WO2022194189A1 - 一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液及其用途 - Google Patents

一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液及其用途 Download PDF

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WO2022194189A1
WO2022194189A1 PCT/CN2022/081079 CN2022081079W WO2022194189A1 WO 2022194189 A1 WO2022194189 A1 WO 2022194189A1 CN 2022081079 W CN2022081079 W CN 2022081079W WO 2022194189 A1 WO2022194189 A1 WO 2022194189A1
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lactobacillus
rhodochrous
fermentation supernatant
purpurea
fermentation
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French (fr)
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姜泽东
董玉婷
倪辉
杜希萍
陈艳红
杨远帆
李志朋
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集美大学
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  • the invention belongs to the technical field of biological resource development, and in particular relates to a fermentation supernatant of Lactobacillus rhodochrous algae and uses thereof.
  • Diabetes is the third largest chronic non-communicable disease after cardiovascular disease and cancer. Among them, more than 90% of diabetic patients suffer from type 2 diabetes mellitus (T2DM).
  • T2DM type 2 diabetes mellitus
  • ⁇ -Glucosidase inhibitors can competitively inhibit the activity of ⁇ -glucosidase in the small intestine, delay or inhibit the absorption of glucose in the intestine, thereby effectively reducing the peak of postprandial blood sugar and adjusting the blood sugar level of the body.
  • Alpha-glucosidase inhibitors play a crucial role in suppressing postprandial hyperglycemia and have long been an ideal drug for the treatment of type II diabetes.
  • Acarbose is a glycosidase inhibitor widely used in clinical practice.
  • ⁇ -glucosidase inhibitors mainly focuses on chemical synthesis or extraction of ⁇ -glucosidase inhibitors from natural substances, but the ⁇ -glucosidase inhibitors obtained by these methods all have certain side effects. Such as causing liver, intestine, stomach and other diseases; at the same time, these inhibitors also have problems such as low extraction amount and high cost.
  • using the method of food lactic acid bacteria fermentation to produce products with ⁇ -glucosidase inhibitory activity has low cost and high safety.
  • Bangiafusco-purpure also known as red hair algae, red cotton algae, etc.
  • red hair algae red cotton algae, etc.
  • the supernatant extracted from the algal body of Rhododendron and the fermentation supernatant of Lactobacillus rhodochrous algae without the optimization of the fermentation process have relatively low inhibitory effects on the enzymatic activity of ⁇ -glucosidase, both less than 40%. Therefore, it is not yet a substitute for alpha-glucosidase inhibitors that are chemically synthesized or extracted from natural substances.
  • one aspect of the present invention provides a Lactobacillus Rhododendron fermentation supernatant with ⁇ -glucosidase inhibitory activity, which has the advantages of low cost, high safety, and high inhibitory activity, and can replace existing Alpha-glucosidase inhibitors in the art, either chemically synthesized or extracted from natural substances.
  • a Lactobacillus rhodochrous fermentation supernatant with ⁇ -glucosidase inhibitory activity comprising the following steps:
  • preparation method of described lactobacillus rhodochrous algae fermentation supernatant also comprises the following steps:
  • Rhododendron was ground with liquid nitrogen before mixing it with water.
  • the step S1 is to mix the red hair algae with water in a ratio of 5% by mass to volume to obtain a red hair algae suspension
  • the step S3 is to inoculate and add Lactobacillus delbrueckii in a ratio of 4% by mass to volume to obtain the milk algae suspension.
  • the bacillus rhodochrous mixed solution is characterized in that: the lactobacillus rhodochrous fermented supernatant is the lactobacillus delbrueckii fermentation supernatant, and the lactobacillus delbrueckii fermentation supernatant is -
  • the inhibition rate of glucosidase was 88.2%.
  • the step S1 is to mix the red hair algae and water in a ratio of 4.45% by mass to volume to obtain a red hair algae suspension
  • the step S3 is to inoculate and add Lactobacillus plantarum by 4% by mass volume ratio to obtain Lactobacillus.
  • the red hair alga mixed solution is characterized in that: the Lactobacillus red hair algae fermentation supernatant is the Lactobacillus plantarum red hair alga fermentation supernatant liquid, and the Lactobacillus plant red hair algae fermentation supernatant liquid has a significant effect on ⁇ -glucoside
  • the inhibition rate of the enzyme was 84.8%.
  • the second purpose of the present invention is to provide a kind of purposes of the Lactobacillus rhodochrous fermentation supernatant with ⁇ -glucosidase inhibitory activity, and its technical scheme is as follows:
  • the purpose of the first invention is to realize the use of the fermentation supernatant of Lactobacillus rhodochrous algae in the preparation of an ⁇ -glucosidase inhibitor.
  • the fermentation supernatant of Lactobacillus rhodochrous algae provided by the present invention is a natural, safe and economical ⁇ -glucosidase inhibitor, the inhibition rate of the ⁇ -glucosidase is more than 80%, and can replace the existing technology of alpha-glucosidase inhibitors, either chemically synthesized or extracted from natural substances.
  • FIG. 1 is a comparison diagram of the results of the inhibition test of the fermentation supernatant obtained in Example 1 and Example 2 of the present invention and the positive control acarbose on ⁇ -glucosidase.
  • Acarbose is acarbose
  • DF is the fermentation supernatant of Lactobacillus delbrueckii
  • PF is the fermentation supernatant of Lactobacillus plantarum.
  • Example 1 Preparation of Lactobacillus delbrueckii fermentation supernatant and ⁇ -glucosidase inhibition rate test
  • the red hair algae are ground with liquid nitrogen;
  • red hair algae suspension add 20 grams of glucose according to the mass volume ratio, and then pasteurize;
  • Lactobacillus delbrueckii Inoculate and add 40 grams of Lactobacillus delbrueckii according to the mass volume ratio to obtain the Lactobacillus delbrueckii mixed solution;
  • the Lactobacillus delbrueckii mixed liquid was fermented at 37°C for 48 hours to obtain the Lactobacillus delbrueckii mixed fermentation liquid;
  • the mixed fermentation broth of Lactobacillus delbrueckii was centrifuged to remove algal residue and Lactobacillus bacteria to obtain Lactobacillus delbrueckii fermentation supernatant.
  • the formula for calculating the inhibition rate is:
  • A1 blank group, with PBS buffer instead of Lactobacillus delbrueckii fermentation supernatant after the reaction
  • A2 sample group, adding Lactobacillus delbrueckii fermentation supernatant or acarbo The absorbance of the sugar aqueous solution sample after the reaction
  • A3 the background control group, the absorbance after the reaction with PBS buffer instead of the ⁇ -glucosidase solution.
  • the concentration of the positive control acarbose aqueous solution is the IC50 concentration of 0.64 mg/mL.
  • the purpose of setting the positive control is to test and compare the supernatant of the Lactobacillus delbrueckii fermented red hair algae and the positive control in this example. The second is to provide a reference for others to ensure the consistency of the experiment when repeating this example.
  • Example 2 Preparation of Lactobacillus plantarum Rhododendron fermentation supernatant and ⁇ -glucosidase inhibition rate test
  • the red hair algae are ground with liquid nitrogen;
  • red hair algae suspension add 20 grams of glucose according to the mass volume ratio, and then pasteurize;
  • the mixed liquid of Lactobacillus plantarum rhodochrous algae was fermented at 37°C for 48 hours to obtain the mixed fermented liquid of Lactobacillus plantarum rhodochrous algae;
  • the mixed fermentation broth of Lactobacillus plantarum Rhododendron was centrifuged to remove algal residue and Lactobacillus cells, and the fermentation supernatant of Lactobacillus plantarum Rhododendron was obtained.
  • test method of this embodiment is the same as that of embodiment 1. After testing, the inhibition rate of ⁇ -glucosidase by the fermentation supernatant of Lactobacillus plantarum rhodochrous algae obtained in this example was 84.8%.

