CN114568663B - 一种解郁宁心的益生菌发酵物及其制备方法和应用 - Google Patents
一种解郁宁心的益生菌发酵物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种解郁宁心的益生菌发酵物及其制备方法和应用,由益生菌对水果中药组合物进行发酵而成;水果中药组合物包括枸杞8‑12份,龙眼肉8‑12份,余甘子8‑12份,百合8‑12份,蜂蜜8‑12份,桑葚6‑12份,玫瑰花6‑12份,玉竹4‑12份,大枣4‑12份,莲子5‑8份,百香果6‑10份,苹果3‑5份,葡萄8‑12份。本发明所提供的益生菌发酵物具有解郁宁心、行气养血的功效,通过乳杆菌对水果中药组合物进行深度发酵,极大地提升了水果中药组合物中解郁宁心、行气养血的功效成分含量,人体服用此发酵物后,可以更好地吸收水果中药组合物中的营养成分和功效因子,加速调节心情,舒缓情绪、行气养血。
Description
技术领域
本发明涉及益生菌发酵物技术领域,尤其涉及一种解郁宁心的益生菌发酵物及其制备方法和应用。
背景技术
“抑郁”起源于拉丁文“Deprimere”一词,意指“下压”。在17世纪便被用来描述情绪状态,其核心症状被称为“缺乏快乐”(anhedonia,源于希腊文),意指丧失体验快乐的能力。美国当代心理学家Angold对抑郁作了如下描述:(1)抑郁为正常心境向情绪低落方面的波动,即每天出现情绪恶劣的一面;(2)抑郁为不愉快、悲伤或精神痛苦,是对一些不良情景或事件的一种反应;(3)抑郁作为一种特征,是指个体持久的、相对稳定的愉快感的缺乏;(4)抑郁作为一种症状,是指心境处于病理性的低下或恶劣。我国学者孟昭兰则将抑郁界定为一种复合性的情绪体验:抑郁同一般的悲伤不同,抑郁的体验比任何一种单一负性情绪更为强烈,而且持续时间更长,给人带来的痛苦更大;它除包含悲伤外,还伴有痛苦、愤怒、自罪感、羞愧等情绪。心理学研究领域内,抑郁常被视作抑郁倾向或抑郁情绪,心理学家更偏重于研究抑郁情绪。
本发明所涉及的抑郁状况也是指这种抑郁情绪,其反映的是普通个体的主观感受,并非特指临床上的抑郁症。这是一种以情绪低落为主要特点的复杂情绪,其强度可以从轻微的失落感到极度的悲哀和绝望。抑郁情绪主要有以下四组特征:悲观、悲哀、失望、无助乃至冷漠或绝望的心境;消极的自我概念和自我评价,自信心下降,有无价值、无用和自卑感,严重者有自罪甚至自杀;睡眠多有障碍,食欲、性欲减退,兴趣索然;活动水平多下降,当事人从社交、工作和家务中退缩出去,回避他人。抑郁在一般人群中的发生率为13%-20%,重度抑郁约5%,它可以影响到任何一个人,无论其地位、种族、年龄、性别、婚姻状况或文化程度如何。近年来,抑郁的发生率有小幅上升,这一领域的研究已经引起了广泛的关注。
心悸是指患者自觉心中急剧跳动,不能自主的一种病证,因多伴惊慌不安,故又称惊悸,重者又称怔忡。由于心悸多见于各种心脏器质性疾病,故长期以来一直将其作为心系病证看待。此外,心悸还可伴发于全身其他系统疾病,更多见于情志性郁证疾病。本研究所涉及的心悸,是由情志因素引起,属于因郁致病,主要表现有心虚胆怯证、心脾两虚证、心阴亏虚证,或有心阳不振证、肝肾阴虚证,甚至有部分痰浊阻滞证和心血瘀阻证。据统计,门诊患者心悸发病率高达16%。
历代医籍均有大量以解郁安神定志方药治疗郁证性心悸的记载,所用处方均含养心安神定志及调理情志类药物。如:唐代孙思邈《千金翼方·妇人三·心悸》载: “治产后忽苦,心中冲悸,或志意不定,恍恍惚惚,言语错谬,心虚所致方: 人参、茯苓(各三两) ,茯神(四两) ,大枣(三十枚,擘) ,生姜(八两) ,芍药、当归、桂心、甘草(各二两) 。”他如远志汤(《千金翼方·卷第十五·补益》) 、大定心汤(《备急千金要方·卷十四·小肠腑方》) 等,均运用了茯神、远志、酸枣仁、龙骨、牡蛎、柏子仁等安神定志类药物。宋代《太平圣惠方·治心脏风虚惊悸方》载: “治心脏风虚,惊悸心忪,常多健忘,宜服茯神丸方”。
现代医学治疗中,镇静安神、抗抑郁、抗焦虑药物用于治疗郁证性心悸见诸许多临床报道。例如,以盐酸氟西汀胶囊(百优解)、氟哌噻吨美利曲辛片(黛力新)治疗心脏神经官能症之抑郁情绪和躯体症状(包括心悸) ,以抗抑郁药物联合心理疗法治疗心脏神经官能症。此外还有以疏肝解郁和(或) 安神定志方药,联合抗抑郁、抗焦虑药物等中西医结合的方法治疗心脏神经官能症之心悸,如柴胡疏肝散合黛力新、逍遥散合帕罗西汀等。
对于市面上缓解抑郁、心悸等症状的药物,虽然有不同程度的效果,但是任存在一定的缺陷,如:中药疗效慢,短期内服用效果不够显著,生物利用度较低,长期服用容易积累毒素。西药疗效较好,但同时毒性更强,长期服用对肝脏的损伤更大。因此,需要一款疗效显著,健康安全的解郁宁心产品来迎合市场需求。
发明内容
为了克服现有技术的不足,本发明的目的之一在于提供一种解郁宁心的益生菌发酵物,疗效显著,健康安全。
本发明的目的之二在于提供一种解郁宁心的益生菌发酵物的制备方法,利用副干酪乳杆菌K9对水果中药组合物进行发酵,所得的发酵物能提高发酵制品解郁宁心、行气养血的功效,同时所得的发酵物为活菌型。
本发明的目的之一采用如下技术方案实现:
一种解郁宁心的益生菌发酵物,由益生菌对水果中药组合物进行发酵而成,所述益生菌包括乳杆菌;所述水果中药组合物由按照重量份的以下组分组成:枸杞8-12份,龙眼肉8-12份,余甘子8-12份,百合8-12份,蜂蜜8-12份,桑葚8-12份,玫瑰花8-12份,玉竹8-12份,大枣8-12份,莲子5-8份,百香果6-10份,苹果3-5份,葡萄8-12份。
优选地,所述水果中药组合物包括枸杞8-12份,龙眼肉8-12份,余甘子8-12份,百合8-12份,蜂蜜8-12份,桑葚8-12份,玫瑰花8-12份,玉竹8-12份,大枣8-12份,莲子5-8份,百香果6-10份,苹果3-5份,葡萄8-12份。
