CN113456757B - 一种复配石斛原浆及其制备方法和应用 - Google Patents
一种复配石斛原浆及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种复配石斛原浆及其制备方法和应用,属于功能食品领域。复配石斛原浆由石斛鲜条、新鲜桂圆、干燥桑叶和和枸杞经过热水提取、酶解、絮凝以及低温沉降等多步操作获得的澄清液。本发明的复配石斛原浆通过抑制α‑葡萄糖苷酶活性、上调乳酸菌属、双歧杆菌属肠道细菌丰度等机制增强降糖效果,并改善糖尿病患者口服α‑葡萄糖苷酶抑制剂类药物引起的腹胀、腹痛等副作用。
Description
技术领域
本发明属于功能食品技术领域,具体涉及一种复配石斛原浆及其制备工艺,该石斛原浆具有增强降糖效果和改善糖尿病患者口服α-葡萄糖苷酶抑制剂类药物后引起腹胀、腹泻副反应的功效。
背景技术
石斛(Dendrobium)属多年生草本植物,包括铁皮石斛、金钗石斛、霍山石斛、鼓槌石斛、流苏石斛、紫皮石斛、铜皮石斛、环草石斛、马鞭石斛、黄草石斛等,主要分布于广西、广东、贵州、云南、福建、浙江、江西、湖南、安徽等地。石斛的主要成分是多糖、黄酮和生物碱,其中多糖成分最为重要。石斛的药用价值和保健功效也有确切报道,古代医学名著和典籍均有记载,奉其为“药中之上品”,其活性物质的提取和高值化利用一直是目前保健食品开发的热门课题。传统的石斛食用方法以枫斗冲泡和鲜条榨汁为主,其中鲜榨存在保存时间短、容易变质等缺点,并且单纯的鲜榨石斛饮品易带青草味,口感不佳、胃寒者常有不适感,影响饮用。由于石斛中存在木质纤维素、果胶、淀粉、粘液质类成分,沸水提取易导致石斛淀粉糊化,水提液很难过滤,所以目前的石斛浆液产品常以混浊液形式存在。这类产品因木质素等不溶性纤维的存在,增加痔疮、***、脐疝以及小腿静脉曲张的风险(国外医学卫生学分册1983,2,85),因此去除石斛浆液的不溶物,获得澄清度好的石斛原浆具有重要意义。
糖尿病是全球范围内一种常见的全身性代谢疾病,它以血糖的慢性增高为主要特征,严重影响着人们的身体健康。随着人们生活水平的提高、饮食结构的改变、体力活动的减少以及吸烟等不良生活习惯,糖尿病的发病率正逐年升高。目前,除注射胰岛素外,口服降血糖/抗糖尿病药物是糖尿病患者最主要的治疗方式。双胍类和磺酰脲类药物是治疗糖尿病最常用的药物,但这些合成药物存在一定副作用,如加重肝肾负担、导致血糖过低或引起胃肠道不适等。与之相比,药食同源植物的有效成份不仅能够有效降低血糖,还具有安全性好的特点,越来越受到人们的重视。石斛在糖尿病防治等相关代谢性疾病方面已有报道,如高剂量的石斛多糖(450mg/kg)具有显著降低2型糖尿病鼠血糖的作用(食品科学2020,41,127-132)。然而单用石斛的降糖作用偏弱,石斛复配产品的开发受到重视,如中国发明专利CN 104784505公开了含有石斛、西洋参、葛根、天花粉和五味子的组合物,经过醇提和水提条件的优化,确定了最佳提取条件,并证实使用最佳提取条件制备的组合物对小鼠具有降糖作用,但该组合物的降糖作用依然偏弱;也有报道加入黄芪、五味子等中药成份后,石斛水提液对α-葡萄糖苷酶的抑制活性有所提高,但提高的幅度不大(福建中医学院学报2004,14:37-38),改善石斛单用功效不足的复配石斛功能产品,仍有待进一步研究和开发。