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Abstract

本发明提供一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液，所述乳杆菌红毛藻发酵上清液的制备方法包括以下步骤：S1：将红毛藻与水按质量体积比4.45％-5％的比例混合得到红毛藻悬液；S2：在所述红毛藻悬液中按质量体积比加入2％的葡萄糖，再进行巴氏杀菌；S3：按质量体积比4％接种加入德氏乳杆菌或植物乳杆菌，得到乳杆菌红毛藻混合液；S4：将所述乳杆菌红毛藻混合液在37℃下静置发酵48小时，得到乳杆菌红毛藻混合发酵液；S5：将所述乳杆菌红毛藻混合发酵液进行离心去除藻渣和乳杆菌菌体，获得所述乳杆菌红毛藻发酵上清液。本发明提供的乳杆菌红毛藻发酵上清液是一种天然、安全、经济的α-葡萄糖苷酶抑制剂。

Description

一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液及其用途 技术领域
本发明属于生物资源开发技术领域，特别涉及一种具乳杆菌红毛藻发酵上清液及其用途。
背景技术
糖尿病是继心血管病、肿瘤之后的第三大慢性非传染性疾病。其中，超过90％的糖尿病患者患有2型糖尿病(T2DM)。α-葡萄糖苷酶抑制剂可竞争性地抑制小肠内α-葡萄糖苷酶的活性活性,延缓或抑制葡萄糖在肠道内的吸收,从而有效地降低餐后血糖的峰值,调整机体血糖的水平。α-葡萄糖苷酶抑制剂在抑制餐后高血糖的过程中起着至关重要的作用，一直以来都作为治疗II型糖尿病的理想药物。阿卡波糖是目前临床应用广泛的糖苷酶抑制剂，对血糖的控制效果较好，但长期服用存在一定的副作用，如对肝脏造成损害、引起肠梗阻等。因此，寻找天然、安全、经济的α-葡萄糖苷酶抑制剂是延缓或辅助治疗II型糖尿病的较好途径。目前，对α-葡萄糖苷酶抑制剂的研究主要集中在化学合成或从天然物质中提取α-葡萄糖苷酶抑制剂，但利用这些方法获得的α-葡萄糖苷酶抑制剂均具一定的副作用，如引起肝、肠、胃等疾病；同时，这些抑制剂也存在提取量少，成本较高等问题。然而，采用食品乳酸菌发酵的方法生产具备α-葡萄糖苷酶抑制活性的产品，成本较低，安全性高。
红毛藻(Bangiafusco-purpure)，又称红毛菜、红棉藻等，是我国福建沿海特有的经济红藻资源，鲜香美味的红毛藻具有显著的降低血压、改善血虚症、滋阴降火以及预防血管疾病等功效。现有技术中利用红毛藻藻体提取的上清液和未经过发酵工艺优化的乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶酶活力的抑制效果较低，均低于40％。因此，其还无法替代从化学合成或从天然物质中提取的α-葡萄糖苷酶抑制剂。
发明内容
有鉴于此，本发明一方面提供了一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液，其具有成本低、安全性高、抑制活性高的优点，可以替代现有技术中的从化学合成或从天然物质中提取的α-葡萄糖苷酶抑制剂。
本发明是通过如下技术方案实现上述第一发明目的的：
一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液，所述乳杆菌红毛藻发酵上清液的制备方法包括以下步骤：
S1：将红毛藻与水按质量体积比4.45％-5％的比例混合得到红毛藻悬液；
S2：在所述红毛藻悬液中按质量体积比加入2％的葡萄糖，再进行巴氏杀菌；
S3：按质量体积比4％接种加入德氏乳杆菌或植物乳杆菌，得到乳杆菌红毛藻混合液；
S4：将所述乳杆菌红毛藻混合液在37℃下静置发酵48小时，得到乳杆菌红毛藻混合发酵液；
S5：将所述乳杆菌红毛藻混合发酵液进行离心去除藻渣和乳杆菌菌体，获得所述乳杆菌红毛藻发酵上清液。
进一步的，所述乳杆菌红毛藻发酵上清液的制备方法还包括以下步骤：
S0:在将红毛藻与水混合前将红毛藻用液氮研磨处理。