所述水果中药组合物中各原料均为食品材料或药食同源材料。其中,枸杞含有多种微量元素,在人体内与酶、激素以及维生素等共同保持生命的代谢过程和肌体的免疫能力,提高皮肤吸收氧分的能力以及明目安神;龙眼肉含有丰富的葡萄糖、蔗糖、酒石酸、维生素A、维生素B类等物质,这些物质能营养神经和脑组织,缓解焦虑,安神,从而有利于调整大脑皮层功能,使思维变得敏捷,并能增强记忆力;余甘子含有丰富的酚类物质和维生素素,具有缓解压力、抗疲劳的功效;百合中含有甾体皂苷、多糖、酚酸甘油酯、生物碱、黄酮、氨基酸、磷脂及其他烷烃等成分,主要为多糖及甾体皂苷,具有抗抑郁、镇静安神的作用;蜂蜜富含葡萄糖和果糖,这两者都能够快速生成能量,恢复机体的健康、缓解疲劳、舒缓压力;桑葚含有丰富的氨基酸、锌、锰、铁和维生素C,具有养血、安神的功效;玫瑰花的药性非常温和,能够温养人的心肝血脉,舒发体内郁气,具有镇静、安抚、抗抑郁的功效;玉竹含有强心苷、生物碱和维生素A和玉竹果聚糖等物质,有改进心肌缺氧和肾上腺皮质激素样效果,可安神、助眠、强心、预防心脑血管疾病;大枣富含多种维生素和微量元素,具有补中益气、养血安神的功效;莲子含有丰富的蛋白质、淀粉、磷脂、莲子碱、芳香甙、类黄酮以及多种维生素等营养保健成分,而且其铁的含量也非常丰富,具有清热降火、补脾止泻、养心安神的功效;百香果含有丰富的维生素、超纤维和蛋白质等上百中对人体非常有益的元素,是一种天然的镇静剂,具有缓解压力、镇静安神的功效;苹果中含有一些果糖,可以使人心情舒畅,可以使抑郁和焦虑等不健康的心理状态能够得到缓解;葡萄含逆转酶,这种酶可以通过减缓动脉壁上胆固醇的堆积而保护心脏,可软化血管、活血化淤,防止血栓形成,具有保护心脏的功效。
益生菌可以通过产生次级代谢产物、代谢前体、免疫信号和迷走神经信号,以及激活下丘脑-垂体-肾上腺(HPA)轴,深入影响大脑的生理和功能。通过益生菌发酵中药,既可降低中药的毒性和不良风味,又可直接与中药反应,提升功效成分的含量,并提高生物利用度。益生菌作用高效、温和、安全且持续,本发明所提供的解郁宁心的益生菌发酵物是一款疗效显著、健康安全的产品,可以很好地满足市场需求。
进一步地,所述乳杆菌为保藏编号为CGMCC No.15025的副干酪乳杆菌(Lactobacillus paracasei)K9。本发明提供的副干酪乳杆菌K9能够发酵生产功效成分,利用该副干酪乳杆菌对水果中药组合物进行发酵时,能提高发酵制品解郁宁心、行气养血的功效,同时所得的发酵物为活菌型。
本发明的副干酪乳杆菌(CGMCC No.15025)的表型特征为:G+,杆状,菌落圆形,乳脂色,表面湿润,边缘整齐。
本发明的副干酪乳杆菌(CGMCC No.15025)为自主通过可培养方法于2005年分离自广西壮族自治区河池市巴马瑶族自治县的健康人体肠道。
本发明的副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)的生长条件可以为常规条件,生长条件例如可以为:生长温度30-37°C,兼性厌氧,生长时间24-48h。
本发明的副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)能够提高发酵制品解郁宁心、行气养血的功效,因此可用于相关用途。
进一步地,所述水果中药组合物为经CO2浸渍发酵后的水果中药组合物。
CO2浸渍是一种独特的酿造工艺,它与传统的借助酵母来完成发酵的方式不一样。一般情况下,果汁的发酵需要酵母的作用,而二氧化碳浸渍法则不需要酵母的参与,操作方式是将完整原料放入发酵罐中,注入二氧化碳后密封。在这种环境下,水果和其他植物成分会进行无氧代谢,释放出自身所含的酶,将糖分转化为酒精。这个过程被称为果内发酵。当酒精度到达2%时,表皮会破裂,并释放出汁液。在浸渍过程中,水果和其他植物成分与表皮接触时间较短,萃取的色素和芳香物质较多,而单宁的萃取较少,因此发酵成品单宁含量较低,口感柔顺,香气馥郁。此外,在果内发酵过程中,各种酶将糖分转化成酒精的同时也会分解尖锐的苹果酸,使得发酵成品酸度柔和,口感清新。一般,通过二氧化碳浸渍法酿制而成的发酵物会有草莓、香蕉、泡泡糖和樱桃酒的香气。
进一步地,所述水果中药组合物由按照重量份的以下组分组成:枸杞8份,龙眼肉8份,余甘子8份,百合8份,蜂蜜8份,桑葚8份,玫瑰花9份,玉竹8份,大枣8份,莲子6份,百香果8份,苹果4份,葡萄9份。
本发明的目的之二采用如下技术方案实现:
一种解郁宁心的益生菌发酵物的制备方法,包括以下步骤:
浸渍发酵步骤:将所述水果中药组合物放到充满CO2的密闭罐中浸渍,得到预处理后的水果中药组合物;
榨汁步骤:将所述预处理后的水果中药组合物进行破碎、压榨,得到原料汁;
益生菌发酵步骤:制备包含所述原料汁的发酵培养基,按预设量将乳杆菌种子液接入所述发酵培养基中,发酵,得到发酵液体制品;
干燥步骤:将所述发酵液体制品进行干燥,得到发酵固体制品;
粉碎过筛步骤:将所述发酵固体制品进行粉碎、过筛,即得所述解郁宁心的益生菌发酵物。
本发明采用副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)对水果中药组合物进行发酵时,对发酵培养基和发酵条件不作严格限制,只要是利于上述副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)发酵水果中药组合物,能提高发酵制品解郁宁心、行气养血的功效即可。
在本发明中,可采用常规方式对原料进行榨汁,从而获得原料汁。
进一步地,还包括调汁步骤:经过所述榨汁步骤获取所述原料汁后,向所述原料汁中加入纯化水进行调浆,控制所述原料汁与纯化水的体积比为1:(1-4),可视实际需要对原料汁进行调浆。