近年来,肠道菌群调控对包括糖尿病、肥胖在内的代谢性疾病、神经***疾病的防治作用越来越受到人们重视,例如,给糖尿病动物或患者移植乳酸菌属和双歧杆菌属微生物具有降糖作用,能够改善患者糖脂代谢功能,调节胰岛素代谢通路,缓解胰岛素抵抗,提高胰岛素敏感性(疑难病志,2019,18(09):882-886;中国微生态学杂志,2018,30:690-692;JCI Insight,2018,3:95-107;Benef Microbes,2017,8:421-432)。铁皮石斛对肠道菌群的调控作用已有报道,无论是高剂量(6g/ml)还是低剂量(1.5g/ml)的铁皮石斛水提物给药,都会使小鼠的双歧杆菌和乳酸菌的丰度有不同程度的下降(中医药信息2019,36,44-49);铁皮石斛水提物(2.37g/kg)同样也能降低高脂饲料喂养小鼠的双歧杆菌的丰度(3Biotech2021,11:22)。从菌群调控的角度看,铁皮石斛水提物降低双歧杆菌和乳酸菌的丰度对降糖不利。目前尚无文献报道,如何改善铁皮石斛对双歧杆菌和乳酸菌的调控进而增强降糖作用。
α-葡萄糖苷酶抑制剂类药物(如阿卡波糖等)能够通过抑制哺乳动物小肠中α-葡萄糖苷酶活性,阻止淀粉水解为葡萄糖,从而防止糖尿病人的血糖升高。该类药物具有不易引起低血糖的特点,是糖尿病人普遍服用的首选治疗药物。但阿卡波糖等α-葡萄糖苷酶抑制剂类药物对α-淀粉酶的抑制作用较强,服用后会使食物中淀粉在体内大量累积,肠道微生物在体内发酵淀粉产生过量气体,会引起胃肠胀气,并诱发腹痛、腹泻等症状(FEBSLetters 2009,583:2157-2159)。肠道菌群体外培养表明,阿卡波糖通过抑制α-葡萄糖苷酶形成的未消化淀粉积累是造成肠道微生物产气的主要原因(黄芩素和阿卡波糖体外调控糖尿病鼠肠道菌群,大连理工大学硕士论文2019年)。开发具有抑制α-葡萄糖苷酶作用活性,并能减轻阿卡波糖等药物引起的腹胀、腹痛副作用的药物或功能食品具有重要的意义。
发明内容
本发明目的是提供一种复配石斛原浆及其制备方法和在辅助降糖和改善腹胀副作用的用途,以解决现有石斛饮品澄清度不高、降糖作用不佳和缺乏缓解糖尿病患者服用阿卡波糖等α-葡萄糖苷酶抑制剂类药物后腹胀、腹痛副作用的缺陷。
本发明目的通过以下方式实现:
本发明提供一种复配石斛原浆的制备方法,其主要包括以下步骤:
(1)将预处理后的2-40wt%石斛鲜条、0.1-10wt%新鲜桂圆、0.5-10wt%桑叶和0.1-10wt%枸杞放入粉碎机,加入适量的水,粉碎至匀浆;
(2)将步骤(1)所得的匀浆液置于浸提罐中补足水,加热至50-150℃,提取0.5-10h,将所得提取液过筛,得滤液;
(3)向步骤(2)所得的滤液中加入果胶酶和几丁质酶,0.5-5h后加入壳聚糖絮凝剂,搅拌处理0.5-5h,得絮凝液;
(4)将步骤(3)所得的絮凝液加热至60-150℃,处理10-200min后,冷却,静置12-48h,离心,得到澄清液;
(5)将步骤(4)所得的澄清液罐装、灭菌、包装,得到复配石斛原浆产品。
进一步地,步骤(1)中所述石斛鲜条的含水量为60-90%,新鲜桂圆的含水量为50-80%。
进一步地,步骤(1)中所述石斛选自铁皮石斛、金钗石斛、霍山石斛、鼓槌石斛、流苏石斛、紫皮石斛、铜皮石斛、环草石斛、马鞭石斛、黄草石斛。
进一步地,步骤(1)中石斛鲜条、新鲜桂圆、桑叶和枸杞的投料比例如下:石斛鲜条5-30wt%,桑叶0.5-5wt%,新鲜桂圆0.1-2wt%,枸杞0.1-2wt%。
进一步地,步骤(1)中所述预处理的具体过程为将新鲜的石斛和桂圆、干燥的桑叶和枸杞洗净,用切断机将石斛切成长度均一的小段,将桂圆去皮去核。