进一步的，所述步骤S1为将红毛藻与水按质量体积比5％的比例混合得到红毛藻悬液，所述步骤S3为按质量体积比4％接种加入德氏乳杆菌，得到乳杆菌红毛藻混合液，其特征在于：所述乳杆菌红毛藻发酵上清液为德氏乳杆菌红毛藻发酵上清液，所述德氏乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶的抑制率为88.2％。
进一步的，所述步骤S1为将红毛藻与水按质量体积比4.45％的比例混合得到红毛藻悬液，所述步骤S3为按质量体积比4％接种加入植物乳杆菌，得到乳杆菌红毛藻混合液，其特征在于：所述乳杆菌红毛藻发酵上清液为植物乳杆菌红毛藻发酵上清液，所述植物乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶的抑制率为84.8％。
本发明的第二发明目的是提供一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液的用途，其技术方案如下：
实现第一发明目的的乳杆菌红毛藻发酵上清液在制备α-葡萄糖苷酶抑制剂中的用途。
本发明提供的乳杆菌红毛藻发酵上清液是一种天然、安全、经济的α-葡萄糖苷酶抑制剂，其对α-葡萄糖苷酶的抑制率大于80％，可以替代现有技术中的从化学合成或从天然物质中提取的α-葡萄糖苷酶抑制剂。
附图说明
图1是本发明实施例1和实施例2得到的发酵上清液与阳性对照阿卡波糖对α-葡萄糖苷酶的抑制测试结果对比图。
图1中，Acarbose为阿卡波糖，DF代表德氏乳杆菌红毛藻发酵上清液，PF代表植物乳杆菌红毛藻发酵上清液。
具体实施方式
实施例1：德氏乳杆菌红毛藻发酵上清液的制备及α-葡萄糖苷酶抑制率测试
制备：
将红毛藻用液氮研磨处理；
取50克研磨好的红毛藻与1000毫升水混合得到红毛藻悬液；
在红毛藻悬液中按质量体积比加入20克的葡萄糖，再进行巴氏杀菌；
按质量体积比接种加入40克德氏乳杆菌，得到德氏乳杆菌红毛藻混合液；
将德氏乳杆菌红毛藻混合液在37℃下静置发酵48小时，得到德氏乳杆菌红毛藻混合发酵液；
将德氏乳杆菌红毛藻混合发酵液进行离心去除藻渣和乳杆菌菌体，获得德氏乳杆菌红毛藻发酵上清液。
测试：
取100μl德氏乳杆菌发酵红毛藻上清液和250μlα-葡萄糖苷酶液混合，置于37℃水浴锅中温育10min，再向其中添加250μl底物溶液起始反应，在37℃下反应15min后加入1mol/L的Na 2CO 3溶液250μL终止反应，于405nm波长下测定其吸光度值。以阿卡波糖(Acarbose)作为阳性对照，以同样体积PBS缓冲液代替发酵液作为空白组，以同样体积PBS缓冲液代替α-葡萄糖苷酶溶液作为背景对照组，每组实验体系设定3个平行实验。
抑制率计算公式为：
Figure PCTCN2022081079-appb-000001
式中，A1：空白组，以PBS缓冲液代替德氏乳杆菌发酵红毛藻上清液反应后的吸光度；A2：样品组，加入德氏乳杆菌红毛藻发酵上清液或阿卡波糖水溶液样品反应后的吸光度；A3：背景对照组，以PBS缓冲液代替α-葡萄糖苷酶溶液反应后的吸光度。
本实施例中阳性对照阿卡波糖水溶液的浓度为IC50浓度0.64mg/mL，设置该阳性对照的目的一是测试比较本实施例的德氏乳杆菌发酵红毛藻上清液与该阳性对照抑制效果大小，二是为他人重复本实施例时提供如何保证试验一致性的参考。
如图1所示，本实施例的德氏乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶的抑制率88.2％
实施例2：植物乳杆菌红毛藻发酵上清液的制备及α-葡萄糖苷酶抑制率测试
制备：
将红毛藻用液氮研磨处理；
取44.5克研磨好的红毛藻与1000毫升水混合得到红毛藻悬液；
在红毛藻悬液中按质量体积比加入20克的葡萄糖，再进行巴氏杀菌；
按质量体积比接种加入40克植物乳杆菌，得到植物乳杆菌红毛藻混合液；
将植物乳杆菌红毛藻混合液在37℃下静置发酵48小时，得到植物乳杆菌红毛藻混合发酵液；
将植物乳杆菌红毛藻混合发酵液进行离心去除藻渣和乳杆菌菌体，获得植物乳杆菌红毛藻发酵上清液。
测试：
本实施例的测试方法同实施例1。经检测，本实施例得到的植物乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶的抑制率为84.8％。

