CN115299593B - 一种具有解酒功能的葛根枳椇子洛神花酵素及其制备方法 - Google Patents
一种具有解酒功能的葛根枳椇子洛神花酵素及其制备方法 Download PDFInfo
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Abstract
本发明涉及酵素技术领域，具体为一种具有解酒功能的葛根枳椇子洛神花酵素及其制备方法。包括以下原料：葛根30‑40g/L，枳椇子25‑35g/L，洛神花20‑30g/L，菊花5‑10g/L，低聚异麦芽糖10‑30g/L，红糖8‑15g/L，木糖醇5‑10g/L。本发明还提供一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法。本发明所制酵素产品功效更强，对白酒造模的小鼠醉酒模型具有明显的解酒效果，具有明显的加速清除大鼠血液中乙醇的作用。
Description
技术领域
本发明涉及酵素技术领域，具体为一种具有解酒功能的葛根枳椇子洛神花酵素及其制备方法。
背景技术
近年来，饮酒成为一种风尚，无论是朋友聚会还是业务洽谈不免要饮酒，因此酒精迫切被人们需要，随之给身体带来不良反应。酒精进入人体后，经过乙醇脱氢酶的作用转化为乙醛，乙醛经过乙醛脱氢酶氧化为乙酸，氧化为二氧化碳和水。当人体一次性摄入70g以上酒精时会引起中毒，饮酒250g以上会有死亡的风险，中国每年因酒精中毒致死人数达70万。急性酒精中毒是指一次酒精摄入量过高导致血液内乙醇含量升高，导致头晕、神志不清、恶心呕吐等症状发生。酒精摄入后主要通过肝脏进行代谢分解，多次酒精中毒导致肝功能受损发生肝病。目前解酒药主要为西药且有副作用，还有可能造成肝脏负担，中药见效慢且口感差。
具有解酒功能的中药原料中，葛根为豆科植物野葛的块根部位，枳椇子是枳椇的肉质果柄和干燥的成熟种子，葛根和枳椇子内部含有大量的黄酮类化合物，如葛根素、槲皮素和二氢杨梅素等活性成分，黄酮类化合物通过抑制机体吸收乙醇，加速乙醇分解代谢从而起到解酒保肝的作用。洛神花可以起到护肝作用，内部花青素可以抑制氧化的伤害保肝护肝。但目前对其应用大多采用直接发酵或酶解法处理，其中直接发酵法药物的有效成分利用率低，而酶解技术能够使药物当中的一些成分发生化学变化，使得药物成分原有的结构遭到破坏，有效成分结构的破坏大大降低了药物有效成分的纯度，致使终产品解酒效果不尽人意。
本发明采用特殊二次蒸煮浸提方法处理原料，并应用固定菌种发酵，制得酵素发酵液，解酒效果更好。
发明内容
针对现有情况，本发明的主要目的是提供一种具有解酒功能的葛根枳椇子洛神花酵素及其制备方法，旨在提供一种解酒的酵素。
为实现上述目的，本发明采用的技术方案为：一种具有解酒功能的葛根枳椇子洛神花酵素，包括以下原料：葛根30-40g/L，枳椇子25-35g/L，洛神花20-30g/L，菊花5-10g/L，低聚异麦芽糖10-30g/L，红糖8-15g/L，木糖醇5-10g/L。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素，包括以下原料：葛根30g/L，枳椇子25g/L，洛神花20g/L，菊花5g/L，低聚异麦芽糖10.2g/L，红糖8.2g/L，木糖醇5.1g/L。
上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，包括如下步骤：
1)原料预处理：将葛根、枳椇子粉碎，洛神花、菊花用水浸透，加水蒸煮2次后冷却得滤液；向滤液中加入低聚异麦芽糖混合均匀；115℃灭菌30min，灭菌后冷却至37℃；
2)菌种活化：将菌粉用无菌水溶解，以0.06％的接种量接种到培养基中，进行培养；
3)发酵：将活化后的菌种接种于步骤1)料液进行发酵，过滤后离心、浓缩得到葛根枳椇子洛神花酵素原液；
