CN117567586A - 一种富含唾液酸化肽的水解酪蛋白的制备方法及其在调节肠道菌群中的应用 - Google Patents
一种富含唾液酸化肽的水解酪蛋白的制备方法及其在调节肠道菌群中的应用 Download PDFInfo
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Abstract
本发明属于功能性寡肽技术领域，公开了一种富含唾液酸化肽的水解酪蛋白的制备方法及其在调节肠道菌群中的应用，本发明从牛羊来源的酪蛋白中制备得到了富含唾液酸化肽的水解酪蛋白，该产品中的肽含量高达60％‑90％，唾液酸含量在1.6‑2.2g/kg，分子量分布90％以上的比例≤3000D，游离氨基酸≤3‑5％。产品低苦味、活性高(500mg/天/人以上有效)，白色粉末、冲调性/溶解性好(溶液澄清透明)、流动性好、耐高温(满足生产高温灭菌需求)、酸溶解。含有唾液酸化肽的水解酪蛋白，具有改善肠道菌群多样性、促进肠道粘膜屏障的构建及促进调节性T细胞增殖、调节免疫，预防肠道炎症发生的作用等。同时，该水解酪蛋白可促进钙铁锌硒锰等矿物质的吸收。
Description
技术领域
本发明属于功能性寡肽技术领域，更具体的，涉及一种富含唾液酸化肽的水解酪蛋白的制备方法及其在调节肠道菌群中的应用。
背景技术
唾液酸是一类极其重要的神经氨酸衍生物，它通常连接在聚糖的末端，是聚糖结构和功能多样化的重要物质基础之一，广泛参与信号分子的靶向传递，如配体/受体、抗原/抗体、以及酶/底物的靶向识别与黏附。
而唾液酸化肽(SGP)是一种含有唾液酸寡糖链的肽，由于其分支型糖链的结构和唾液酸化糖链的存在，SGP及其相关物质有较好的抗细菌和抗病毒作用。而从乳源中提取唾液酸化肽，并开发唾液酸化肽的新功能的研究较少，从而限制了其应用范围。
发明内容
本发明所要解决的技术问题是克服现有技术中存在的上述问题，首先提供唾液酸化肽水解酪蛋白的应用。
本发明的目的通过以下技术方案实现：
本发明首先提供唾液酸化肽水解酪蛋白在调节肠道菌群中的应用，所述水解酪蛋白的氨基酸序列为TSTP。
本发明还提供唾液酸化肽水解酪蛋白的制备方法，包括以下步骤：
(1)加酪蛋白3-4倍质量的水，在40℃-50℃条件下搅拌溶解；
(2)加入胰凝乳蛋白酶进行酶解，加酶量为酪蛋白质量的0.1％-0.2％，反应温度40℃-50℃，反应时间1h-2h，pH 7.0-9.0；
(3)将温度调节至80-90℃，保温20min-30min，进行灭菌；
(4)调pH值4.0-5.0，过滤除去未酶解的大分子蛋白沉淀；
(5)上清液利用超滤膜2000D分离，得到唾液酸化肽水解酪蛋白溶液；
(6)经过冷冻干燥、喷雾干燥、真空干燥的任意一种，对得到的产物进行干燥成粉。
利用本发明方法获得的唾液酸化肽水解酪蛋白的产品肽含量60％-90％，唾液酸含量为1.6-2.2g/kg，分子量≤3000D。产品低苦味、活性高(500mg/天/人以上有效)，白色粉末、冲调性/溶解性好(溶液澄清透明)、流动性好、耐高温(满足生产高温灭菌需求)、酸溶解(果汁pH4.0左右，一般的蛋白/肽会沉淀)、含量(适合特医用)。
优选的，所述唾液酸化肽水解酪蛋白能够增加肠道菌群丰度和肠道菌群α多样性。
优选的，所述唾液酸化肽水解酪蛋白能够增加产短链脂肪酸(SCFAs)的细菌的丰度。
优选的，所述肠道菌群为Bacteroides uniformis、Bifidobacterium longum、Ruminococcus albus、Faecalibacteriumprausnitzii、Aminipila butyrica、Bacteroidesuniformis、Intestinibacillus massiliensis、Agathobaculum desmolans、Blautiafaecis、Faecalibacteriumprausnitzii、Blautia obeum、Eubacterium rectale、Bifidobacterium bifidum、Faecalibacteriumprausnitzii、Streptococcusparasanguinis。