进一步地,在所述调汁步骤中,还向所述原料汁中加入70~100ml/L的S02,原料汁含有多种功效成分,如:生物碱类、黄酮类、多糖、挥发油类、甾醇类化合物、微量元素等,这些物质具有较强的嗜氧性,在与空气接触时很容易被氧化生成棕色聚合物,使原料汁的性状发生劣变,加入S02可最大程度减少上述不利情况的发生。
进一步地,在所述调汁步骤中,还向所述原料汁中加入0.4~0.6%的小苏打,发酵过程中,益生菌持续产生有机酸,容易导致培养基的pH波动。因此,可以根据实际情况加入小苏打,小苏打作为缓冲物质,可以保持培养基的pH稳定。
进一步地,解郁宁心的益生菌发酵物的制备方法包括以下步骤:
浸渍发酵步骤:将所述水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为20-25℃,时间为3-7d,得到预处理后的水果中药组合物;
榨汁步骤:将所述预处理后的水果中药组合物进行破碎、压榨,得到原料汁;
调汁步骤:向所述原料汁中加入纯化水进行调浆,控制所述原料汁与纯化水的体积比为1:(1-4),并向所述原料汁中加入70~100ml/L的S02和0.4~0.6%的小苏打,得到处理后原料汁;接着向每升所述处理后原料汁中加入0-100g碳源、5-10g氮源和1-15g无机盐,随后调节pH值至5.5-6.5,得到发酵培养基;
益生菌发酵步骤:按照0.3~2.0%(v/v)的接种量将乳杆菌种子液接入所述发酵培养基中进行发酵培养,控制发酵温度为30~32℃,转速为90~120r/min,发酵周期为5~9,得到发酵液体制品;
干燥步骤:将所述发酵液体制品放入真空冷冻干燥机内进行冻干处理,冷冻流程依次为-45~-35℃保持1.8~2.2h、-2~2℃保持19~21h、28~32℃保持 13~18h,总冻干时间为35~40h,终点时物料温度和搁板温度之差不高于5℃,得到发酵固体制品;
粉碎过筛步骤:将所述发酵固体制品采用超微粉碎方式处理3~5min,然后过筛,即得所述解郁宁心的益生菌发酵物。
在所述调汁步骤中,本发明对碳源、氮源和无机盐不作严格限制,只要适于所述副干酪乳杆菌(Lactobacillus paracasei)发酵所述水果中药组合物即可。具体地,碳源可以采用蔗糖、葡萄糖、糖蜜等中的至少一种,氮源可以采用蛋白胨、牛肉膏、酵母浸粉等其中的至少一种,无机盐可以采用硫酸锰、硫酸镁、磷酸氢二钾等中的至少一种。
进一步地,所述乳杆菌种子液的获取方法如下:制备基础培养基:将6.0~10.0g酵母水解物、2.0~3.0g玉米浆、3.0~4.0g花生粉、10.0~12.0g糖蜜、2.0~3.0gKCl、0.1~0.3gZnSO4·8H2O、2~2.2gFeSO4·4H2O、8.0~10.0gCaCO3溶解于1L纯化水中,调节pH值至5.8~6.0,制得液体基础培养基;制备固体培养基时,向所述液体基础培养基内加入12~18g琼脂;
菌种活化:将保存于斜面上的保藏编号为CGMCC No.15025的副干酪乳杆菌K9接种于所述固体培养基中,在厌氧条件下静置培养20~30h,得到活化菌种;
制备种子液:将所述活化菌种接种于所述液体基础培养基中,在厌氧条件下静置培养20~48h,得到乳杆菌种子液。
本发明的目的之三采用如下技术方案实现:
一种解郁宁心的益生菌发酵物在制备具有解郁宁心作用的产品中的应用,所述具有解郁宁心作用的产品可以包括保健品、药品,产品形态可以为液态或固态,可以为口服液、悬浮液、膏状物、片剂、颗粒剂等。所述益生菌为副干酪乳杆菌(Lactobacillusparacasei,CGMCC No.15025)。
本发明对副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)发酵物(解郁宁心的益生菌发酵物)的应用方式不作严格限制,既可以生产包含有机酸的发酵制品,还可以根据需要用该发酵制品制成干粉,提供下游的应用。
相比现有技术,本发明的有益效果在于:
(1)本发明所提供的解郁宁心的益生菌发酵物具有解郁宁心、行气养血的功效,通过乳杆菌对水果中药组合物进行深度发酵,极大地提升了水果中药组合物中解郁宁心、行气养血的功效成分含量,花色苷含量高,总糖含量高,提高生物利用度。花色苷主要是通过糖苷键将花青素与糖进行结合而形成的化合物,具有多种生物活性,如清除自由基、抗氧化、抗炎症、缓解多种并发症等。多糖类物质是由醛糖和酮糖通过糖苷键连接而成的一类高分子化合物,可通过修复小肠肠道屏障,调控肠道促炎因子, 进而缓解免疫应激,同时可通过有效调节免疫释放因子和免疫细胞,来调节免疫功能。
人体服用此发酵物后,可以更好地吸收水果中药组合物中的营养成分和功效因子,加速调节心情,舒缓情绪、安抚内心、行气养血。此外,我们通过细菌粘附性实验发现具有解郁宁心、行气养血的益生菌能高效定殖于服用者的肠道内,此外利用副干酪乳杆菌K9发酵筛选出的水果中药组合物后,能最大程度地产生大量活性益生菌和功能性成分(如:花色苷、多糖类物质等),实现双重调节,能够改善服用者肠道微环境,抑制肠道有害菌的生长繁殖,肠道内毒素水平降低,有效减少了炎症与应激反应,释放有机酸以及其他微量元素,从根源处实现解郁宁心、行气养血。
(2)本发明所提供的解郁宁心的益生菌发酵物的制备方法,利用副干酪乳杆菌K9对水果中药组合物进行发酵,所得的发酵物能提高发酵制品解郁宁心、行气养血的功效,同时所得的发酵物为活菌型。
生物材料保藏信息:
本发明所提供的菌株,副干酪乳杆菌K9,保藏编号为CGMCC No.15025,分类命名为:副干酪乳杆菌Lactobacillus paracasei,已于2017年12月01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 (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,邮政编码:100101),保藏单位的缩写为CGMCC。