4)调配：在葛根枳椇子洛神花酵素原液中加入低聚异麦芽糖、红糖、木糖醇，搅拌均匀后蒸煮灭菌、灌装。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，步骤2)中，所述的菌粉为植物乳杆菌HCS03-001、罗伊氏乳杆菌HCS02-001、鼠李糖乳杆菌HCS01-013的混合。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，步骤2)中，按质量比，植物乳杆菌HCS03-001:罗伊氏乳杆菌HCS02-001:鼠李糖乳杆菌HCS01-013＝10:9:1。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，步骤2)中，所述的菌种活化培养基的配方为水900-950g/L、无水葡萄糖30-50g/L、酵母浸出物10-20/L、酵母蛋白胨8-12g/L、L-苹果酸2-4g/L、一水柠檬酸1-3g/L、磷酸二氢钾1-3g/L、氯化钙0.3-0.7g/L、硫酸镁0.04-0.08g/L、硫酸锰0.005-0.015g/L。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，步骤2)中，所述的菌种活化培养基的配方为水927.43g/L、无水葡萄糖40g/L、酵母浸出物15g/L、酵母蛋白胨10g/L、L-苹果酸3g/L、一水柠檬酸2g/L、磷酸二氢钾2g/L、氯化钙0.5g/L、硫酸镁0.06g/L、硫酸锰0.01g/L。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，所述的菌种活化培养，装液量为50～80％，搅动培养，搅拌速度100r/min，通气量为0，初始气压0.02-0.03MPa，培养温度为37℃。培养时间16±1h，发酵进行15h开始，每半小时监控pH、OD，菌液pH值＜4.05，OD600＞9.00。
优选地，上述的一种具有解酒功能的葛根枳椇子洛神花酵素的制备方法，所述的发酵，装液量为50～75％，搅拌速度为30r/min；通气量为0，初始气压0.02-0.03MPa；培养温度为37℃，室温下发酵进行64h开始，每小时监控发酵液pH值，当接连两次pH值相差＜0.10发酵结束，发酵终点pH值＜3.00。
本发明中的水均为符合GB5749《生活饮用水卫生标准》的生活饮用水。
所述的原料均应经过严格筛选的市售符合相应的国家标准或行业标准和国家规定的合格产品。
优选地，葛根为精选葛根，葛根中含葛根异黄酮等生物活性物质，其中葛根素及其衍生物是葛根特有的生物活性物质，能够提高肝脏乙醇脱氢酶和乙醛脱氢酶的活性，具有解酒护肝的作用；葛根还具有抗疲劳、改善心脑血管血流量、改善血管微循环障碍、降血压、降血糖、降血脂及抗心律失常等生理功能；
优选地，菊花为精选贡菊，贡菊中木犀草苷等活性成分较其他品种含量相对较高，木犀草苷具有较强的呼吸道杀菌作用和降低胆固醇在动脉粥样硬化中的作用。另外菊花还含有黄酮类化合物和酚酸类化合物，对治疗心血管疾病、抗氧化、抗炎和抗肿瘤都具有一定的作用；
优选地，洛神花为整朵干燥状，洛神花含花青素、多酚酸、有机酸、黄酮等有效成分，具有抗氧化、抗菌、降血脂、降血压、保护肝脏等作用。
本发明与现有技术相比，具有以下有益效果：
1、本发明酵素发酵液采用药食同源原料，使用安全性高；
2、本发明采用本发明采用特殊二次蒸煮浸提方法处理原料，有效成分利用率高，且结构稳定不会遭到破坏；
3、本发明酵素发酵液采用葛根为原料，葛根中含葛根异黄酮等生物活性物质，其中葛根素及其衍生物是葛根特有的生物活性物质，能够提高肝脏乙醇脱氢酶和乙醛脱氢酶的活性，具有解酒护肝的作用；葛根还具有抗疲劳、改善心脑血管血流量、改善血管微循环障碍、降血压、降血糖、降血脂及抗心律失常等生理功能；
4、本发明酵素发酵液采用枳椇子为原料，枳椇子含有皂苷类、黄酮类及生物碱等活性成分，对急性酒精中毒有预防和治疗及保护胃黏膜作用，黄酮类中二氢杨梅素含量较高，其具有抗菌、调节血糖血脂、护肝、抗氧化、抗肿瘤作用；