本发明还提供所述唾液酸化肽水解酪蛋白在制备调节肠道菌群的功能产品中的应用。
优选的，所述产品为保健品、药品、功能性食品。
优选的，所述唾液酸化肽水解酪蛋白能够增加产短链脂肪酸(SCFAs)的细菌的丰度。
优选的，所述肠道菌群为Bacteroides uniformis、Bifidobacterium longum、Ruminococcus albus、Faecalibacteriumprausnitzii、Aminipila butyrica、Bacteroidesuniformis、Intestinibacillus massiliensis、Agathobaculum desmolans、Blautiafaecis、Faecalibacteriumprausnitzii、Blautia obeum、Eubacterium rectale、Bifidobacterium bifidum、Faecalibacteriumprausnitzii、Streptococcusparasanguinis。
与现有技术相比，本发明具有以下有益效果：
本发明从乳源原料中制备得到了唾液酸化肽水解酪蛋白，该唾液酸化肽水解酪蛋白的产品肽含量60％-90％，唾液酸含量为1.6-2.2g/kg，分子量≤3000D。产品低苦味、活性高(500mg/天/人以上有效)，白色粉末、冲调性/溶解性好(溶液澄清透明)、流动性好、耐高温(满足生产高温灭菌需求)、酸溶解(果汁pH4.0左右，一般的蛋白/肽会沉淀)、含量(适合特医用)。含有唾液酸化肽等活性肽的酪蛋白水解物，具有改善肠道菌群多样性、促进机体非特异性防御屏障的构建，维持肠道屏障功能等功效。
附图说明
图1为实验技术路线图；
图2为实验研究流程图；
图3为服用水解酪蛋白或安慰剂两周后肠道菌群α多样性的变化。服用水解酪蛋白或安慰剂两周后肠道菌群α多样性变化，***组内比较P<0.001，#组间比较P<0.05(LDB：低剂量组基线、HDB：高剂量组基线、PB：安慰剂组基线、LDS：低剂量组干预后、HDS：高剂量组干预后、PS：安慰剂干预后)；
图4为服用水解酪蛋白或安慰剂两周后肠道菌群构成变化。A、随机森林分类器显示低、高剂量水解酪蛋白、安慰剂干预后对粪便菌群结构；B、对随机森林分类器贡献的特征细菌物种Top15；C、OPLSDA显示14天干预后各组间细菌分类的差异；D、各组间肠道菌群物种相对丰度(LDS：低剂量组干预后、HDS：高剂量组干预后、PS：安慰剂干预后)。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1唾液酸化肽水解酪蛋白的制备
包括以下步骤：
(1)向烧杯中加纯水400mL，开搅拌200rpm，向烧杯中缓慢投入酪蛋白100g，在50℃条件下搅拌溶解；
(2)加入胰凝乳蛋白酶进行酶解，加酶量为酪蛋白质量的0.1％，反应温度40℃，反应时间1h，pH 7.0；
(3)将温度调节至80℃，保温20min，进行灭菌；
(4)调pH值5.0，过滤除去未酶解的大分子蛋白沉淀；
(5)上清液利用超滤膜2000D分离，得到唾液酸化肽水解酪蛋白溶液；
(6)经过冷冻干燥、喷雾干燥、真空干燥的任意一种，对得到的产物进行干燥成粉，得到唾液酸化肽水解酪蛋白。
测定唾液酸化肽水解酪蛋白的氨基酸序列为：TSTP。
实施例2唾液酸化肽水解酪蛋白的应用
样品：广州绿萃生物科技有限公司提供的唾液酸化肽水解酪蛋白，添加辅料制成片剂。规格：400mg/片，水解酪蛋白的有效量为250mg/片，60片/瓶。安慰剂在剂型、口感、外观和包装上与样品相同。
1、志愿者招募
在青岛大学、青岛大学附属医院开展现场志愿者招募，研究方案经青岛大学医学部伦理委员会批准(批准号：QDU-HEC-2022001)。
志愿者纳入标准：
1)年龄18-65岁。
2)性别不限。
3)身体质量指数18.5-23.9公斤/平方米。
4)生命体征正常(血压低于140/90mmHg(即收缩压低于140mmHg，舒张压低于90mmHg，心率在60-100bpm之间)。
5)符合原发性失眠症的症状。
6)已签署书面知情同意书。
排除标准：
1)已确诊患有重度焦虑、抑郁症患者。
2)酒精或药物依赖者。