进一步地,步骤(2)中所述筛为60-80目振动筛。
进一步地,步骤(3)中所述果胶酶的加入量为0.01-1wt%,所述几丁质酶的加入量为0.01-1wt%。
进一步地,步骤(3)中酶解和絮凝处理的温度为30-40℃。
进一步地,步骤(3)中壳聚糖絮凝剂的加入量为0.01%-1wt%。
进一步地,步骤(4)的具体步骤为将步骤(3)所得的絮凝液加热至80-100℃,处理10-60min后,冷却至2-8℃,静置24-48h,转移至管式离心机中离心,得到澄清液。
进一步地,步骤(4)中所述离心的条件为5,000-10,000g条件下离心10-30min。
本发明还提供了一种由上述制备方法获得的复配石斛原浆。
进一步地,所述的复配石斛原浆在860nm下的透光率大于60%。
本发明还提供了上述的复配石斛原浆在辅助降糖或/和改善腹胀副作用中的应用。
进一步地,所述复配石斛原浆通过抑制α-葡萄糖苷酶活性来增强降糖效果。
进一步地,所述复配石斛原浆通过提高乳酸菌属、双歧杆菌属等肠道细菌丰度增强降糖效果。
进一步地,所述复配石斛原浆能够改善糖尿病患者服用α-葡萄糖苷酶抑制剂引起的腹胀、腹泻等副作用。
本发明的有益效果
提供了一种复配石斛原浆及其制备方法和在增效、减毒方面的用途,以解决现有石斛饮品浑浊不清、降糖作用不佳和缺乏改善糖尿病患者服用阿卡波糖等α-葡萄糖苷酶抑制剂类药物后腹胀、腹痛等副反应的问题。
附图说明:
图1.复配石斛原浆对α-葡萄糖苷酶的抑制作用;
图2.复配石斛原浆对乳酸菌和双歧杆菌丰度的影响;
图3.复配石斛原浆对肠道菌群产气量的影响;
图4.产气相关肠道微生物在复配石斛原浆、石斛原浆和对照组的相对丰度。
具体实施方式
下面结合非限制性实施例对本发明的技术方案做进一步的说明,以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
如无特别说明,本说明书述及液体溶液的浓度时均指质量浓度。本发明中提取用溶剂均为分析纯试剂。
1.多糖的分析方法:
采用苯酚—硫酸法测定,取样品0.2mL,于2mL离心管内,缓慢加入1mL无水乙醇,同时使用涡旋震荡器振摇,使混合均匀,冷藏1h,于4000r/min离心10min,弃去上清液。不溶物用1mL无水乙醇溶液洗涤、离心2次。用1mL水将上述不溶物溶解,将上清转移至500mL容量瓶中,残渣洗涤2~3次,洗涤液转移至容量瓶中,加水定容。此溶液为样品溶液,如颜色过深,可通过C18 SPE小柱等进行脱色处理。
分别吸取0、0.2、0.4、0.6、0.8和1.0mL的标准葡萄糖工作溶液置于20mL具塞试管中,用蒸馏水补至1.0mL。向试液中加入1.0mL苯酚溶液,然后快速加入5.0mL硫酸,静置10min。使用涡旋振荡器使反应液充分混合,然后将试管放置于30℃水浴中反应20min,490nm测吸光度。以葡萄糖质量浓度为横坐标(x),吸光值为纵坐标(y),进行线性拟合,得到多糖的标准曲线(以葡萄糖计)、线性相关系数以及有效线性范围。
y=0.0094x+0.158 (1)
R2=0.9954线性范围0-25μg/mL
吸取1.00mL样品液于20mL具塞试管中,按上述标准液相同方法测定吸光度。与试样的测定平行进行空白试验,取相同量的所有试剂,采用相同的分析步骤,但不加试样。样品中多糖含量以质量分数ω计算,单位以克每毫升(mg/mL)表示,按照式(2)计算,重复三次。
ω=m1V1/(V2×106) (2)