5、本发明酵素发酵液采用洛神花、菊花为原料，洛神花含花青素、多酚酸、有机酸、黄酮等有效成分，具有抗氧化、抗菌、降血脂、降血压、保护肝脏等作用。菊花精选贡菊品种，较高含量的木犀草苷具有较强的呼吸道杀菌作用和降低胆固醇在动脉粥样硬化中的作用。菊花清热解毒的作用，降低酒精对肝脏的伤害，另外还含有黄酮类化合物和酚酸类化合物，对心血管疾病、抗氧化、抗炎和抗肿瘤都具有一定的作用；
6、本发明酵素发酵液选用低聚异麦芽糖为原料，低聚异麦芽糖能有效的促进人体内有益细菌双歧杆菌的生长繁殖，故又称为“双歧杆菌生长促进因子”，简称“双歧因子”。维持肠道正常细菌群平衡，尤其是老年和婴儿。降低血中胆固醇水平，防治高血压。改善乳制品消化率，提高耐乳糖性。增强人体免疫功能，预防抗生素类对人体各种不良副作用。具有预防龋齿功能，低聚异麦芽糖不被龋齿链球菌利用，不被口腔酶液分解，因而能防止龋齿。具有异麦芽糖残基低聚异麦芽糖与蔗糖结合使用时会强烈抑制不溶性葡聚糖合成，从而阻止齿垢形成，使蛀芽菌不能在牙齿上附着生长繁殖。
7、本发明采用特定菌株及特定比例进行发酵，发酵速度快，发酵程度彻底，发酵时间短。
8、本发明所制酵素产品功效更强，对白酒造模的小鼠醉酒模型具有明显的解酒效果，具有明显的加速清除大鼠血液中乙醇的作用。
具体实施方式
实施例1具有解酒功能葛根枳椇子洛神花酵素的制备
1、原料预处理
1.1原料粉碎、投料、蒸煮：称量原料葛根30g/L，枳椇子25g/L，洛神花20g/L，菊花5g/L，将葛根、枳椇子分别粉碎，葛根粉碎至10目左右，枳椇子破壳粉碎即可。菊花，洛神花用水全部浸透，连同葛根、枳椇子直接投入多功能提取罐中，加水量为发酵重量的1.02倍。100℃提取2次：第一次加总水量的2/3，蒸煮60min；第二次加总水量的1/3，蒸煮30min；蒸煮过程中禁止向物料里面通蒸汽。
1.2发酵培养基制备
(1)将1.1经过二次蒸煮的料液冷却后打入二级发酵罐，滤液量为发酵重量；
(2)将低聚异麦芽糖30g/L投入滤液中，和料液混合均匀。
1.3灭菌：115℃灭菌30min，取样检测无杂菌，物料通过夹套升温加热，禁止向物料内通蒸汽，灭菌后冷却至37℃，测定灭菌后培养基pH值(正常范围3.10-3.30)。
2、菌种活化
2.1菌种制备：菌粉称量所用器具115℃灭菌30min，称量所需菌粉用无菌水进行充分溶解无结块。所述菌种及质量比例为植物乳杆菌HCS03-001:罗伊氏乳杆菌HCS02-001:鼠李糖乳杆菌HCS01-013＝10:9:1。
2.2培养基制备：所述培养基配方为：水927.43g/L、无水葡萄糖40g/L、酵母浸出物15g/L、酵母蛋白胨10g/L、L-苹果酸3g/L、一水柠檬酸2g/L、磷酸二氢钾2g/L、氯化钙0.5g/L、硫酸镁0.06g/L、硫酸锰0.01g/L。将称量好的物料投入水中溶解，搅拌均匀，调节pH值为6.60；115℃灭菌30min，冷却至37℃备用。
2.3培养：采用无菌接种方式，接种量为0.06％，将菌种接种到培养基中，装液量为50～80％，搅动培养，搅拌速度100r/min，通气量为0，初始气压0.02-0.03MPa，培养温度为37℃。培养时间16±1h，发酵进行15h开始，每半小时监控pH、OD，菌液pH值＜4.05，OD600＞9.00；要求菌液活菌数≥1.0×109CFU/mL，菌液无杂菌(不包括菌落总数)。
3发酵
3.1接种：无菌接种，将2.3得到的菌液接种到1.3得到的物料中，要求接种量为5％。
3.2培养：设置装液量为50～75％，搅拌速度为30r/min；通气量为0，初始气压0.02-0.03MPa；培养温度为37℃，培养时间要求68±4h，发酵进行64h开始，每小时监控发酵液pH值，当接连两次pH值相差＜0.10发酵结束，发酵终点pH值＜3.00(发酵液温度为常温)。
