CN108936577A - 一种具有心血管保护作用的天然可食用化合物群制备方法 - Google Patents
一种具有心血管保护作用的天然可食用化合物群制备方法 Download PDFInfo
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- CN108936577A CN108936577A CN201810855689.4A CN201810855689A CN108936577A CN 108936577 A CN108936577 A CN 108936577A CN 201810855689 A CN201810855689 A CN 201810855689A CN 108936577 A CN108936577 A CN 108936577A
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- A—HUMAN NECESSITIES
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
本发明涉及一种可用于食品加工的红曲霉菌及银杏叶、茯苓等中药,结合鲜红薯、大米粉、大豆粉等天然食材,基于工业化液体发酵法制备的一种具有心血管保护作用的食品。本发明公开了红曲霉菌与鲜红薯、大米粉、大豆粉等液态发酵原料按一定比例混合并处理后,采用一定的工业液态发酵方法发酵一段时间后,收集发酵液加入茯苓提取物、银杏叶提取物等,混合、离心、过滤后制备的一种饮料。本发明制备的天然化合物群含有可保护心肌、降低血脂、抗氧化的Monacolin‑K、黄酮、多糖类物质等多种活性物质,具有良好的心血管保护作用效果,既是食品又有较高的保健价值。
Description
技术领域
本发明涉及一种天然化合物群的制备方法,具体涉及一种基于工业化液体发酵法的具有心血管保护作用的天然可食用化合物群的制备方法。
背景技术
随着我国社会经济的快速发展和生活方式的改变,居民的营养与健康状况处于快速变迁时期。自20世纪90年代中期起,成人超重、肥胖呈现加快增长趋势。据国家卫生计生委疾病预防控制局公布的《中国居民营养与慢性病状况报告(2015)》显示,2012年18岁及以上成人的血脂异常发生率为40.4%,其也是诱发心血管病的危险因素。研究可具有心血管保护作用的天然可食用化合物群,能安全辅助治疗、控制心血管慢性病,对于确保我国人群健康、控制心脏血管病发生率具有极其重要的意义。
红薯又称红苕,含有丰富的淀粉;维生素;纤维素;镁、磷、钙等矿物元素和亚油酸等人体必需的营养成分,能保持血管弹性,具有心血管辅助保护作用;且红薯的多糖类成分具有特殊的甜味,可代替蔗糖等甜味剂。
茯苓Poria cocos(Schw.)Wolf.为多孔菌科卧菌属真菌的干燥菌核,有健脾补中、养心安神、利水渗湿功能;茯苓中茯苓多糖、三萜皂苷等成分特别是预防和治疗高脂血症引发的动脉硬化、冠心病、脑梗塞等心血管病。银杏叶为银杏科植物银杏Ginkgo biloba L.的干燥叶,能活血化瘀,通络止痛,化浊降脂;银杏叶提取物中的黄酮类成分能增加脑血管血液流量,改善脑血管血液循环功能,保护脑细胞,扩张冠状动脉,预防心绞痛及心肌梗塞,预防血栓形成,提高机体免疫能力。以上两者均属于国家规定的药食同源类既是食品也是药品的原料。
红曲是我国传统的食药两用发酵产品,其由红曲霉属真菌接种于大米发酵而成,是一种食疗兼备的传统中药;红曲霉菌(Monascus anka)也在国家卫生部印发的《可用于食品的菌种名单》中。红曲霉菌在发酵过程中能代谢产生Monacolin K等化合物,具有良好的降低胆固醇等作用,疗效显著、毒副作用小、耐受性好。