m1---从标准曲线上查得样品测定液中含糖量,单位为微克每毫升μg/mL;
V1---样品定容体积,单位为毫升mL;
V2---取样品体积,单位为毫升mL;
106---换算系数;
2.黄酮的分析方法:
吸取5mL~10mL样品置于100mL烘干恒重三角瓶中(称重精确到1mg,供后续测定使用),加入约30mL无水乙醇,充分摇匀样品﹐将摇匀样品置于超声清洗器中超声浸提1h,其间每20min摇匀溶液一次。提取液过滤至50mL容量瓶中,使用无水乙醇冲洗滤纸、三角瓶,合并溶液,待溶液冷却至室温,用无水乙醇定容至50mL待测。
标准曲线的绘制:精密吸取芦丁标准品工作溶液l mL,2mI,3mL,4mL,5mL分别置于50mL容量瓶中。加无水乙醇至总体积为15mL,依次加入硝酸铝溶液1mL,醋酸钾溶液1mL,摇匀,加水至刻度﹐摇匀。静置1h,用1cm比色皿于420nm处,以30%乙醇溶液为空白,测定吸光度。以50mL容量瓶中芦丁质量(mg)为横坐标(x),吸光值为纵坐标(y),进行线性拟合,得到黄酮的标准曲线(以芦丁计)、线性相关系数以及有效线性范围。
y=0.0018x+0.0565 (3)
R2=0.9996线性范围0-200μg/mL
空白试验除不加试样外,均按上述测定步骤进行。
精密吸取待测样品溶液1.0mL,置于50mL容量瓶中,按上述方法进行操作。
以空白试液作参比,用l cm比色杯,在波长420nm处测定试料溶液的吸光度。
查标准曲线或通过回归方程计算,求出样品溶液中的黄酮类化合物含量(mg)。在标准曲线上求得样液中的浓度,其吸光度值应在标准曲线的线性范围内。
样品中黄酮含量以质量分数ω计算,单位以毫克每毫升(mg/mL)表示,按照式(3)计算,重复三次。
ω=(m/(V1×d×1000))×100% (4)
m---从标准曲线上查得样品测定液中芦丁量,单位为毫克每mg;
V1---样品体积,单位为毫升mL;
d—稀释比例;
1000---换算系数。
3.澄清度的表征方法:
通过透光率反映澄清度(药酒生产实用技术,中国轻工出版社,2001.400~403),具体测定方法为:样品在分光光度计860nm波长处测定其吸光度T,按照式(5)计算透光率T,重复三次。
T=10-A×100% (5)
4.α-葡萄糖苷酶抑制活性测定方法:
小鼠禁食不禁水24h后断颈处死,取小肠段称重,剪碎,按0.02g/L的比例置于4℃的PBS缓冲液中,用玻璃匀浆器在冰上研磨至均匀。得到的小肠匀浆液在4℃下12000g/min离心5min,取上清即为α-葡萄糖苷酶溶液,-20℃保存。反应在PBS缓冲液中进行,总体积为250μL(125μLPBS缓冲液+50μL酶溶液+25μL样品液+50μL 1g/20mL麦芽糖底物),空白组的酶溶液用等体积的PBS缓冲液代替。原酶组的样品液用等体积的PBS缓冲液代替。体系在37℃孵育20min,沸水浴5min终止反应。冷却至室温用葡萄糖测定试剂盒中的葡萄糖氧化酶法测定反应生成的葡萄糖浓度,在505nm处测定其OD值表示生成的葡萄糖浓度,计算按照式(6)计算α-葡萄糖苷酶抑制率,重复三次。
抑制率=(OD原酶-OD样品)/(OD原酶-OD空白)×100%(6)
5.肠道菌群培养方法:
根据杨帆等方法进行适当修改(黄芩素和阿卡波糖体外调控糖尿病鼠肠道菌群,大连理工大学硕士论文2019年),主要包括:
1)体外淀粉消化和未消化淀粉中各成分的分析:体外淀粉消化实验在PBS缓冲液中进行,总体系为150μL,体系中DMSO终浓度为10%。50μL酶溶液(含0.52U/mLα-葡萄糖苷酶)和50μL阿卡波糖,在37℃共同孵育30min,加入50μL 5%(w/v)淀粉溶液。37℃反应30min,沸水浴5min终止反应。用高效液相-蒸发光检测(HPLC-ELSD)法测定葡萄糖、麦芽糖、麦芽三糖、麦芽四糖的含量(色谱柱:YMC-Pack Polyamine-Ⅱ柱(250×4.6mm,5μm)。流动相为(A)水,(B)乙腈。洗脱条件是等度洗脱,0-25min,60%B。流速是0.8mL/min,进样量是20μL。ELSD参数:增益50,载气为空气,气压20psi,漂移管温度是45℃,喷雾器模式为50%加热)。淀粉含量用I2-KI法测定,往200μL待测淀粉溶液中加入20μL 0.2%I2-KI溶液,混匀,测定在最大吸收波长610nm处的吸光度。由上述条件测得,阿卡波糖抑制α-葡萄糖苷酶水解淀粉形成的未消化淀粉中主要成份的含量比例为,葡萄糖:麦芽糖:麦芽三糖:麦芽四糖:淀粉=1:0.4:1.6:3.1:10,表明阿卡波糖抑制后,未消化淀粉中淀粉含量较高,以此比例进行后续肠道菌群培养,模拟患者口服阿卡波糖等α-葡萄糖苷酶抑制剂后情况。
2)肠道菌群培养:GAM培养基(上述未消化淀粉3g/L(葡萄糖:麦芽糖:麦芽三糖:麦芽四糖:淀粉=1:0.4:1.6:3.1:10),蛋白胨10g/L,大豆胨3g/L,月示胨10g/L,血清消化粉13.5g/L,牛肉浸粉2.2g/L,酵母浸粉5g/L,牛肝浸粉1.2g/L,氯化钠3g/L,磷酸二氢钾2.5g/L,L-半胱氨酸0.3g/L,硫代乙醇酸钠0.3g/L)用去离子水充分溶解,加热煮沸溶解,冷却后,加入5mg氯化血红素和1mg维生素K,调节pH=7.2±0.2,定容至1L。分装后高压蒸汽灭菌115℃,15min。冷却后于厌氧工作站中接种小鼠肠道菌群,接种量1%,在转速为200rpm/min,37℃的恒温培养箱中培养24h。
3)肠道菌群产气量测定方法:用无菌注射器将厌氧瓶中的气体收集,当厌氧瓶中的气体压强和大气压相等时,即注射器的活塞不因气压差异移动而达到平衡,记录活塞最低点对应注射器的刻度,即为产气体积,单位为mL。
4)肠道菌群丰度的测定方法:上述培养后的菌经离心后收集离心管底部菌体,送样至杭州谷禾生物科技有限公司测序,并各菌的相对丰度绘制三角相图表征特定微生物在不同组中的丰度占比。
实施例1 复配石斛原浆的制备
将新鲜铁皮石斛(含水量为80%)和桂圆(含水量为65%)、干燥的桑叶和枸杞洗净,用切断机将石斛切成长度均一的小段,将桂圆去皮去核;称取质量份数为40%的石斛、2%的桑叶、1%的枸杞和1%的桂圆一起放入粉碎机,加入适量的水,粉碎至匀浆;将所得匀浆液置于浸提罐中补足水分,加热至95℃,提取1h。用60目振动筛过滤所得提取液,得滤液;向滤液中加入0.02%果胶酶和0.02%几丁质酶在30℃下处理0.5h后,加入0.01%壳聚糖絮凝剂,30℃搅拌0.5h,将絮凝后的溶液加热至90℃,搅拌30min后,冷却至4℃静置24h。将冷却的絮凝液置于管式离心机中离心,在7,000g下离心15min,得到澄清液,样品标号为S1。
将新鲜石斛的重量分数调整为20%和10%,桑叶、枸杞和桂圆的质量分数不变,按照上述相同的方法进行制备,获得的样品分别编号为S2和S3。
按照上述方法制备石斛原浆样品石斛的质量分数为40%,20%和10%,但不加入桑叶、枸杞和桂圆,分别编号为T1、T2和T3。
对比例1 非澄清原浆的制备