具体实施方式
下面,结合具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
验证体系一:筛选水果中药组合
首先,本发明需要研发出合适的水果中药组合物,在实验过程中,按照下表1的配方分别取原料进行组合。
表1 水果中药组合物配方
分别取表1中的水果中药(组合1~7),分别加入60%的水,煮沸并保持5min,制成水果中药组合茶饮。招募21名心情烦闷的志愿者,分成7组,每组3人,分别服用组合1~7的水果中药组合茶饮,坚持10天,每日2杯,每杯300ml,每天的水果中药茶饮均当天熬制。测试者需自我评估心情改善、嘴唇红润情况,评价指标分别见下表2-3。评价结果取该组3名志愿者的平均值,结果见下表4。
表2 心情改善情况标准
表3 嘴唇颜色情况标准
表4 水果中药组合物人群功效验证结果
由表4可得,水果中药组合物2和组合物5的分值较高,对志愿者的心情和嘴唇颜色改善程度相对较大。而组合1、组合3和组合4中,部分原料的用量经过调整,试用效果较差;而组合6和组合7,部分原料的缺少,试用结果最差。
初步确定本发明的水果中药组合物由按照重量份的以下组分组成:枸杞8-12份,龙眼肉8-12份,余甘子8-12份,百合8-12份,蜂蜜8-12份,桑葚6-12份,玫瑰花6-12份,玉竹4-12份,大枣4-12份,莲子5-8份,百香果6-10份,苹果3-5份,葡萄8-12份。
更加优选地,水果中药组合物由按照重量份的以下组分组成:枸杞8-12份,龙眼肉8-12份,余甘子8-12份,百合8-12份,蜂蜜8-12份,桑葚8-12份,玫瑰花8-12份,玉竹8-12份,大枣8-12份,莲子5-8份,百香果6-10份,苹果3-5份,葡萄8-12份。
并且,较佳组合为:枸杞8份,龙眼肉8份,余甘子8份,百合8份,蜂蜜8份,桑葚8份,玫瑰花9份,玉竹8份,大枣8份,莲子6份,百香果8份,苹果4份,葡萄9份。
验证体系二:筛选益生菌
以水果中药组合2为实验对象,选取副干酪乳杆菌K9、植物乳杆菌FEED8、乳双歧杆菌Y6、鼠李糖乳杆菌YGG、长双歧杆菌W68、发酵乳杆菌FM12、戊糖片球菌LN-PT16以及青春双歧杆菌Y54等8种益生菌进行发酵实验,以筛选出较佳地益生菌,实验中所采用的益生菌均来自益可维生物技术有限公司。筛选方式如下:
种子活化扩培:在-80 ℃的菌种库中,每种菌株分别取保藏甘油管2支,在无菌操作条件下,按照10%接种量接入10 mL试管中进行培养,培养条件为37℃厌氧静置培养,培养时间24 h。待成熟后按2%接种量继续接入200 mL三角瓶,培养条件为37℃厌氧静置培养,培养时间16 h,之后继续扩培至3代备用。
水果中药组合物制备:取“枸杞8份,龙眼肉8份,余甘子8份,百合8份,蜂蜜8份,桑葚8份,玫瑰花9份,玉竹8份,大枣8份,莲子6份,百香果8份,苹果4份,葡萄9份”,将按比例制成的水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为20℃,时间为3d,然后破碎、压榨,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:4,加入S02 80ml/L和小苏打0.5%,制得水果中药组合物原料汁。
细菌粘附性实验:
一、细胞培养:
( 1)细胞培养液。DMEM(高糖) 培养基粉末一袋溶于950 mL去离子水,加入3.7gNaHCO3, 用NaOH调pH=7.2,去离子水定容至1 000 mL, 0.22 μm微孔滤膜过滤除菌,分装,4 ℃冰箱保存备用。
( 2) PBS液。PBS贮存液 ( 10×) : NaCl 8.5 g, KCl 0.2 g, Na2HPO4.12H2O2.85 g, KH2PO4 0.27 g,溶于100 mL去离子水中。PBS使用液:PBS贮存液用去离子水稀释10倍,NaOH调pH值至7.4,经121℃,15 min灭菌,4 ℃保存备用。
( 3) 细胞消化液(胰酶- EDTA消化液)。用PBS液溶解胰酶和EDTA,使最终质量分数为胰酶0.25%,EDTA质量分数为0.02%。0.22 μm过滤除菌,分装小瓶, - 20℃保存备用。
( 4) 细胞培养物制备。HT- 29细胞用含16%胎牛血清的DMEM培养液培养,CO2质量分数为10%,温度为37 ℃。每48 h换培养液1次,长成单层后传代接种于含盖玻片的六孔组织细胞培养板中,于质量分数为5%的CO2, 95%空气,37 ℃恒温培养48 h后进行粘附性实验。
二、细菌粘附性实验:用生理盐水将益生菌培养斜面洗下,用PBS溶液清洗,离心后制成一定浊度的菌悬液。加入到培养好的细胞中( HT- 29单层细胞),37 ℃培养,再用37℃的PBS溶液清洗,放置到12孔培养板中,自然干燥,甲醇(丙酮) 固定,Gram染色,显微观察。具体步骤为:
1) 试验前将菌株活化成斜面备用;
2) 用生理盐水将斜面洗下,用PBS溶液清洗2次,离心后制成一定浊度的菌悬液;
3) 加入到培养好的细胞中( HT- 29单层细胞),37 ℃培养60 min;4) 再用37 ℃的PBS溶液洗3~4次,放置到12孔培养板中,自然干燥;5) 甲醇(丙酮) 固定,Gram染色,显微观察。
三、细菌粘附指数计算:于油镜下( 1500×)随机选取20个视野,计数细胞上粘附的细菌个数,粘附指数=细菌数/ 10个细胞(即平均每10个细胞粘附的细菌个数)。测定结果进行统计学处理,如下表5所示。
耐水果中药组合物菌株的测定:将经过扩大培养的菌液,分别按照5%的接种量接入水果中药组合物原料汁添加浓度为2%的培养基中,培养条件:37℃,时间12h,获得水果中药发酵组合物,根据“GB 4789.35—2016”分别测定水果中药发酵组合物的活菌数,测定结果如下表6所示。