优选地，发酵结束时发酵原液为红棕色液体，微微粘稠，入口葛根味儿浓郁，后带有洛神花、菊花和枳椇子的味道，口感酸。
4过滤：采用布袋过滤目数为100目，过滤后料液状态略浑浊，无大块明显残渣。
5浓缩：设置浓缩倍数为1.5倍，浓缩温度为60-70℃。发酵原液呈深红棕色，略浑浊，有明显的葛根味道，伴有淡淡的枳椇子味，较发酵液酸。
6离心：转速13000r/min(频率42Hz)，发酵原液呈浅黄色，溶液均一，无杂质，得到葛根枳椇子洛神花酵素原液。
7调配：将步骤6中的发酵原液，经过调配得到葛根枳椇子洛神花酵素，具体配方为葛根枳椇子洛神花酵素原液76.5g/L，低聚异麦芽糖10.2g/L，红糖8.2g/L，木糖醇5.1g/L。
8蒸煮灭菌：灭菌条件为95℃，20min；灭菌后料液呈浅黄色，澄清，无杂质，取样检测结果无杂菌。
9无菌灌装：灌装量符合产品规格要求。
实施例2一种具有解酒功能的葛根枳椇子洛神花酵素发酵菌种选择
采用相同的原料蒸煮处理方式(具体步骤同实施例1)，接种不同的菌种发酵68h后测发酵原液pH，并观察其感官状态，经过滤，浓缩及离心后测其乳酸含量，结果如表1。按G组菌种和比例发酵所得的发酵原液呈红棕色，葛根味道浓郁，经检测其乳酸含量最高，发酵底物能力最强。
表1不同发酵菌种的筛选结果
实施例3一种具有解酒功能的葛根枳椇子洛神花酵素有益成分分析
一种具有解酒功能的葛根枳椇子洛神花酵素的有益成分如表2所示。酵素中的有机酸具有抑菌、消炎、抗氧化以及调节机体免疫等作用，能够软化血管，加强钙吸收，促进消化，具有预防疾病和促进新陈代谢的作用。粗多糖既可抑菌，又能促进肠道蠕动，起到润肠通便的功效。游离氨基酸具有抗疲劳，调整亚健康状态，改善睡眠质量的作用。
表2一种具有解酒功能的葛根枳椇子洛神花酵素的有益成分分析
实施例4一种具有解酒功能的酵素产品的氨基酸成分
益生菌发酵能够产生的有机酸、氨基酸及粗多糖等有益成分。有机酸能够减少酒精对人体的伤害，产生的氨基酸在解酒过程中发挥着重要作用，如氨基酸可激活乙醇代谢关键酶ADH3(乙醇脱氢酶3)，进一步促进乙醇的代谢；丙氨酸在体内发挥着将氨基和含碳物从肌肉转运到肝脏的作用，因此血浆中丙氨酸浓度的增加可补充足够的NAD+(烟酰胺腺嘌呤二核苷酸)，并激活三羧酸循环。粗多糖具有能够降低血浆总胆固醇、甘油三酯、抗血凝抗血栓、促进单核巨噬细胞系统吞噬功能增强机体免疫等作用，一种具有解酒功能的酵素产品的氨基酸成分如表3所示。
表3一种具有解酒功能的酵素产品的氨基酸成分
实施例5一种具有解酒功能的酵素产品小鼠醉酒时间试验
试验选用辽宁长生生物技术有限公司提供的清洁级ICR小鼠24只，18-22g，雄性。无菌块状鼠料由辽宁长生生物技术有限公司提供。检验条件为清洁级动物实验室，温度22±1.5℃，湿度50％±10％，工作照度160-280lx，噪音<60dB。
1、试验方法
1.1、试验共用24只动物，雄性，平衡饲养后按照体重随机分为2组，模型对照组及试验样品组，每组12只；
1.2、模型对照组和试验样品组分别给予纯净水和酵素样品，给样量均为10mL/kg，给样前禁食禁水6h；
1.3、给予样品后30min，各组按照15mL/kg体积灌胃给予52度白酒，以翻正反射为指标观察小鼠开始醉酒时间及醉酒恢复时间。
2、试验结果
模型对照组和试验样品组的实验动物开始醉酒时间及醉酒恢复时间见表4。
表4小鼠醉酒时间试验结果
*为与模型对照组比较P＜0.05
根据表4试验结果，试验样品组小鼠平均开始醉酒时间较模型对照组延后达20min左右，醉酒恢复时间也显著短于模型对照组。在本试验条件下，酵素产品对白酒造模的小鼠醉酒模型有明显的解酒功能。
实施例6一种具有解酒功能的酵素产品血醇浓度试验
试验选用辽宁长生生物技术有限公司提供的清洁级SD大鼠90只，180-230g，雄性。无菌块状鼠料由辽宁长生生物技术有限公司提供。检验条件为清洁级动物实验室，温度22±1.5℃，湿度50％±10％，工作照度160-280lx，噪音<60dB。