因此,基于工业化的液体发酵及现代生物技术,利用红薯、茯苓、银杏叶、红曲霉菌制备一种用于保护心血管功能的天然可食用化合物群,综合发挥茯苓、银杏叶、红曲霉菌的效用,安全有效地辅助治疗心血管慢性病,是本发明解决的关键问题。
发明内容
本发明针对现有技术中存在的技术问题,提供一种具有心血管保护作用的天然可食用化合物群制备方法。在使用超过上千年的传统中医药中筛选出可当食用使用的茯苓、银杏叶等;并以红曲霉为菌种,加入大米、大豆、红薯等,运用现代工业化液体发酵技术,制备一种天然化合物群,作为一种原料可广泛用于具有保健功能的食品。
本发明解决上述技术问题的技术方案如下:本发明解决上述技术问题的技术方案如下:一种具有心血管保护作用的天然可食用化合物群制备方法,包括以下步骤:
将所述鲜红薯蒸汽锅蒸制去皮,加水粉碎匀浆;
将所述大米淀粉、大豆蛋白粉、红薯浆、淀粉酶等按一定比例混合并搅拌均匀;
加入红曲霉菌于上所述均匀液进行液态发酵处理;
制备茯苓、银杏叶纯水提取物;
将液态发酵后液体与茯苓、银杏叶提取物混合,离心、过滤,获取澄清液体。
进一步,所述红薯的加工方法,精选红安地理标志产品--红薯的鲜品,去除泥土、黑斑等杂质并清洗。将所述鲜红薯用密闭蒸汽压力锅于30-70KPa蒸制10-20分钟至熟至透芯,取出冷至常温却后去皮。粗粉碎后,与水按1:2-5(质量:体积)比例混合,机械搅拌形成红薯混悬液。
进一步,所述大米粉、大豆粉按一定比例混合,并粉碎过100目筛。按食品级α-淀粉酶(20000-100000u/g)与浆液中大米粉、大豆粉、固态红薯等固态物质质量比(1-5:100)的比例混合,并加入一定纯化水并调pH至6.0±0.2,置于密闭均质搅拌罐中,保持温度70-100℃,并按1000-3000转/分进行搅拌2-4小时,形成匀浆液。于匀浆液中加入硝酸钠、硫酸镁、二氢磷酸钾、硫酸锌等盐类;推荐加入比例为:NaNO3:0.18-0.22%;MgSO4·7H2O:0.08-0.12%;KH2PO4:0.18-0.22%;ZnSO4·7H2O:0.34-0.38%。所述原浆液加入纯化水稀释,于发酵罐中搅拌均匀,并使用乳酸调节pH值至4.9-5.1。培养基灭菌,温度123℃,灭菌时间30-45min,形成液态发酵基础原液。
进一步,所述的红曲霉菌在进入液态发酵罐之前经过发酵培养。将红曲霉菌移种于红曲霉菌培养基中,于生物恒温培养摇床中进行培育;摇床温度控制在30-35℃,摇床速度控制在160-200转/分钟,发酵时间控制在24-72小时;以菌丝体生长速度为参照,记录每毫升培养液中孢子数目,并于光学显微镜下观察菌丝体浓度,控制原菌液中所述菌丝体的折算浓度为5.0-8.0×102株/毫升,得到红曲霉菌原菌液。推荐红曲霉菌培养基组成:70-100g/L葡萄糖,5-15g/L蛋白胨,2-5g/L磷酸二氢钾,1-3g/L七水硫酸镁,1-3g/L七水硫酸锌,3-5g/L的柠檬酸钠,调pH至6.0-6.2,纯化水定容至一定体积,于121℃、0.1Mpa压力下灭菌15-30分钟。培养基成分可根据红曲霉菌的生长做微调,不排除加入3-5%含量的甘油等。
进一步,将红曲霉菌原菌液以一定比例加入常温液态发酵基础原液。推荐按与终液态发酵基溶液体积比(1-3:100)加入。发酵期间,根据发酵液溶氧数据调控:a)无菌空气进入罐中的流速;b)发酵罐中有上下2套搅拌桨速可独立控制的搅拌系统,搅拌速度可根据需要独立调整。具体的,所述根据发酵液溶氧数据调控,包括:根据红曲霉菌的生长特点,发酵期间,发酵罐中传感器所测定的溶解氧浓度应控制在20%~30%。
