CN116083252B - 一种高产降脂天然莫拉可林k的红曲米发酵制备方法 - Google Patents
一种高产降脂天然莫拉可林k的红曲米发酵制备方法 Download PDFInfo
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Abstract
本发明公开了一种高产降脂天然莫拉可林K的红曲米发酵制备方法,属于生物发酵技术领域,为了解决红曲米中莫拉可林K含量低且存在桔霉素的问题,本发明提出通过在发酵过程中外源添加γ‑丁内酯的方式,促进了菌体生长和能量代谢,进而提高莫拉可林K的产量,抑制桔霉素的生成;同时,以藜麦粉和糙米粉为发酵原料,二者搭配,协同促进,糙米粉中的种皮可以起到疏松、保湿作用,使发酵周期缩短,藜麦粉所含脂肪中多不饱和脂肪酸占83%,对胆固醇合成的抑制活性更强。
Description
技术领域
本发明属于生物发酵技术领域,具体是指一种高产降脂天然莫拉可林K的红曲米发酵制备方法。
背景技术
随着人们生活水平的提高和人口老龄化,因脂质代谢紊乱引起的高脂血症,已成为脂肪肝、冠心病和动脉粥样硬化等常见病的重要发病因素。
红曲是我国常见的传统中药材,是现存已知能够有效降低高脂血症的药物之一,其含有的莫拉克林K能够与羟甲戊二酰辅酶A还原酶直接结合,从而抑制胆固醇在体内的合成,且不需要体内羟基酸脂酶参与水解,不增加肝脏负担,即能发生降脂作用,但现有工艺制得的红曲米中的有效成分莫拉克林K的含量较低,以至于影响产品的整体药效;且红曲菌发酵过程中易生成桔霉素,具有肾毒性和致畸性,会对人体产生危害;此外,目前降脂红曲米多采用固态发酵法生产,专利CN102406127中降脂红曲的制备方法包括浸米、洗米、蒸煮装袋、培养过程喷水管理等步骤,专利CN105349438中功能红曲制备方法包括浸米、沥干、蒸米等步骤,其缺点是:人工制备程序繁杂,制备效率低,周期长,而关于外源添加对红曲菌发酵产物影响的研究仍欠缺,因此,寻找既能促进红曲菌高产莫拉克林K,不产桔霉素但又相对简单的发酵调控方法是首要任务。
发明内容
针对上述情况,为克服现有技术的缺陷,本发明提供了一种高产降脂天然莫拉可林K的红曲米发酵制备方法,为了解决红曲米中莫拉克林K含量低且存在桔霉素的问题,本发明提出通过在发酵过程中外源添加γ-丁内酯的方式,促进了菌体生长和能量代谢,进而提高莫拉可林K的产量,抑制桔霉素的生成;同时,以藜麦粉和糙米粉为发酵原料,二者搭配,协同促进,糙米粉中的种皮可以起到疏松、保湿作用,使发酵周期缩短,藜麦粉所含脂肪中多不饱和脂肪酸占83%,对胆固醇合成的抑制活性更强;此外,与固态发酵法相比,本发明采用的液态发酵方式具有周期短,杂质少,产品质量稳定等优点,有助于实现生产自动化,提升品质,扩大生产规模。
为了实现上述目的,本发明采取的技术方案如下,本发明提出了一种高产降脂天然莫拉可林K的红曲米发酵制备方法,具体包括如下步骤:
(1)制备PDA培养基:将马铃薯洗净去皮,称取200g,切成2cm3的小块,加入1000mL蒸馏水后煮沸30min,用八层纱布过滤,滤液用无菌水定容至1L,得到马铃薯汁,加入20g葡萄糖和15g琼脂粉,加热至溶解,调节pH值至7,于1×105Pa灭菌20min;
(2)菌种活化:将菌种转接至PDA斜面培养基中,30℃恒温培养4d后,于PDA培养基平板画线,30℃孵育5-7天;
(3)发酵培养:将种子液接种于发酵培养基中,加入γ-丁内酯,进行摇床培养,得到红曲发酵产物。
优选地,步骤(1)中所述菌种来源于中国工业微生物菌种保藏管理中心,菌株编号为CICC5046。
优选地,步骤(3)中所述种子液的接种量为8%(v/v)。
优选地,步骤(3)中所述种子液的制备方法如下:用无菌水冲洗PDA培养基上的红曲菌孢子,调整孢子浓度为106CFU/mL,以10%(v/v)的接种量接入种子液培养基中,于30℃、120r/min培养48h。