水果中药发酵组合物花色苷含量测定(pH示差法):取两个10ml棕色容量瓶,加入1ml蒸馏水,分别加入9ml pH 1.0缓冲液1和pH 4.5缓冲液2,摇匀,静置2h,分别测定510nm和700nm处的吸光度,作为对照组。而对于样品测定时,分别将上述水果中药发酵组合物离心,取1ml水果中药发酵组合物上清液,代替对照组中1ml的蒸馏水,其余操作均与对照组一样。
花色苷含量测定按照公式(1)进行计算,计算结果如下表7所示。
花色苷含量(mg/ml)=A × MW × DF/ε× I 公式(1)
(1)式中:A =(A510-A700)pH1.0-(A510-A700)pH4.5; MW为矢车菊素-3-葡萄糖苷的分子量(在此取449.2);
DF为稀释因子; ε为矢车菊素-3-葡萄糖苷的摩尔消光系数(在此取26900); I为比色皿光程(1cm)。
水果中药发酵组合物总糖含量的测定(蒽酮硫酸法):
标准曲线的制作:精密称取105℃烘千至恒重的葡萄糖0.1000g,置于100ml容量瓶中,加水使其溶解,稀释至刻度摇匀,得到1mg/ml葡萄糖溶液,再将其稀释至0.1mg/ml。配制成质量浓度为0、10、20、30、40、50、60、70、80mg/L的葡萄糖溶液,标号分别为1到9号管,分别取1ml于试管中,延壁加入4ml蒽酮硫酸试剂(2.0g/L),盖塞摇勾,置于沸水浴中10min。取出,待冰水浴至室温,在620nm波长下,以1号管为空白,测定其余各管吸光度。以葡萄糖溶液浓度(mg/L)为横坐标,OD620为纵坐标,制作标准曲线。
总糖含量的测定:将水果中药发酵组合物样品稀释至合适梯度,取1ml样品进行测定,根据葡萄糖含量的标准曲线,由样品溶液的吸光度值计算溶液中糖的含量,计算结果如下表8所示。
表5 8种益生菌益生菌对HT- 29细胞的粘附指数
从表5中的数据可得,进行试验的菌株中有6株菌株具有较好的吸附能力,10个细胞所吸附菌数总和大于20个,占受试菌株的75%。其中副干酪乳杆菌K9吸附细菌数最多达到286个,表现出相当强的对肠上皮细胞的吸附能力。
表6 8种益生菌在水果中药组合物的活菌含量CFU/ml
由表6可得,副干酪乳杆菌K9在水果中药组合物的培养基中存活率最高,表示活力最强,能更好地对水果中药组合物进行发酵。
表7 花色苷含量mg/L
表8 总糖含量g/L
从表7和表8的结果可得,采用副干酪乳杆菌K9对本发明的水果中药组合进行发酵,发酵组合物中的花色苷含量和总糖含量较高,有效成分含量高,发酵效果好。综上所述,选定副干酪乳杆菌K9为发酵菌。花色苷主要是通过糖苷键将花青素与糖进行结合而形成的化合物,具有多种生物活性,如清除自由基、抗氧化、抗炎症、缓解多种并发症等,提高血管的弹性,加速身体血液循环,有效避免人体郁气凝结,缓解人体焦虑,实现解郁宁心、行气养血。多糖类物质是由醛糖和酮糖通过糖苷键连接而成的一类高分子化合物,可通过修复小肠肠道屏障,调控肠道促炎因子,进而缓解免疫应激,同时可通过有效调节免疫释放因子和免疫细胞,来调节免疫功能,构建机体血脑免疫屏障。
验证体系三:筛选发酵条件
本发明实施例所提供的解郁宁心的益生菌发酵物,利用副干酪乳杆菌K9对水果中药组合物(表1中的组合2)进行发酵,所得的发酵物能提高发酵制品解郁宁心、行气养血的功效,同时所得的发酵物为活菌型。
实施例1
一、制备培养基
1)基础培养基
将6.0g酵母水解物、2.0g玉米浆、3.0g花生粉、10.0g糖蜜、3.0gKCl、0.1gZnSO4·8H2O、2gFeSO4·4H2O、8.0gCaCO3溶解于1L纯化水中,调节pH值至5.8,制得基础培养基(制备固体培养基时,加入15g琼脂)。
2)发酵培养基
将水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为20℃,时间为3d,然后破碎、压榨,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:4,加入S02 80ml/L和小苏打0.5%,制得水果中药组合物原料汁。
向1L上述原料汁中加入50g糖蜜、6g酵母水解物、2g硫酸铁和8.0gCaCO3,调节pH值至5.8,得到发酵培养基。
二、发酵
1)菌种活化
将保存于斜面上的副干酪乳杆菌(Lactobacillus paracasei)菌种(CGMCCNo.15025)接种于上述固体基础培养基中,在厌氧条件下静置培养24h,得到活化菌种。
2)制备种子液
将上述活化的菌种接种于上述液体基础培养基中,在厌氧条件下静置培养20h,得到种子液。
3)发酵
按照0.5%(v/v)的接种量,将上述种子液接种于上述发酵培养基中进行发酵培养,控制发酵温度为32°C,转速为100r/min,发酵周期为5天,得到水果中药组合物发酵液体制品。
三、真空冷冻干燥预处理
在无菌操作的条件下,将上述的水果中药组合物发酵液体制品放入真空冷冻干燥机内进行冻干处理,即直接置于箱内抽真空,操作方法为-40℃保持2h、0℃保持20h、30℃保持13~18h即可冻干,总冻干时间为35h到40h,终点时物料温度和搁板温度之差不高于5℃。
四、粉碎与过筛
采用超微粉碎方式处理4min,得到水果中药组合物发酵制品,即发酵水果中药组合物冻干粉。
根据中华人民共和国国家标准GB 4789.35—2016对发酵水果中药组合物冻干粉中的副干酪乳杆菌(乳酸菌)进行含量检测,结果见表9。
按照PH示差法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的花色苷进行含量检测,结果见表10。
按照蒽酮硫酸法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的总糖进行含量检测,结果见表11。
实施例2:
一、制备培养基