Claims (5)

  1. 一种具α-葡萄糖苷酶抑制活性的乳杆菌红毛藻发酵上清液，其特征在于，所述乳杆菌红毛藻发酵上清液的制备方法包括以下步骤：
    S1：将红毛藻与水按质量体积比4.45％-5％的比例混合得到红毛藻悬液；
    S2：在所述红毛藻悬液中按质量体积比加入2％的葡萄糖，再进行巴氏杀菌；
    S3：按质量体积比4％接种加入德氏乳杆菌或植物乳杆菌，得到乳杆菌红毛藻混合液；
    S4：将所述乳杆菌红毛藻混合液在37℃下静置发酵48小时，得到乳杆菌红毛藻混合发酵液；
    S5：将所述乳杆菌红毛藻混合发酵液进行离心去除藻渣和乳杆菌菌体，获得所述乳杆菌红毛藻发酵上清液。
  2. 根据权利要求1所述的乳杆菌红毛藻发酵上清液，其特征在于，所述乳杆菌红毛藻发酵上清液的制备方法还包括以下步骤：
    S0:在将红毛藻与水混合前将红毛藻用液氮研磨处理。
  3. 根据权利要求2所述的乳杆菌红毛藻发酵上清液，所述步骤S1为将红毛藻与水按质量体积比5％的比例混合得到红毛藻悬液，所述步骤S3为按质量体积比4％接种加入德氏乳杆菌，得到乳杆菌红毛藻混合液，其特征在于：所述乳杆菌红毛藻发酵上清液为德氏乳杆菌红毛藻发酵上清液，所述德氏乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶的抑制率大于80％。
  4. 根据权利要求2所述的乳杆菌红毛藻发酵上清液，所述步骤S1为将红毛藻与水按质量体积比4.45％的比例混合得到红毛藻悬液，所述步骤S3为按质量体积比4％接种加入植物乳杆菌，得到乳杆菌红毛藻混合液，其特征在于：所述乳杆菌红毛藻发酵上清液为植物乳杆菌红毛藻发酵上清液，所述植物乳杆菌红毛藻发酵上清液对α-葡萄糖苷酶的抑制率大于80％。
  5. 根据权利要求1所述的乳杆菌红毛藻发酵上清液在制备α-葡萄糖苷酶抑制剂中的用途。
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