进一步地,所述种子液培养基的配方为:碳源,30g/L;氮源,25g/L;甘油,70mL/L;KH2PO4,2g/L;NaNO3,2g/L,MgSO4•7H2O,1g/L,用马铃薯汁定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min。
进一步地,所述碳源为葡萄糖、蔗糖、甘油、糖蜜中的一种或几种,所述氮源为玉米浆、酵母膏、豆粕、牛肉膏、蛋白胨中的一种或几种。
进一步地,所述碳源为葡萄糖,所述氮源为蛋白胨和豆粕,质量比为1:2。
优选地,步骤(3)中所述发酵培养基的配方为:藜麦粉,8-12g/L;糙米粉,20-26g/L;MgSO4•7H2O,1-3g/L;ZnSO4•7H2O,0.7-2.2g/L;KH2PO4,2.3-2.8g/L;甘油,86-95g/L;NaNO3,3-6g/L;蛋白胨,8-12g/L,用无菌水定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min。
优选地,步骤(3)中所述γ-丁内酯的浓度为4%(v/v)。
优选地,步骤(3)中所述摇床培养的条件为先于30℃、200r/min培养3d,再于25℃,150r/min培养8d。
本发明取得的有益效果如下:
(1)本发明提供了一种高产降脂天然莫拉可林K的红曲米发酵制备方法,首次提出以γ-丁内酯为外源添加,改变红曲霉菌丝体和孢子的形态,使菌丝表面更粗糙蓬松,表现出明显的褶皱和膨胀,促进了菌体生长和能量代谢,莫拉可林K的含量可高达16.3mg/g,同时提高了红曲色素的含量,抑制桔霉素的生成;
(2)豆粕作为大豆加工后的副产物,富含蛋白质、膳食纤维等成分,具有很高的利用价值,本实验利用红曲菌产生的复杂酶系,将豆粕与蛋白胨作为氮源,降低生产成本;
(3)与固态发酵法相比,本发明采用的液态发酵方式具有周期短,杂质少,产品质量稳定等优点,有助于实现生产自动化,提升品质,扩大生产规模;
(4)本发明首次使用藜麦粉和糙米粉为发酵原料,更易被红曲菌分解利用,糙米粉中的种皮可以起到疏松、保湿作用,使发酵周期缩短;藜麦粉所含脂肪中多不饱和脂肪酸占83%,具有双键的不饱和脂肪酸比饱和脂肪酸对胆固醇合成的抑制活性更强,二者搭配,协同促进,不仅能够提高红曲中降血脂成份莫拉可林K的含量,同时带来另一种降脂成分多不饱和脂肪酸的积累;
(5)本发明具有制备工艺简单,成本低廉、生产效率高的优点,且降脂活性物质更多,非常适宜用做降脂功能保健产品的原料,多种活性成分具有协同作用,克服了现有药物洛伐他汀需联合用药、药价高、副作用大的缺点。
附图说明
图1红曲发酵液中的生物量;
图2γ-丁内酯对红曲霉菌丝体和孢子形态的影响;
图3大鼠血清血脂TC、LDL-C、TG、HDL-C的含量。
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例;基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
除非另行定义,文中所使用的所有专业与科学用语与本领域技术人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用,但不能限制本申请的内容。
下述实施例中的实验方法,如无特殊说明,均为常规方法;下述实施例中所用的试验材料及试验菌株,如无特殊说明,均为从商业渠道购买得到的。
实施例1
本发明提供了一种高产降脂天然莫拉可林K的红曲米发酵制备方法,具体包括如下步骤:
(1)制备PDA培养基:将马铃薯洗净去皮,称取200g,切成2cm3的小块,加入1000mL蒸馏水后煮沸30min,用八层纱布过滤,滤液用无菌水定容至1L,得到马铃薯汁,加入20g葡萄糖和15g琼脂粉,加热至溶解,调节pH值至7,于1×105Pa灭菌20min;
(2)菌种活化:将菌种转接至PDA斜面培养基中,30℃恒温培养4d后,于PDA培养基平板画线,30℃孵育5-7天;