1)基础培养基
将10.0g酵母水解物、3.0g玉米浆、4.0g花生粉、10.0g糖蜜、2.0gKCl、0.3gZnSO4·8H2O、2.2gFeSO4·4H2O、10.0gCaCO3溶解于1L纯化水中,调节pH值至6.0,制得基础培养基(制备固体培养基时,加入15g琼脂)。
2)发酵培养基
将水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为22℃,时间为4d,然后破碎、压榨,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:3,加入S02 80ml/L和小苏打0.5%,制得水果中药组合物原料汁。
向1L上述原料汁中加入60g糖蜜、10g酵母水解物、2.2g硫酸铁和10.0gCaCO3,调节pH值至6.0,得到发酵培养基。
二、发酵
1)菌种活化
将保存于斜面上的副干酪乳杆菌(Lactobacillus paracasei)菌种(CGMCCNo.15025)接种于上述固体基础培养基中,在厌氧条件下静置培养24h,得到活化菌种。
2)制备种子液
将上述活化的菌种接种于上述液体基础培养基中,在厌氧条件下静置培养30h,得到种子液。
3)发酵
按照1.0%(v/v)的接种量,将上述种子液接种于上述发酵培养基中进行发酵培养,控制发酵温度为30°C,转速为100r/min,发酵周期为7天,得到水果中药组合物发酵液体制品。
三、真空冷冻干燥预处理
将上述的水果中药组合物发酵液体制品无菌操作放入真空冷冻干燥机内进行冻干处理,即直接置于箱内抽真空,操作方法为-40℃保持2h、0℃保持20h、30℃保持13~18h即可冻干,总冻干时间为35h到40h,终点时物料温度和搁板温度之差不高于5℃。
四、粉碎与过筛
采用超微粉碎方式处理4min,得到水果中药组合物发酵制品,即发酵水果中药组合物冻干粉。
根据中华人民共和国国家标准GB 4789.35—2016对发酵水果中药组合物冻干粉中的副干酪乳杆菌(乳酸菌)进行含量检测,结果见表9。
按照PH示差法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的花色苷进行含量检测,结果见表10。
按照蒽酮硫酸法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的总糖进行含量检测,结果见表11。
实施例3:
一、制备培养基
1)基础培养基
将10.0g酵母水解物、3.0g玉米浆、4.0g花生粉、10.0g糖蜜、2.0gKCl、0.3gZnSO4·8H2O、2.2gFeSO4·4H2O、10.0gCaCO3溶解于1L纯化水中,调节pH值至6.0,制得基础培养基(制备固体培养基时,加入15g琼脂)。
2)发酵培养基
将水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为24℃,时间为5d,然后破碎、压榨,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:2,加入S02 80ml/L和小苏打0.5%,制得水果中药组合物原料汁。
向1L上述原料汁中加入60g糖蜜、10g酵母水解物、2.2g硫酸铁和10.0gCaCO3,调节pH值至6.0,得到发酵培养基。
二、发酵
1)菌种活化
将保存于斜面上的副干酪乳杆菌(Lactobacillus paracasei)菌种(CGMCCNo.15025)接种于上述固体基础培养基中,在厌氧条件下静置培养24h,得到活化菌种。
2)制备种子液
将上述活化的菌种接种于上述液体基础培养基中,在厌氧条件下静置培养30h,得到种子液。
3)发酵
按照1.0%(v/v)的接种量,将上述种子液接种于上述发酵培养基中进行发酵培养,控制发酵温度为30°C,转速为100r/min,发酵周期为7天,得到水果中药组合物发酵液体制品。
三、真空冷冻干燥预处理
将上述的水果中药组合物发酵液体制品无菌操作放入真空冷冻干燥机内进行冻干处理,即直接置于箱内抽真空,操作方法为-40℃保持2h、0℃保持20h、30℃保持13~18h即可冻干,总冻干时间为35h到40h,终点时物料温度和搁板温度之差不高于5℃。
四、粉碎与过筛
采用超微粉碎方式处理4min,得到水果中药组合物发酵制品,即发酵水果中药组合物冻干粉。
根据中华人民共和国国家标准GB 4789.35—2016对发酵水果中药组合物冻干粉中的副干酪乳杆菌(乳酸菌)进行含量检测,结果见表9。
按照PH示差法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的花色苷进行含量检测,结果见表10。
按照蒽酮硫酸法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的总糖进行含量检测,结果见表11。
实施例4:
一、制备培养基
1)基础培养基
将8.0g酵母水解物、2.0g玉米浆、3.0g花生粉、12.0g糖蜜、3.0gKCl、0.2gZnSO4·8H2O、2gFeSO4·4H2O、8.0gCaCO3溶解于1L纯化水中,调节pH值至6.5,制得基础培养基(制备固体培养基时,加入15g琼脂)。
2)发酵培养基
将水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为24℃,时间为5d,然后破碎、压榨,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:1,加入S02 80ml/L和小苏打0.5%,制得水果中药组合物原料汁。
向1L上述原料汁中加入60g糖蜜、10g酵母水解物、2.2g硫酸铁和10.0gCaCO3,调节pH值至6.5,得到发酵培养基。