将新鲜铁皮石斛和桂圆、干燥的桑叶和枸杞洗净,用切断机将石斛切成长度均一的小段,将桂圆去皮去核。称取重量分数为20%的石斛、2%的桑叶、1%的枸杞和1%的桂圆一起放入粉碎机,加入适量的水,粉碎至匀浆;将所得匀浆液置于浸提罐中补足水分,加热至95℃,提取1h。用60目振动筛过滤所得提取液,得滤液;将滤液置于管式离心机中离心,在7,000g下离心15min,得到非澄清液,样品标号为T4。
实施例2 复配石斛原浆的澄清度
根据上述澄清度的测定方法,测定实施例1获得样品S1-S3和对比例1样品T4的透光率,结果如表1所示:
表1不同石斛质量份数和处理方法对复配石斛原浆透光率的影响
结果表明,本发明所述方法处理不同质量份数的石斛均可以得到澄清度非常好的复配石斛原浆,而采用常规方法得到的复配石斛浆液透光率较低,澄清度较差。
实施例3 复配石斛原浆的有效成份含量
根据上述多糖测定方法和黄酮测定方法,测定实施例1获得的样品S2的总多糖的含量为8.95mg/mL,总黄酮含量为0.014mg/mL;测定对比例1样品T4的总多糖的含量为9.41mg/mL,总黄酮含量为0.015mg/mL,以上结果表明经过本发明的制备方法,有效成份含量基本保持不变,即本发明的制备方法能在保证有效成份不降低的情况下,获得澄清度好的复配石斛原浆。
实施例4 提取物温度对澄清度的影响
按照实施例1中样品S1的方法制备复配石斛原浆,只是将铁皮石斛换为金钗石斛,将粉碎后样品的提取温度由95℃调整为60℃、80℃、100℃以及121℃(利用高压蒸汽锅加热到1.05kg/cm2),样品编号分别为S4-S7,测定制备的复配石斛原浆的透光率,结果如表2所示。
表2提取温度对复配石斛原浆透光率的影响
结果表明,使用本发明的制备方法,不同品种的石斛和不同的提取温度均可以得到澄清度较好的复配石斛原浆。
实施例5 提取时间对澄清度的影响
按照实施例1中样品S1的方法制备复配石斛原浆,只是将粉碎后样品的提取时间由1h调整为0.5h,1.5h和2h,样品编号分别为S8-S10,测定其透光率,结果如表3所示。
表3提取时间对复配石斛原浆透光率的影响
结果表明,使用本发明的制备方法,不同的提取时间均可以得到澄清度较好的复配石斛原浆。
实施例6 絮凝时间对澄清度的影响
按照实施例1中样品S1的方法制备复配石斛原浆,只是将絮凝时间调整为1h,1.5h和3h,样品编号分别为S11-S13。分别测定其透光率,结果如表4所示。
表4絮凝时间对复配石斛原浆透光率的影响
结果表明,使用本发明的制备方法,不同的絮凝时间均可以得到澄清度较好的复配石斛原浆。
实施例7 酶浓度对澄清度的影响
按照实施例1中样品S1的制备方法制备复配石斛原浆,只是将过滤后样品的酶解过程中酶浓度由0.02%的果胶酶和0.02%的几丁质酶调整为0.01%的果胶酶和1%的几丁质酶、1%的果胶酶和0.02%的几丁质酶、0.5%的果胶酶和0.5%的几丁质酶,样品编号分别为S14-S16。分别测定其透光率,如表5所示。
表5酶浓度对复配石斛原浆透光率的影响
结果表明,使用本发明的制备方法,不同的酶浓度均可以得到澄清度较好的复配石斛原浆。
实施例8 复配石斛原浆制备
按照实施例1中样品S1的方法制备复配石斛原浆,只是将石斛、桑叶、桂圆和枸杞的质量分数按照下表配制,样品编号分别为S17-S21。另外制备一个只含有20%石斛的样本编号为T5。
表6复配石斛原浆的质量分数表
实施例9 复配石斛原浆对α-葡萄糖苷酶抑制作用