3)每天吸烟超过10支。
4)对牛奶蛋白或水解酪蛋白成分过敏。
5)继发性失眠患者。
6)纳入前一个月使用过治疗失眠的药物。
7)正在进行失眠的非药物治疗(如认知行为疗法、放松疗法等)。
8)怀孕或哺乳期。
9)有癌症或其他重大疾病者。
10)不愿或不能完成整个试验者。
2、样本含量计算
按以下公式计算样本含量。设置检验水准α＝0.05，检验效能β＝0.8，计算得75人，考虑10％的失访率，样本含量为82人。
3、试验分组和技术路线
符合条件的所有志愿者被随机分配。基于外部独立第三方集中生成随机列表，唾液酸化肽水解酪蛋白与安慰剂包装分别分布独立编号。在治疗阶段，受试者与研究实施人员对研究治疗分组盲目，除非在特殊的医疗情况下，否则不能接触到随机化代码。每个受试者按分组结果参与治疗，包括安慰剂(压片载体，2片)、低剂量水解酪蛋白(500mg/次，2片)、高剂量水解酪蛋白(1000mg/次，4片)，要求志愿者每天在睡觉前30分钟内口服。技术路线图见图1。
4、试验方法
水解酪蛋白和安慰片剂的尺寸、外观、颜色、气味和味道一致。均以药瓶形式提供给志愿者。每个药瓶中都装够足量片剂(60片)。采用Excel随机数字表法对纳入的志愿者进行随机分组，分别为安慰剂组、低剂量水解酪蛋白组、高剂量水解酪蛋白组。志愿者入组后第0、14天进行粪便样本采集。
5、粪便样本的采集和检测
使用无菌粪便采集管(30ml)进行粪便样本采集，采样完成后完成定量分装、预处理，冻存于-80℃冰箱待测。利用第三代16s rRNA扩增子测序技术分析肠道菌群构成，利用基于UPLC-QTOF的非靶向代谢组学分析技术分析血清和粪便代谢组学信息，分析挖掘水解酪蛋白作用相关生物标识。
6、试验结果
本次试验招募志愿者100人，随机化分配至低剂量组33人、高剂量组33人、安慰剂组34人。其中低剂量组因样本采集不全，流失5人，完成随访28人；高剂量组因疫情原因未及时采样，流失7人，完成随访26人；安慰剂组因样本采集不全，流失6人，完成随访28人。完成随访及进入数据分析的志愿者共82人。
6.1、肠道菌群多样性分析--增加了肠道菌群的多样性
与基线相比，14天干预后，安慰剂组、高剂量组粪便菌群chao1指数下降(P<0.01)。与安慰剂组比较，低、高剂量组shannon指数显著上升(P<0.05)，Chao1指数和shannon指数均属于α多样性指标，显示个体内不同微生物群落之间的多样性，即肠道内每个微生物群落内的菌种数。说明低、高剂量水解酪蛋白肽处理一定程度上增加了肠道菌群α多样性(图3)。
研究结果显示：低、高剂量水解酪蛋白组处理后，均能显著增加肠道菌群α多样性，而高多样性的微生物群落与代谢健康、免疫健康等因素有关。其中α多样性是指在个体内不同微生物群落之间的多样性，即肠道内每个微生物群落内的菌种数。
6.2、肠道菌群构成--促进更多的有益菌产生
随机森林分类器显示低、高剂量水解酪蛋白干预相对于安慰剂对粪便菌群结构产生影响(图4A、4B)。对随机森林分类器贡献的特征细菌物种Top15分别是低剂量组Bacteroides uniformis、Bifidobacterium longum、Ruminococcus albus、Faecalibacteriumprausnitzii、Aminipila butyrica、Bacteroides uniformis、Intestinibacillus massiliensis、Agathobaculum desmolans。高剂量组Blautiafaecis、Faecalibacteriumprausnitzii、Blautia obeum。偏最小二乘回归(OPLS-DA)显示了14天干预后组间细菌分类的差异。低剂量组与安慰剂组、高剂量组与安慰剂组之间均存在明显的差异，不同剂量的水解酪蛋白处理均可以改变肠道菌群的组成，对肠道菌群的调节产生积极的影响(图4C)。低、高剂量水解酪蛋白干预与安慰剂组比较，细菌物种相对丰度比较显示，低剂量组富集的细菌物种是Faecalibacterium prausnitzii。高剂量组富集的细菌物种是Oscillibacter ruminantium、PAC001208_s、Lachnospira eligens、Hungatellaeffluvii(图4D)。