1、试验方法
1.1将90只雄性大鼠平衡饲养后按照体重随机分为3组，分别为空白组、模型对照组和试验样品组，每组30只；
1.2空白组和模型对照组均以10mL/kg给予纯净水，试验样品组给予酵素样品，给样量10mL/kg；
1.3给样后30分钟，空白组给予等体积纯净水，模型对照组及试验样品组按照8mL/kg体积灌胃给予52度白酒；
1.4给予白酒后1h、2h、3h、4h、6h各组分别取6只大鼠采集血液，分离血清后，生化比色法测定血清乙醇浓度。
2、试验结果
一种具有解酒功能的酵素产品大鼠血醇浓度试验结果见表5。
表5酵素产品大鼠血醇浓度试验结果
*为与模型对照组比较P＜0.05
根据表5试验结果，酵素试验样品组实验动物在2、3、4h时血醇浓度均表现出更明显的下降趋势，其中4h血醇浓度显著低于模型对照组(P＜0.05)。这说明，在本试验条件下，酵素试验样品具有加速清除血液中乙醇的作用。
Claims (5)
1.一种具有解酒功能的葛根枳椇子洛神花酵素，其特征在于，所述具有解酒功能的葛根枳椇子洛神花酵素的制备方法，步骤如下：
1)原料预处理：称量原料葛根30g/L，枳椇子25g/L，洛神花20g/L，菊花5g/L，将葛根、枳椇子粉碎，洛神花、菊花用水浸透，加水蒸煮2次后冷却得滤液；向滤液中加入30g/L低聚异麦芽糖混合均匀；115℃灭菌30min，灭菌后冷却至37℃；
2)菌种活化：将菌粉用无菌水溶解，以0.06％的接种量接种到培养基中，进行培养；
3)发酵：将活化后的菌种接种于步骤1)料液进行发酵，过滤后离心、浓缩得到葛根枳椇子洛神花酵素原液；
4)调配：在葛根枳椇子洛神花酵素原液中加入低聚异麦芽糖、红糖、木糖醇，搅拌均匀后蒸煮灭菌、灌装得到葛根枳椇子洛神花酵素，具体配方为葛根枳椇子洛神花酵素原液76.5g/L，低聚异麦芽糖10.2g/L，红糖8.2g/L，木糖醇5.1g/L；
步骤2)中，所述的菌粉为植物乳杆菌HCS03-001、罗伊氏乳杆菌HCS02-001、鼠李糖乳杆菌HCS01-013的混合；按质量比，植物乳杆菌HCS03-001:罗伊氏乳杆菌HCS02-001:鼠李糖乳杆菌HCS01-013＝10:9:1。
2.根据权利要求1所述的具有解酒功能的葛根枳椇子洛神花酵素，其特征在于，步骤2)中，所述培养基的配方为水900-950g/L、无水葡萄糖30-50g/L、酵母浸出物10-20/L、酵母蛋白胨8-12g/L、L-苹果酸2-4g/L、一水柠檬酸1-3g/L、磷酸二氢钾1-3g/L、氯化钙0.3-0.7g/L、硫酸镁0.04-0.08g/L、硫酸锰0.005-0.015g/L。
3.根据权利要求2所述的具有解酒功能的葛根枳椇子洛神花酵素，其特征在于，步骤2)中，所述培养基的配方为水927.43g/L、无水葡萄糖40g/L、酵母浸出物15g/L、酵母蛋白胨10g/L、L-苹果酸3g/L、一水柠檬酸2g/L、磷酸二氢钾2g/L、氯化钙0.5g/L、硫酸镁0.06g/L、硫酸锰0.01g/L。
4.根据权利要求1所述的具有解酒功能的葛根枳椇子洛神花酵素，其特征在于，步骤2)中，培养条件为：装液量为50～80％，搅动培养，搅拌速度100r/min，通气量为0，初始气压0.02-0.03MPa，培养温度为37℃，培养时间16±1h，发酵进行15h开始，每半小时监控pH、OD，菌液pH值＜4.05，OD600＞9.00。
5.根据权利要求4所述的具有解酒功能的葛根枳椇子洛神花酵素，其特征在于，步骤3）中，发酵条件为：装液量为50～75％，搅拌速度为30r/min；通气量为0，初始气压0.02-0.03MPa；培养温度为37℃，室温下发酵进行64h开始，每小时监控发酵液pH值，当接连两次pH值相差＜0.10发酵结束，发酵终点pH值＜3.00。
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