二、发酵
1)菌种活化
将保存于斜面上的副干酪乳杆菌(Lactobacillus paracasei)菌种(CGMCCNo.15025)接种于上述固体基础培养基中,在厌氧条件下静置培养24h,得到活化菌种。
2)制备种子液
将上述活化的菌种接种于上述液体基础培养基中,在厌氧条件下静置培养48h左右,得到种子液。
3)发酵
按照0.9%(v/v)的接种量,将上述种子液接种于上述发酵培养基中进行发酵培养,控制发酵温度为30°C,转速为100r/min,发酵周期为9天,得到水果中药组合物发酵液体制品。
三、真空冷冻干燥预处理
将上述的水果中药组合物发酵液体制品无菌操作放入真空冷冻干燥机内进行冻干处理,即直接置于箱内抽真空,操作方法为-40℃保持2h、0℃保持20h、30℃保持13~18h即可冻干,总冻干时间为35h到40h,终点时物料温度和搁板温度之差不高于5℃。
四、粉碎与过筛
采用超微粉碎方式处理4min,得到水果中药组合物发酵制品,即发酵水果中药组合物冻干粉。
根据中华人民共和国国家标准GB 4789.35—2016对发酵水果中药组合物冻干粉中的副干酪乳杆菌(乳酸菌)进行含量检测,结果见表9。
按照PH示差法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的花色苷进行含量检测,结果见表10。
按照蒽酮硫酸法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的总糖进行含量检测,结果见表11。
对比例1:
一、制备空白培养基
将水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为20℃,时间为3d,然后破碎、压榨,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:4,加入S02 80ml/L和小苏打0.5%,制得水果中药组合物原料汁。
向1L上述原料汁中加入50g糖蜜、6g酵母水解物、2g硫酸铁和8.0gCaCO3,调节pH值至5.8,得到发酵培养基。
二、发酵
按照0.0%(v/v)的接种量,将种子液接种于上述发酵培养基中进行发酵培养,控制发酵温度为32°C,转速为100r/min,发酵周期为5天,得到水果中药组合物发酵液体制品。
三、真空冷冻干燥预处理
将上述的水果中药组合物发酵液体制品无菌操作放入真空冷冻干燥机内进行冻干处理,即直接置于箱内抽真空,操作方法为-40℃保持2h、0℃保持20h、30℃保持13~18h即可冻干,总冻干时间为35h到40h,终点时物料温度和搁板温度之差不高于5℃。
四、粉碎与过筛
采用超微粉碎方式处理4min,得到水果中药组合物发酵制品,即发酵水果中药组合物冻干粉。
根据中华人民共和国国家标准GB 4789.35—2016对发酵水果中药组合物冻干粉中的副干酪乳杆菌(乳酸菌)进行含量检测,结果见表9。
按照PH示差法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的花色苷进行含量检测,结果见表10。
按照蒽酮硫酸法(具体方法如验证体系二中所述)对上述发酵水果中药组合物冻干粉中的总糖进行含量检测,结果见表11。
对比例2
对比例2与实施例1的不同之处在于:发酵培养基的不同,水果中药组合物不是采用CO2浸渍发酵,而是采用酵母菌进行发酵。具体操作如下:
将水果中药组合物破碎、压榨,然后将酵母与5%的糖水按质量比1∶10混合,于40℃水浴锅内活化30 min,向水果中药组合物中接入活化后的酵母菌,发酵,得原料汁基料;向原料汁基料中加入纯化水进行调浆,控制原料汁基料与纯化水的体积比为1:4,加入S0280ml/L和小苏打0.5%,制得水果中药组合物原料汁。
向1L上述原料汁中加入50g糖蜜、6g酵母水解物、2g硫酸铁和8.0gCaCO3,调节pH值至5.8,得到发酵培养基。
表9 活菌含量CFU/g
表10 花色苷含量mg/L
表11 总糖含量g/L
由表9、表10、表11可以发现,实施例3在活菌数、花色苷、总糖等方面含量均最高,因此可以得出副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)发酵水果中药组合物的最佳工艺条件为实施例3所述。
人体实验
试验过程:设计双盲试验,招募21位创业型公司的健康志愿者,年龄在25−60岁之间,测试周期30天。将测试组分为7组,每组3人,分别使用实施例1-4、对比例1-2、市售产品,测试者午餐、晚餐饭后半小时服用该组合物各1袋。测试结束后,测试者需自我评估心理情况、负面情绪情况、睡眠质量、嘴唇颜色等情况,并对所服用样品的口感进行评价,每组结果取该组3名志愿者的平均值。
其中,市售样品为Garden of Life的情绪健康益生菌保健品,这是一款美国的热门产品,含有蓝莓、人参、瑞氏乳杆菌、长双歧杆菌、阿拉伯树胶益生元,以硬胶囊形式吞服实现情绪健康。
表12 心理情况标准
表13负面情绪标准
表14睡眠质量
表15嘴唇颜色
表16 解郁宁心益生菌发酵物人群功效验证
从表16的记录可得,实施例3的解郁宁心效果均优于市售产品。服用30天后,受试者在心理情况、负面情绪、睡眠质量以及嘴唇颜色这几个方面均有较好的体验感,普遍反馈解郁宁心、行气养血效果显著,因此总有效率达到100%以上。其中实施例3心理情况的分值高达8.5分,表明了受试者服用益生菌期间心情舒畅、没有抑郁情况、身轻体健、血气十足。
结合验证体系一表4中的记录可得,相对于未发酵的水果中药组合物,利用本发明的副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)进行发酵的发酵制品中,能提高发酵制品解郁宁心、行气养血的功效,同时所得的发酵物为活菌型。