根据上述α-葡萄糖苷酶抑制活性的测定方法,测定了复配石斛原浆样品S17-S21和石斛原浆样品T5的α-葡萄糖苷酶抑制活性,结果如图1所示,复配石斛原浆样品S17-S21对哺乳动物的α-葡萄糖苷酶均显示较好的抑制作用,抑制率高达81.8%~100%,而对照的石斛原浆T5的抑制率仅为11.4%,本发明制备方法获得的复配石斛原浆抑制率较对照组提高了7.2-8.8倍,表明本发明制备方法获得的复配石斛原浆能够高效抑制α-葡萄糖苷酶的活性,进而有效降低淀粉水解为葡萄糖的含量,起到增强降糖效果的作用。
实施例10 复配石斛原浆对肠道菌群的结构和丰度的影响
按照上述肠道菌群培养方法,培养2型糖尿病小鼠的粪便,并加入培养液体积20%的复配石斛原浆样品S1-S3和T1,另取一个样品,不加入石斛原浆或复配石斛原浆样品,只加入相同体积的无菌水做对照,编号为C,培养结束后对各组肠道菌群结构和丰度进行分析。每个样品3个重复。
对C、T1、S1、S2和S3样品的肠道菌群结构和丰度分析,结果如图2所示,可以发现,石斛单用组(T1)对乳酸菌(Lactobacillus)的上调作用较弱,复配组(S1、S2、S3)对乳酸菌丰度的上调作用分别是石斛单用组(T1)的2.8倍、11.0倍和82.1倍,石斛单用组(T1)对双歧杆菌(Bifidobacterium)的丰度呈现下调作用,复配组(S1、S2、S3)对双歧杆菌丰度具有明显的上调作用。该结果表明,本发明制备方法获得的复配石斛原浆能够通过上调乳酸菌和双歧杆菌肠道细菌的丰度,发挥增强降糖效果的作用。
实施例11 复配石斛原浆对肠道菌群产气量和产气相关菌的影响
对实施例9培养后的肠道菌群产气量进行分析,结果如图3所示,对照组(C组)的产气量为12.5mL,表明未消化淀粉积累致使肠道菌群发酵增加产气量,进而引起腹胀、腹痛等副作用;加入石斛原浆的各组(T1-T3)可以显著降低产气量,并且有剂量依赖性,其中高剂量组(T1)产气量为1.8mL,仅为对照组的14.1%。而本发明制备方法获得的复配石斛原浆各组的产气量进一步降低,高剂量、中剂量和低剂量石斛的复配石斛原浆的产气量分别为相应的石斛原浆产气量的58.3%(S1 vs T1),26.8%(S2 vs T2)和10.4%(S3 vs T3),表明复配石斛后,具有更好的降低肠道菌群产气量的作用。利用三元相图分析了对照组(C)、石斛原浆组(T3)和复配石斛原浆组(S3)中和产气相关的主要肠道细菌相对丰度占比(如图4所示),结果显示,相比于对照组和石斛原浆组,复配石斛原浆组中乳酸菌和双歧杆菌的相对丰度占比显著提高,其中乳酸菌在对照组、石斛原浆组和复配石斛原浆组三组中的占比分别为0.01、0.04、0.95;双歧杆菌在三组中的占比分别为0.05、0.003、0.94;而Parabacteroides、Ruminococcus、Rikenellaceae、Enterobacter等菌的相对丰度占比明显降低,其中Parabacteroides菌在对照组、石斛原浆组和复配石斛原浆组三组中的相对丰度占比分别为0.19、0.81、0.002;Ruminococcus菌在三组中的相对丰度占比分别为0.5、0.5、0;Rikenellaceae菌在三组中的相对丰度占比分别为0.51、0.49、0;Enterobacter菌在三组中的占比分别为0.30、0.63、0.07。