研究结果显示：低、高剂量水解酪蛋白均较显著的增加了肠道中更多的有益菌种类，如促进了产丁酸等短链脂肪酸的Faecalibacteriumprausnitzii、Lachnospiraeligens等的相对丰度，以及促进了产双歧杆菌Bifidobacterium longum等的相对丰度，进而促进肠道粘膜屏障的构建。此外，Faecalibacterium prausnitzii和Bifidobacteriumlongum有潜在促进调节性T细胞增殖，调节免疫，预防肠道炎症发生的作用。
6.3、依从性分析
三组患者依从性均为100％，差异无统计学意义。依从性良好。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
Claims (9)
1.富含唾液酸化肽的水解酪蛋白在调节肠道菌群中的应用，其特征在于，所述水解酪蛋白的氨基酸序列为TSTP。
2.权利要求1所述唾液酸化肽的水解酪蛋白的制备方法，其特征在于，包括以下步骤：
(1)加酪蛋白8-10倍质量的水，在40℃-50℃条件下搅拌溶解；
(2)加入碱性蛋白酶进行酶解，加酶量为酪蛋白质量的0.5％，反应温度40℃-50℃，反应时间1h-2h，pH 7.0-9.0；
(3)将温度调节至80-90℃，保温20min-30min，进行灭菌；
(4)调pH值4.0-5.0，过滤除去未酶解的大分子蛋白沉淀；
(5)上清液利用超滤膜3000D分离，得到富含唾液酸化肽的水解酪蛋白溶液；
(6)经过冷冻干燥、喷雾干燥、真空干燥的任意一种，对得到的产物进行干燥成粉。
3.根据权利要求1所述的应用，其特征在于，所述富含唾液酸化肽的水解酪蛋白能够增加肠道菌群丰度和肠道菌群α多样性。
4.根据权利要求3所述的应用，其特征在于，所述富含唾液酸化肽的水解酪蛋白能够增加产短链脂肪酸(SCFAs)的细菌的丰度。
5.根据权利要求3所述的应用，其特征在于，所述肠道菌群为Bacteroides uniformis、Bifidobacterium longum、Ruminococcus albus、Faecalibacteriumprausnitzii、Aminipila butyrica、Bacteroides uniformis、Intestinibacillus massiliensis、Agathobaculum desmolans、Blautiafaecis、Faecalibacteriumprausnitzii、Blautiaobeum、Eubacterium rectale、Bifidobacterium bifidum、Faecalibacteriumprausnitzii、Streptococcusparasanguinis。
6.权利要求1所述唾液酸化肽的水解酪蛋白在制备调节肠道菌群的功能产品中的应用。
7.根据权利要求6所述的应用，其特征在于，所述产品为特殊医学食品、保健品、药品、功能性食品。
8.根据权利要求6所述的应用，其特征在于，所述富含唾液酸化肽的水解酪蛋白能够增加产短链脂肪酸(SCFAs)的细菌的丰度。
9.根据权利要求7所述的应用，其特征在于，所述肠道菌群为Bacteroides uniformis、Bifidobacterium longum、Ruminococcus albus、Faecalibacteriumprausnitzii、Aminipila butyrica、Bacteroides uniformis、Intestinibacillus massiliensis、Agathobaculum desmolans、Blautiafaecis、Faecalibacteriumprausnitzii、Blautiaobeum、Eubacterium rectale、Bifidobacterium bifidum、Faecalibacteriumprausnitzii、Streptococcusparasanguinis。
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