进一步,液态发酵共分2个阶段进行:细胞生长阶段与生物代谢物生物合成的阶段。其中,第一阶段温度控制在30-32℃,搅拌桨速控制在230-250r/min;通气量1-3wm,WM表示每分钟通气量与罐体实际料液体积的比值,根据溶解氧浓度调整通气量;并根据罐中取样液菌丝体生长状况及稠度调整桨速。第二阶段,温度控制在22-25℃;搅拌桨速控制在190-210r/min;通气量1-3wm,根据罐中取样液菌丝体生长状况及稠度调整桨速。发酵罐中液态发酵第一阶段时间控制在2-6天,第二阶段控制在4-10天。第二阶段根据功能红曲主要活性成分莫纳可林K(MK)的含量为终止发酵的参考条件,测定条件为公认的高效液相法测定,通过MK含量—发酵时间绘制曲线。建议终止发酵时MK含量不低于0.02%。发酵期间,不排除在第一阶段末期加入3-5%含量的甘油等。
进一步,根据《中国药典》2015版,对两个药材银杏叶、茯苓完成对应指标的检测。所述银杏叶、茯苓提取物,其特征在于:将银杏叶、茯苓药材按质量比1:1混合,粗粉碎过4号筛。将筛后的粗粉与去离子纯化水按1:8-1:10(质量:体积)混合,冷浸30分钟后,加热煮沸并保持微沸提取1-1.5小时,趁热过滤;滤后药材再加入1:6-1:8(质量:体积)的去离子纯化水混合,加热煮沸并保持微沸提取0.75-1小时,趁热过滤。收集滤液,减压浓缩成膏状,密度为1.10-1.30g/cm3。将提取物膏减压干燥成干膏,并粉碎成粉,并称量其质量,并折算成原药材质量,备用。
进一步,从发酵罐中取出发酵液,并过滤形成发酵后清液。按一定比例加入茯苓、银杏叶提取物搅拌并致完全溶解。推荐:依据《中国药典》2015版,按100mL含有折算后银杏叶、茯苓原药材不高于9g。滤过除菌后,灌装成饮料;或喷雾干燥成粉,按比例添加适当的食用辅料,制备片剂、颗粒剂、软胶囊剂、丸剂或口服液等天然食材类保健品领域常用制剂。
进一步,根据原料投料计量及现有测量方法测定Monacolin-K、黄酮等成分后,按比例添加适当的食用辅料,制备片剂、颗粒剂、软胶囊剂、丸剂或口服液。
本发明所述的液态发酵原料里,包括国家卫计委规定的药食同源原料银杏叶、茯苓提取物;国家卫生部的《可用于食品的菌种名单》中的红曲霉菌;以及红薯、大米粉、大豆粉等。根据本领域的公知常识和辅料的成分,本发明的天然化合物群可以制成本领域常用的各种剂型。
具体实施方式
以下结合实例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
实施例1
将银杏叶、茯苓药材按质量比1:1混合,粗粉碎过4号筛,称量。将筛后的原料与去离子纯化水,按1:8(质量:体积)混合,冷浸30分钟后,加热煮沸并保持微沸提取1小时,趁热过滤;滤后药材再加入去离子纯化水按1:6混合,加热煮沸并保持微沸提取0.75小时,趁热过滤。收集滤液,将滤液减压浓缩成膏状,密度为1.10-1.20g/cm3。将提取物膏减压干燥成干膏,粉碎成粉,并称量其质量;干浸膏折算成原药材质量,备用,其中山楂、茯苓混合药材干浸膏粉出膏率为19.6%。
制备红曲霉菌培养基:按1L算,取葡萄糖70g,蛋白胨10g,磷酸二氢钾4.0g,七水硫酸镁2.5g,七水硫酸锌2g,柠檬酸钠3.5g,甘油50g。调pH至6.0-6.2并用纯化水定容至1L,于121℃、0.1Mpa压力下灭菌30分钟。冷却至常温。