(3)发酵培养:按8%(v/v)的接种量将种子液接种于发酵培养基中,加入4%(v/v)γ-丁内酯,先于30℃、200r/min培养3d,再于25℃,150r/min培养8d,得到红曲发酵产物。
其中,步骤(3)中种子液的制备方法如下:用无菌水冲洗PDA培养基上的红曲菌孢子,调整孢子浓度为106CFU/mL,以10%(v/v)的接种量接入种子液培养基中,于30℃、120r/min培养48h。
其中,种子液培养基的配方为:碳源,30g/L;氮源,25g/L;甘油,70mL/L;KH2PO4,2g/L;NaNO3,2g/L,MgSO4•7H2O,1g/L,用马铃薯汁定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min。
其中,碳源为葡萄糖,氮源为蛋白胨和豆粕,蛋白胨和豆粕质量比为1:2。
其中,步骤(3)中发酵培养基的配方为:藜麦粉,10g/L;糙米粉,22g/L;MgSO4•7H2O,2.5g/L;ZnSO4•7H2O,2g/L;KH2PO4,2.5g/L;甘油,90g/L;NaNO3,5g/L;蛋白胨,10g/L,用无菌水定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min。
γ-丁内酯购自贝斯特试剂;红曲菌种来源于中国工业微生物菌种保藏管理中心(CICC),菌株编号为CICC5046;藜麦粉、糙米粉购自江苏海奥生物科技有限公司;豆粕购自济南鑫宏化工有限公司;其他试剂均为国产分析纯。
实施例2
本实施例提供一种高产降脂天然莫拉可林K的红曲米发酵制备方法,与实施例1的区别仅在于,步骤(3)中所述发酵培养基的配方为:藜麦粉,8g/L;糙米粉,20g/L;MgSO4•7H2O,1g/L;ZnSO4•7H2O,0.7g/L;KH2PO4,2.3g/L;甘油,86g/L;NaNO3,3g/L;蛋白胨,8g/L,用无菌水定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min,其余组分、组分含量与实施例1相同。
实施例3
实施例提供一种高产降脂天然莫拉可林K的红曲米发酵制备方法,与实施例1的区别仅在于,步骤(3)中所述发酵培养基的配方为:藜麦粉,12g/L;糙米粉,26g/L;MgSO4•7H2O,3g/L;ZnSO4•7H2O,2.2g/L;KH2PO4,2.8g/L;甘油,95g/L;NaNO3,6g/L;蛋白胨,12g/L,用无菌水定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min,其余组分、组分含量与实施例1相同。
对比例1
本对比例提供一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其与实施例1的区别仅在于步骤(3)中不加γ-丁内酯,其余组分、组分含量与实施例1相同。
对比例2
本对比例提供一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其与实施例1的区别仅在于所述氮源为蛋白胨,其余组分、组分含量与实施例1相同。
对比例3
本对比例提供一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其与实施例1的区别仅在于步骤(3)中所述发酵培养基的配方为:米粉,28g/L;MgSO4•7H2O,1g/L;ZnSO4•7H2O,0.7g/L;KH2PO4,2.3g/L;甘油,86g/L;NaNO3,3g/L;蛋白胨,8g/L,用无菌水定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min,其余组分、组分含量与实施例1相同。
实验例1
(一)红曲发酵产物中莫拉可林K含量的测定
以实施例1、实施例2、实施例3、对比例1、对比例2、对比例3中制得的红曲发酵产物进行实验,具体包括如下步骤:
(1)在50mL的离心管中加入0.3g红曲发酵产物和10mL的75%(v/v)乙醇,振荡混匀,超声提取1h,提取液于8000r/min离心10min,所得上清液过0.22μm滤膜;
(2)用高效液相色谱(HPLC)法测定莫拉可林K的含量,HPLC系统为Agilent-1260,色谱柱为InertsilODS-3(250mm×4.6mm,5μm),流动相体积比为乙腈:水:0.5%(v/v)H3PO4=60:37:3,流速1mL/min,柱温25℃,检测波长238nm,进样量20μL。
红曲发酵产物中莫拉可林K的含量计算公式如下:
莫拉可林K含量/(mg·g-1)=[(酸式MK峰面积+内酯式MK峰面积)×稀释倍数×标准品浓度×10]/(标准品峰面积×0.3×1000)
(二)红曲发酵产物中桔霉素含量的测定
以实施例1、实施例2、实施例3、对比例1、对比例2、对比例3中制得红曲发酵产物进行实验,具体包括如下步骤:
(1)称取红曲发酵产物0.2g,加入0.5mL盐酸水溶液(pH2)酸化10min,再加入甲苯、乙酸乙酯混和提取剂2mL,超声提取15min,6000rpm离心10min,吸取溶剂层置另一离心管中,45℃水浴上通空气挥干,残渣用100μL甲醇溶解,6000rpm离心10min,取上清备用;
(2)色谱柱:Irrogulour-HC18柱(250×4.6mm,10μm),预柱:PhenomenexC18,流动相:乙腈-水(15:85),磷酸调节pH至2.8,检测波长:紫外检测器,λ=254nm,荧光检测器:λex=331nm,λem=500nm,流速:1mL/min,柱温:25℃;
(3)称取桔霉素对照品14.64mg,用甲醇溶解并定容至10.0mL,浓度为1.464mg/mL,作为对照品贮备液,精密吸取贮备液,用甲醇稀释定容,配置成浓度为7.32μg/mL,21.96μg/mL,65.88μg/mL,144.94μg/mL,219.6μg/mL的标准溶液,均以10μL进样,以峰面积对标准品浓度进行线性回归,回归方程为Y=1078+4098X,r=0.99989。
(三)红曲发酵液中生物量的测定
发酵液中的菌丝用蒸馏水冲洗三次,过滤后收集,将菌丝在60℃干燥至恒重,测定生物量,生物量(g/L)以单位体积发酵液菌丝干重表示。
表1红曲发酵产物中莫拉可林K和桔霉素的含量
结果分析,如表1所示,实施例1中莫拉可林K的含量可达16.3mg/g,且不含桔霉素,对比例1中莫拉可林K的含量最少,同时桔霉素含量最高;由图1所示,实施例1中生物量最大,实施例2、实施例3次之,对比例1发酵液中生物量最少,表明本发明外源添加γ-丁内酯,促进了菌体生长和能量代谢,提高了莫拉可林K的含量,抑制了桔霉素的生成。
实验例2
γ-丁内酯对红曲霉菌丝体和孢子形态的影响
在2.5%戊二醛溶液中收获菌丝并固定12h,用0.1mol/L磷酸盐缓冲液(pH7.2)洗涤菌丝两次,用一系列浓度的乙醇溶液(30、50、70、80、90和100%)脱水,重复两次,丢弃上清液,12000rpm离心5min,然后加入六甲基二硅氮烷,60℃干燥成粉末,用扫描电镜观察菌丝结构和孢子形态。
结果分析,图2中a、b分别为实施例1和对比例1中红曲霉菌丝体和孢子在5000×下的SEM图像,c、d分别为实施例1和对比例1中红曲霉菌丝体和孢子在30000×下的SEM图像,经γ-丁内酯处理的实施例1和未经γ-丁内酯处理的对比例1的菌丝体表面结构差异明显,对比例1菌丝表面光滑平坦,而实施例1菌丝表面粗糙蓬松,表现出明显的褶皱和膨胀,菌丝表面的合成产物积累量远远大于对比例1。
实验例3
红曲发酵产物对大鼠食物性高脂血症的影响
