:
将益生菌组合物和功能性物质混合均匀,即得到益生菌剂,其中益生菌组合物为0.4g植物乳杆菌Lp3a冻干菌粉和0.4g短双歧杆菌BB033冻干菌粉的混合菌粉,功能性物质为4g沙棘提取物、2g低聚异麦芽糖和2g低聚半乳糖的混合物。
小鼠悬尾试验、强迫游泳试验是验证给药后是否具有明显缓解焦虑、抑郁症的常规动物实验。雄性ICR小鼠,18~22g,适应性喂养1周以后,随机分成9组,每组6只,分别喂给本实施例1-4和对比例1-4所制备的益生菌剂,剂量为0.05g/kg,药物用生理盐水配成混悬液灌胃给药,空白对照组在给药期间给与等体积生理盐水。每日1次,连续灌胃给药7天,末次给药1h后进行测试。
小鼠悬尾试验:主要是通过固定小鼠尾部使其头向下悬挂,小鼠在该环境中处于恐慌而拼命挣扎逃跑,又无法逃脱的状态,给小鼠提供一个无法回避的压迫环境,一段时间后,记录处于该环境下的小鼠产生绝望的不动状态过程的不动时间,以观察给药后的治疗效果。将小鼠尾端粘在悬尾箱上部的支架上,将其成为倒吊状态进行悬尾实验,倒挂小鼠的头部距离箱底10cm,一次悬挂6只小鼠,中间用隔板隔开以免碰撞,小鼠在倒挂后由于恐慌会进行挣扎活动,当挣扎疲劳后会处于失望状态而出现间断性不动,悬挂小鼠10分钟,统计后5分钟内悬尾倒挂小鼠累计不动时间;
强迫游泳实验:主要通过将实验小鼠置于一个局限的水中,小鼠在该环境中拼命挣扎试图逃跑又无法逃脱,提供了一个无可回避的压迫环境,一段时间的实验后,小鼠即表现出典型的行为绝望状态的不动状态,记录小鼠产生绝望的不动状态时的不动时间,以观察用药后的治疗效果,将小鼠放入长50cm,宽30cm,高20cm的塑料游泳箱中做强迫性游泳试验,从小鼠放入水中后计时10分钟,记录后5分钟内小鼠的不动时间(指小鼠在水中停止挣扎或显示漂浮状态,仅有微小的肢体运动以保持头部浮在水面),实验结果如下表所示:
组别 | 动物数(n) | 悬尾静止时间(s) | 游泳不动时间(s) |
实施例1 | 6 | 69.34±3.45 | 102.67±1.87 |
实施例2 | 6 | 64.76±2.93 | 103.25±2.41 |
实施例3 | 6 | 58.44±1.57 | 97.58±2.05 |
实施例4 | 6 | 60.21±2.56 | 99.32±1.92 |
对比例1 | 6 | 89.56±1.35 | 114.46±2.73 |
对比例2 | 6 | 101.21±2.27 | 123.35±2.46 |
对比例3 | 6 | 98.62±1.98 | 122.13±1.99 |
空白对照组 | 6 | 127.18±3.26 | 148.33±4.65 |
从表中可以看出,本发明所制备的益生菌剂能够明显减少行为绝望动物模型小鼠中的不动时间,可以确定本发明所制备的益生菌剂具有明显的抗抑郁作用,可用于制备预防抑郁症的药物或保健食品。
最后需要说明的是:以上实施例不以任何形式限制本发明。对本领域技术人员来说,在本发明基础上,可以对其作一些修改和改进。因此,凡在不偏离本发明精神的基础上所做的任何修改或改进,均属于本发明要求保护的范围之内。
Claims (2)
1.一种具有预防抑郁和焦虑行为形成的益生菌剂,其特征在于,所述益生菌剂由益生菌组合物和功能性物质所组成,所述益生菌组合物由植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033所组成;
其中,植物乳杆菌Lp3a的生物保藏信息为:CGMCC No.17054;
副干酪乳杆菌LPC45的生物保藏信息为:CGMCC No.22772;
短双歧杆菌BB033的生物保藏信息为:CGMCC No.22777;
所述功能性物质由沙棘提取物、低聚异麦芽糖和低聚半乳糖组成;
所述益生菌组合物中的总活菌数≥2.0×1011CFU/g;
所述益生菌组合物中植物乳杆菌Lp3a、副干酪乳杆菌LPC45和短双歧杆菌BB033的质量比为1-2:1-3:1-2;
所述沙棘提取物、低聚异麦芽糖和低聚半乳糖的质量比为10:5-8:2-6;
所述益生菌剂中益生菌组合物的含量为5-20%。
2.如权利要求1所述具有预防抑郁和焦虑行为形成的益生菌剂在制备药品中的应用。
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