红曲霉菌菌种培养:将红曲霉菌种移种于红曲霉菌培养基中,于生物恒温培养摇床中进行培养;摇床温度控制在32℃,摇床速度控制在180转/分钟,发酵40小时。发酵结束后记录每毫升培养液中孢子数目,实测在原菌液中折算浓度为7.2×102株/毫升。
精选红安地理标志产品--红薯鲜品,去除泥土、黑斑等杂质并清洗。将所述鲜红薯用密闭蒸汽压力锅于50KPa蒸制15分钟至熟至透芯,取出冷至常温却后去皮。常温粗粉碎后,与水按1:4(质量:体积)比例混合,机械搅拌形成红薯混悬液。
大米粉、大豆粉按质量比1:2混合,并粉碎过100目筛。混合粉按混合粉质量:固态红薯质量为5:1比例加入上述红薯混悬液中。按食品级α-淀粉酶(20000-100000u/g)与液体浆液中大米、大豆粉、固态红薯等固态物质质量比(3:100)的比例加入淀粉酶,分次加入一定量纯化水并调pH至6.0±0.2。将搅拌机内温度预加热至80℃,投入物料,搅拌速度2000转/分钟,搅拌时间2-4小时,制成匀浆液。于匀浆液中加入硝酸钠、硫酸镁、二氢磷酸钾、硫酸锌等盐类;推荐加入比例为:NaNO3:0.20%;MgSO4·7H2O:0.10%;KH2PO4:0.20%;ZnSO4·7H2O:0.35%。所述原浆液加入纯化水稀释,于发酵罐中搅拌均匀,并使用乳酸调节pH值致5.0。培养基灭菌,温度123℃,灭菌时间45min,形成液态发酵基础原液。
灭菌后的液态发酵基冷却至常温后,加入红曲霉菌原菌液,按最终液态发酵基溶液体积比(2.5:100)加入。于发酵罐中进行液态条件下发酵。
根据红曲霉菌的生长特点,发酵罐中电子鼻所测定的溶解氧浓度应控制在23-25%。液态发酵共分2个阶段进行:细胞生长阶段与生物代谢物生物合成的阶段。其中,第一阶段温度控制在30℃,时间控制在3天;搅拌桨速控制在230-250r/min,通气量1-3wm(WM表示每分钟通气量与罐体实际料液体积的比值),根据溶解氧浓度调整通气量。根据罐中取样液菌丝体生长状况及稠度,可在桨速范围内调整上下桨速度。第二阶段,温度控制在23℃,搅拌桨速控制在190-210r/min,上下桨速度相同;通气量1-3wm,根据溶解氧浓度调整通气量,根据罐中取样液菌丝体生长状况及稠度调整桨速。在第一阶段发酵初期按50g/L加入甘油并混合均匀;发酵期间每天从发酵罐中取出样液,观察红曲霉菌丝生长状况、Monacolin-K为主的代谢产物浓度。终止发酵时MK含量为0.03%。结束发酵后,从发酵罐中取出发酵液,并过滤形成发酵后清液。
将发酵后清液与前所制备银杏叶、茯苓共提物粉末完全溶解并搅拌均匀;提取物加入量以每100毫升清液含折算后的9克银杏叶、茯苓原药材为标准。滤过除菌后,灌装成饮料。
实施例2
选择了经典药理学实验来确认实施例1所制备的固态细粉的心血管保护作用。实验动物为50只雄性昆明小鼠,体重28±2g,随机分为5组,即,给予一定体积的生理盐水正常组(1)、模型组(2),低剂量组(3)、中剂量组(4)和高剂量组(5);每组十只,2-5号组给予高脂饮食。根据动物与人体表面积比换算法,按每公斤体重小鼠给予的原药材量(银杏叶+茯苓)计算,高剂量组为2.6g/kg(原药材量/公斤体重),中剂量组为1.3g/kg,低剂量组为相当于0.65g/kg;将实例1中的饮料减压浓缩至1g原药材(银杏叶+茯苓)/毫升。1、2号组给予生理盐水,3、4、5号组给予不同剂量浓缩物,均灌胃给药,每天1次,连续服用14天,于14天后小鼠眼眶采血,用全自动生化分析仪测定不同组小鼠血浆中胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C);按ELASA法测定超氧化物歧化酶(SOD)、丙二醛(MDA)的含量。结果见表1、表2。