取60只雄性SD大鼠按空腹血清胆固醇和体重随机分为对照组(10只)和造模组(50只),造模组按以下配方:5%猪油、2%胆固醇、1%胆盐、92%基础饲料喂饲30天,第26晚禁食,次日晨剪尾取血,测定空腹血清TC,和喂饲普通饲料的对照组大鼠的TC比较,差异显著即确定造模成功(TC>6.00mmol/L),共选出40只高脂模型大鼠,然后按血清胆固醇和体重随机的原则分为4组(每组10只),即模型组、实施例1组、对比例1组、洛伐他汀组(2mg/kg),给药方式为灌胃(模型组不给药),10mL/kg,1次/日,在给药期间,除对照组外,其余四组仍予高脂饲料(药物用0.5%CMC悬浮溶解配成8mg/mL浓度冻存备用,临用前加0.5%CMC稀释,配成0.2mg/mL浓度)。
结果分析,如图3所示,实施例1组对高脂血症大鼠血清血脂TC、LDL-C、TG降低活性、HDL-C升高活性明显优于相同剂量的洛伐他汀,表明本发明发酵制备的红曲发酵产物中莫拉可林K用于高脂血症的治疗具有更加显著的药理活性。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
以上对本发明及其实施方式进行了描述,这种描述没有限制性,附图中所示的也只是本发明的实施方式之一,实际的应用并不局限于此。总而言之如果本领域的普通技术人员受其启示,在不脱离本发明创造宗旨的情况下,不经创造性的设计出与该技术方案相似的方式及实施例,均应属于本发明的保护范围。
Claims (4)
1.一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其特征在于,具体包括如下步骤:
(1)制备PDA培养基:将马铃薯洗净去皮,称取200g,切成2cm3的小块,加入1000mL蒸馏水后煮沸30min,用八层纱布过滤,滤液用无菌水定容至1L,得到马铃薯汁,加入20g葡萄糖和15g琼脂粉,加热至溶解,调节pH值至7,于1×105Pa灭菌20min;
(2)菌种活化:将菌种转接至PDA斜面培养基中,30℃恒温培养4d后,于PDA培养基平板画线,30℃孵育5-7天;所述菌种来源于中国工业微生物菌种保藏管理中心,菌株编号为CICC5046;
(3)发酵培养:将种子液接种于发酵培养基中,加入γ-丁内酯,进行摇床培养,得到红曲发酵产物;
所述种子液的制备方法如下:用无菌水冲洗PDA培养基上的红曲菌孢子,调整孢子浓度为106CFU/mL,以10%(v/v)的接种量接入种子液培养基中,于30℃、120r/min培养48h;
所述种子液培养基的配方为:碳源,30g/L;氮源,25g/L;甘油,70mL/L;KH2PO4,2g/L;NaNO3,2g/L;MgSO4•7H2O,1g/L;用马铃薯汁定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min;
所述碳源为葡萄糖,所述氮源为蛋白胨和豆粕,所述蛋白胨和豆粕质量比为1:2;
所述发酵培养基的配方为:藜麦粉,8-12g/L;糙米粉,20-26g/L;MgSO4•7H2O,1-3g/L;ZnSO4•7H2O,0.7-2.2g/L;KH2PO4,2.3-2.8g/L;甘油,86-95g/L;NaNO3,3-6g/L;蛋白胨,8-12g/L;用无菌水定容至1L,用乳酸调节pH值至6,于1×105Pa灭菌20min。
2.根据权利要求1所述的一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其特征在于,步骤(3)中所述种子液的接种量为8%(v/v)。
3.根据权利要求2所述的一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其特征在于:步骤(3)中所述γ-丁内酯的浓度为4%(v/v)。
4.根据权利要求3所述的一种高产降脂天然莫拉可林K的红曲米发酵制备方法,其特征在于:步骤(3)中所述摇床培养的条件为先于30℃、200r/min培养3d,再于25℃,150r/min培养8d。
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