研究表明乳酸菌和双歧杆菌是和肠道产气负相关的细菌,而Parabacteroides、Ruminococcus、Rikenellaceae、Enterobacter等菌是和肠道产气正相关的细菌(BMC microbiology 2020,21:47),因此以上结果显示产气正相关菌的相对丰度占比在对照组和石斛原浆组较高,而在复配石斛原浆组较低。相反的,产气负相关菌的相对丰度占比在对照组和石斛原浆组较低,而在复配石斛原浆组较高。即复配石斛可以通过提高产气负相关的乳酸菌和双歧杆菌等肠道细菌的丰度,降低产气正相关的Parabacteroides、Ruminococcus、Rikenellaceae、Enterobacter等肠道细菌的丰度,进而降低肠道细菌的产气量,降低糖尿病患者腹胀、腹痛的副作用。
Claims (9)
1.一种辅助降糖或/和改善腹胀副作用的复配石斛原浆的制备方法,其特征在于,主要包括以下步骤:
(1)将预处理后的2-40 wt%石斛鲜条、0.1-10 wt %新鲜桂圆、0.5-10 wt %桑叶和0.1-10 wt %枸杞放入粉碎机,加入适量的水,粉碎至匀浆;
(2)将步骤(1)所得的匀浆液置于浸提罐中补足水,加热至50-150℃,提取0.5-10 h,将所得提取液过筛,得滤液;
(3)向步骤(2)所得的滤液中加入果胶酶和几丁质酶,0.5-5 h后加入壳聚糖絮凝剂,搅拌处理0.5-5h,得絮凝液;
(4)将步骤(3)所得的絮凝液加热至60-150℃,处理10-200min后,冷却,静置12-48h,离心,得到澄清液;
(5)将步骤(4)所得的澄清液罐装、灭菌、包装,得到复配石斛原浆产品;
步骤(3)中所述果胶酶的加入量为0.01-1 wt%,所述几丁质酶的加入量为0.01-1 wt%,所述壳聚糖絮凝剂的加入量为0.01%-1 wt %,酶解和絮凝处理的温度为30-40℃。
2.根据权利要求1所述复配石斛原浆的制备方法,其特征在于,步骤(1)中所述石斛选自铁皮石斛、金钗石斛、霍山石斛、鼓槌石斛、流苏石斛、紫皮石斛、铜皮石斛、环草石斛、马鞭石斛、黄草石斛。
3.根据权利要求1所述复配石斛原浆的制备方法,其特征在于,步骤(1)中石斛鲜条、新鲜桂圆、桑叶和枸杞的投料比例为:石斛鲜条5-30 wt%,桑叶0.5-5 wt %,新鲜桂圆0.1-2wt %,枸杞0.1-2 wt %。
4.根据权利要求1所述复配石斛原浆的制备方法,其特征在于,步骤(1)中所述预处理的具体过程为将新鲜的石斛和桂圆、干燥的桑叶和枸杞洗净,用切断机将石斛切成长度均一的小段,将桂圆去皮去核。
5.根据权利要求1所述复配石斛原浆的制备方法,其特征在于,步骤(2)中所述筛为60-80目振动筛。
6.根据权利要求1所述复配石斛原浆的制备方法,其特征在于,步骤(4)的具体步骤为将步骤(3)所得的絮凝液加热至80-100℃,处理10-60min后,冷却至2-8℃,静置24-48h,转移至管式离心机中,5,000-10,000 g条件下离心10-30 min,得到澄清液。
7.一种复配石斛原浆,其特征在于,由权利要求1-6任一项所述的制备方法制得。
8.权利要求7所述复配石斛原浆在制备辅助降糖或/和改善腹胀副作用的药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述复配石斛原浆通过抑制a-葡萄糖苷酶活性来增强降糖效果或/和通过提高肠道益生菌丰度来增强降糖效果;所述复配石斛原浆能够改善糖尿病患者服用α-葡萄糖苷酶抑制剂引起的腹胀、腹泻副作用。
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