表1不同组小鼠血浆中TC、TG、LDL-C、HDL-C比较
表2不同组小鼠血浆中SOD、MDA比较
实验结果显示,本方法制备的天然化合物群与模型组相比,具有良好的降低血液中TC、TG、LDL-C的浓度,升高HDL-C浓度;提高血浆中SOD含量,降低MDA含量,通过降低血脂及提高机体抗氧化作用,达到心血管保护作用。
实施例3
建立高效液相色谱测定Monacolin-K的方法。精密量取一定体积实施例1所述发酵后样品,加酸调整pH至3并定容;加入一定体积三氯甲烷,置于涡旋混匀器混匀15min。静置后去掉上层水相,将三氯甲烷层以3000r/min离心3min。精密取一定体积离心后氯仿层液体,通过微孔滤膜过滤取续滤液制备液相检测样品。使用C18反向色谱柱(4.6mm×250mm),柱温为室温;紫外检测波长238nm;流动相为乙腈:水:0.5%磷酸=60:37:3(体积比);流速为1.0mL min,进样量10微升。测得发酵完毕后未离心前样品Monacolin-K含量为0.03%(内酯式+酸式总和)。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,包括以下步骤:
将鲜红薯蒸制去皮,加水粉碎匀浆,制得红薯悬液;
将大米粉、大豆粉、红薯悬液、淀粉酶按一定比例混合并搅拌均匀成匀浆液,形成液态发酵基础原液;
加入红曲霉菌原菌液于上所述液态发酵基础原液在发酵罐中进行液态发酵处理一段时间,得到液态发酵液;
制备茯苓、银杏叶纯水提取物;
将液态发酵液与茯苓、银杏叶提取物混合,离心、过滤,获取澄清液体。
2.根据权利要求1所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,
所述的红曲霉菌在进入液态发酵罐之前经过发酵培养;
所述红曲霉菌液态培养基组成:70-100g/L葡萄糖,5-15g/L蛋白胨,2-5g/L磷酸二氢钾,1-3g/L七水硫酸镁,1-3g/L七水硫酸锌,3-5g/L的柠檬酸钠,调pH至6.0-6.2,纯化水定容至一定体积,于121℃、0.1Mpa压力下灭菌15-30分钟;将红曲霉菌菌种(Monascus anka)移种于红曲霉菌培养基中,于生物恒温培养摇床中进行培育;摇床温度控制在30-35℃,摇床速度控制在160-200转/分钟,发酵时间控制在24-72小时;以菌丝体生长速度为参照,记录每毫升培养液中孢子数目,并于光学显微镜下观察菌丝体浓度,控制原菌液中所述菌丝体的折算浓度为5.0-8.0×102株/毫升,得到红曲霉菌原菌液。
3.根据权利要求1所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述的将鲜红薯蒸制去皮,加水粉碎匀浆,包括:
将所述鲜红薯去除泥土、黑斑杂质并清洗后,用密闭蒸汽压力锅于30-70KPa蒸制10-20分钟至透芯,取出冷至常温后去皮;
去皮后红薯粗粉碎后,与水按质量体积比为1:2~1:5的比例混合,搅拌形成红薯悬液。
4.根据权利要求1所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述的将大米粉、大豆粉、红薯浆、淀粉酶按一定比例混合并搅拌均匀成匀浆液,包括:
大米粉、大豆粉按一定比例混合,并粉碎过100目筛;
将以上混合粉按一定比例加入所述红薯悬液中并搅拌成浆液;
将食品级α-淀粉酶(20000-100000u/g)与浆液中大米粉、大豆粉、固态红薯固态物质按质量比1-5:100的比例混合,并加入一定纯化水并用磷酸缓冲盐调pH至6.0±0.2,置于密闭均质搅拌罐中;