本发明实施例使用副干酪乳杆菌(Lactobacillus paracasei,CGMCC No.15025)发酵制成的活菌型水果中药组合物冻干粉,发酵制品的解郁宁心、行气养血的功效显著提升,同时,在发酵过程中,益生菌不断与水果中药组合物组织相互作用,既增殖了益生菌的数量,又产生丰富的有机酸、糖类、蛋白等营养物质。服用本品后,能补充丰富的营养成分和功效成分,能拮抗哇巴因、肾上腺素所诱发的心律失常,清热解毒,解郁宁心,体感明显。此外,益生菌定殖后能改善肠道微环境,抑制有害菌,降低肠道内毒素,继续持续生成机体所需的营养物质,协助调节机体内分泌,保持体内激素水平平稳,使人体保持心情舒畅,解郁宁心,血气充足,效果持久。
本发明实施例所提供的具有解郁宁心、行气养血的益生菌发酵物,通过水果结合中药组合物进行深度发酵,极大地提升了水果中药组合物解郁宁心、行气养血的功效成分,提高生物利用度。人体服用此发酵物后,可以更好地吸收水果中药中的营养成分和功效因子,加速调节心情,舒缓情绪、安抚内心、行气养血。此外让具有解郁宁心、行气养血的益生菌高效定殖于肠道,改善了肠道微环境,抑制肠道有害菌的生长繁殖,肠道内毒素水平降低,有效减少了炎症与应激反应,释放有机酸以及其他微量元素,从根源处实现解郁宁心、行气养血。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (8)
1.一种解郁宁心的益生菌发酵物,其特征在于,由益生菌对水果中药组合物进行发酵而成,所述益生菌包括保藏编号为CGMCC No.15025的副干酪乳杆菌K9;所述水果中药组合物为经CO2浸渍发酵后的水果中药组合物;所述水果中药组合物由按照重量份的以下组分组成:枸杞8-12份,龙眼肉8-12份,余甘子8-12份,百合8-12份,蜂蜜8-12份,桑葚6-12份,玫瑰花6-12份,玉竹4-12份,大枣4-12份,莲子5-8份,百香果6-10份,苹果3-5份,葡萄8-12份。
2.如权利要求1所述的解郁宁心的益生菌发酵物,其特征在于,所述水果中药组合物由按照重量份的以下组分组成:枸杞8份,龙眼肉8份,余甘子8份,百合8份,蜂蜜8份,桑葚8份,玫瑰花9份,玉竹8份,大枣8份,莲子6份,百香果8份,苹果4份,葡萄9份。
3.一种如权利要求1-2任一项所述的解郁宁心的益生菌发酵物的制备方法,其特征在于,包括以下步骤:
浸渍发酵步骤:将所述水果中药组合物放到充满CO2的密闭罐中浸渍,得到预处理后的水果中药组合物;
榨汁步骤:将所述预处理后的水果中药组合物进行破碎、压榨,得到原料汁;
益生菌发酵步骤:制备包含所述原料汁的发酵培养基,将副干酪乳杆菌K9种子液接入所述发酵培养基中,发酵,得到发酵液体制品;
干燥步骤:将所述发酵液体制品进行干燥,得到发酵固体制品;
粉碎过筛步骤:将所述发酵固体制品进行粉碎、过筛,即得所述解郁宁心的益生菌发酵物。
4.如权利要求3所述的解郁宁心的益生菌发酵物的制备方法,其特征在于,还包括调汁步骤:经过所述榨汁步骤获取所述原料汁后,向所述原料汁中加入纯化水进行调浆,控制所述原料汁与纯化水的体积比为1:(1-4)。
5.如权利要求4所述的解郁宁心的益生菌发酵物的制备方法,其特征在于,在所述调汁步骤中,还向所述原料汁中加入70~100mL/L的SO2和0.4~0.6%的小苏打。
6.如权利要求3所述的解郁宁心的益生菌发酵物的制备方法,其特征在于,包括以下步骤:
浸渍发酵步骤:将所述水果中药组合物放到充满CO2的密闭罐中浸渍,浸渍温度为20-25℃,时间为3-7d,得到预处理后的水果中药组合物;
榨汁步骤:将所述预处理后的水果中药组合物进行破碎、压榨,得到原料汁;
调汁步骤:向所述原料汁中加入纯化水进行调浆,控制所述原料汁与纯化水的体积比为1:(1-4);并向所述原料汁中加入70~100mL/L的SO2和0.4~0.6%的小苏打,得到处理后原料汁;
益生菌发酵步骤:向每升所述处理后原料汁中加入0-100g碳源、5-10g氮源和1-15g无机盐,随后调节pH值至5.5-6.5,得到发酵培养基;以体积比计按照0.3~2.0%的接种量将副干酪乳杆菌K9种子液接入所述发酵培养基中进行发酵培养,控制发酵温度为30~32℃,转速为90~120r/min,发酵周期为5~9天,得到发酵液体制品;
干燥步骤:将所述发酵液体制品放入真空冷冻干燥机内进行冻干处理,冷冻流程依次为 -45~-35℃保持1.8~2.2h、-2~2℃保持19~21h、28~32℃保持13~18h,总冻干时间为35~40h,终点时物料温度和搁板温度之差不高于5℃,得到发酵固体制品;
粉碎过筛步骤:将所述发酵固体制品采用超微粉碎方式处理3~5min,然后过筛,即得所述解郁宁心的益生菌发酵物。
7.如权利要求3所述的解郁宁心的益生菌发酵物的制备方法,其特征在于,所述副干酪乳杆菌K9种子液的获取方法如下:制备基础培养基:将6.0~10.0g酵母水解物、2.0~3.0g玉米浆、3.0~4.0g花生粉、10.0~12.0g糖蜜、2.0~3.0gKCl、0.1~0.3gZnSO4·8H2O、2~2.2gFeSO4·4H2O、8.0~10.0gCaCO3溶解于1L纯化水中,调节pH值至5.8~6.0,制得液体基础培养基;制备固体培养基时,向所述液体基础培养基内加入12~18g琼脂;
菌种活化:将保存于斜面上的保藏编号为CGMCC No.15025的副干酪乳杆菌K9接种于所述固体培养基中,在厌氧条件下静置培养20~30h,得到活化菌种;
制备种子液:将所述活化菌种接种于所述液体基础培养基中,在厌氧条件下静置培养20~48h,得到副干酪乳杆菌K9种子液。
8.一种如权利要求1-2任一项所述的解郁宁心的益生菌发酵物在制备具有解郁宁心作用的产品中的应用。
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