保持温度70-100℃,并按1000-3000转/分进行搅拌2-4小时,形成匀浆液;
于匀浆液中加入一定比例的硝酸钠、硫酸镁、二氢磷酸钾、硫酸锌;
所述原浆液加入纯化水稀释,于发酵罐中搅拌均匀,并使用乳酸调节pH值致4.9-5.1;
培养基灭菌,温度123℃,灭菌时间30-45min,形成液态发酵基础原液。
5.根据权利要求4所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述硝酸钠、硫酸镁、二氢磷酸钾、硫酸锌的加入比例为:
NaNO3:0.18-0.22%;MgSO4·7H2O:0.08-0.12%;KH2PO4:0.18-0.22%;ZnSO4·7H2O:0.34-0.38%。
6.根据权利要求1所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述红曲霉菌原菌液以体积比为1~3:100的比例加入所述的液态发酵基础原液。
7.根据权利要求6所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述含红曲霉的发酵液于发酵罐中进行液态条件下发酵;发酵期间,根据发酵液中溶氧数据调控:a)无菌空气进入罐中的流速;b)发酵罐中上下2套搅拌桨速可独立控制的搅拌系统,搅拌速度可根据需要独立调整。
8.根据权利要求6所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述发酵液溶氧数据,
根据红曲霉菌的生长特点,发酵期间,发酵罐中电子鼻所测定的溶解氧浓度应控制在20%~30%;
液态发酵共分2个阶段进行:细胞生长阶段与生物代谢物生物合成的阶段;
其中,第一阶段温度控制在30-32℃;搅拌桨速控制在230-250r/min,可上、下桨分开设置桨速;通气量1-3wm,wm表示每分钟通气量与罐体实际料液体积的比值,根据溶解氧浓度调整通气量,并根据罐中取样液菌丝体生长状况及稠度调整桨速;
第二阶段,温度控制在22-25℃;搅拌桨速控制在190-210r/min;通气量1-3wm,根据溶解氧浓度调整通气量,根据罐中取样液菌丝体生长状况及稠度调整桨速;
发酵罐中液态发酵第一阶段时间控制在2-6天,第二阶段控制在4-10天;终止发酵时MK含量不低于0.02%。
9.根据权利要求8所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述银杏叶、茯苓提取物,
将银杏叶、茯苓药材按质量比1:1混合,粗粉碎过4号筛;
将筛后的粗粉与去离子纯化水按质量体积比1:8~1:10混合,冷浸30分钟后,加热煮沸并保持微沸提取1-1.5小时,趁热过滤;滤后药材再加入体积质量比为1:6~1:8的去离子纯化水混合,加热煮沸并保持微沸提取0.75-1小时,趁热过滤;
收集滤液,减压浓缩成膏状,密度为1.10-1.30g/cm3;
将提取物膏减压干燥成干膏,并粉碎成粉,并称量其质量,并折算成原药材质量,备用。
10.根据权利要求9所述一种具有心血管保护作用的天然可食用化合物群制备方法,其特征在于,所述液态发酵液于第二阶段结束后停止发酵,从发酵罐中取出发酵液,并过滤形成发酵后清液;加入所述茯苓、银杏叶提取物,完全溶解并搅拌均匀后,滤过除菌后,灌装成饮料;或喷雾干燥成粉,按比例添加适当的食用辅料,制备片剂、颗粒剂、软胶囊剂、丸剂或口服液制剂。
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