CN111808166B - 一种多级分离纯化工艺制备的黄酒多肽及其应用 - Google Patents
一种多级分离纯化工艺制备的黄酒多肽及其应用 Download PDFInfo
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Abstract
本发明涉及一种多级分离纯化工艺制备的黄酒多肽及其应用,属于生物技术领域。本发明提供了一种将多肽从复杂体系黄酒中多级分离纯化的工艺方法,包括黄酒的预处理除杂、超滤、大孔树脂吸附和葡聚糖凝胶色谱层析。同时根据此工艺方法分离纯化出一种黄酒多肽并进行氨基酸结构鉴定,获得一种黄酒多肽为Leu‑Phe‑Trp(LFW)。同时提供了一种评价多肽免疫调节功能活性的应用方法,在RAW264.7细胞模型中,在LPS刺激下经黄酒多肽组分LFW的预处理明显抑制了促炎因子TNF‑α、IL‑6和IL‑1β的表达,具有免疫调节功能,具备极高的应用前景。
Description
技术领域
本发明涉及一种多级分离纯化工艺制备的黄酒多肽及其应用,属于食品技术领域
背景技术
黄酒的酿造历史悠久,与啤酒、葡萄酒并称世界三大古酒。黄酒中蛋白质、多肽和游离氨基酸的含量丰富,平均氨基酸含量为13.5g/L,含量是啤酒的7.55倍,是白酒的2.09倍。黄酒多肽主要来源于原料分解与微生物代谢积累,酿造黄酒的原料如糯米等富含丰富的蛋白质,微生物体内含有各种代谢酶,因此在发酵过程中可以产生多种不同功能的活性肽。
随着生活水平的提高,人们在饮食和饮酒方面越来越注重健康性,低度、营养、相对健康的黄酒对酒类爱好者的吸引逐渐增加,促进了黄酒行业的迅猛发展。黄酒自古以来具有养生酒的功效,但是黄酒摄入对人体健康的具体影响,如抗氧化、降血脂等功能仍有待研究。并且黄酒中发挥有益作用的功效成分还有待挖掘。黄酒多肽源于食品原料和微生物发酵,天然功效较强,毒副作用较小,具有比药物更高的安全性。因此在大健康背景下,分离纯化出黄酒多肽并评价其功能活性十分必要,具有较高的经济和研究价值。
但是,黄酒成品酒体系复杂,成分多样,分离纯化活性多肽具有难度。同时黄酒多肽的功能活性评价较为欠缺,无法明确黄酒的功能性效果。
发明内容
为解决黄酒体系复杂活性物质难以分离纯化的问题,本发明提供了一种分离纯化和制备高纯度黄酒多肽的工艺,并应用体外细胞实验评价其功能活性。
本发明提供了一种黄酒多肽,所述黄酒多肽的氨基酸序列为亮氨酸-苯丙氨酸-色氨酸。
本发明提供了所述多肽在制备清除自由基、辅助抗癌、辅助抗氧化和/或辅助抗衰老的产品中的应用,利用所述的黄酒多肽清除自由基。
在本发明的一种实施方式中,所述自由基包括超氧阴离子自由基、羟自由基、羧自由基、脂氧自由基、一氧化氮自由基、硝基自由基或超氧化氢自由基中的至少一种。
在本发明的一种实施方式中,所述产品包括功能性食品、功能性饮品和/或药物组合物。
在本发明的一种实施方式中,所述多肽在所述产品的浓度为200~1800μg/mL或200~1800μg/mg。
本发明提供了所述多肽在制备降低促炎症因子的产品中的应用,利用所述的黄酒多肽降低促炎因子。
在本发明的一种实施方式中,所述促炎因子包括TNF-α、TNF-β、IL-6、IL-1β,激肽、组织胺、IL-2或前列腺素中的至少一种。
在本发明的一种实施方式中,所述多肽在产品中的浓度为50~200μg/mL或50~200μg/mg。
本发明提供了一种制备所述多肽的方法,所述方法包括:
(1)黄酒预处理:将黄酒过膜抽滤,得到抽滤液;
(2)将抽滤液经超滤系统分离,取分子量不大于3kDa的组分;
(3)将分子量不大于3kDa的组分通过大孔树脂吸附,得到粗多肽;
(4)将粗多肽经凝胶色谱层析,收集洗脱液。
在本发明的一种实施方式中,步骤(3)中的大孔树脂为DA201-C极性大孔吸附树脂;洗脱液为50%~70%的乙醇。
本发明的有益效果:
本发明提供了一种评价黄酒多肽免疫调节功能活性的应用方法。由于巨噬细胞在急性免疫应答中起关键作用,以LPS为刺激物建立体外炎症模型,通过对LPS诱导的RAW264.7细胞释放细胞因子的水平来评价免疫调节活性的强弱。在人体的疾病发生和发展过程中,当组织或免疫系统发生炎症时,免疫相关细胞会产生大量信号分子使免疫系统做出相应的反应,这些信号分子包括各种促炎因子,如TNF-α、IL-6和IL-1β等。本发明中,经黄酒多肽LFW样品预处理4h后再由LPS刺激,随着样品浓度由50μg/mL提高至200μg/mL,相关促炎因子TNF-α、IL-6和IL-1β的表达量明显得到抑制,表明分离纯化得到的黄酒多肽LFW具有明显的调节免疫因子的作用。因此,本发明所分离纯化得到的黄酒多肽组分具有免疫调节功能,具备极高的应用前景。
附图说明
图1为黄酒多肽LFW的质谱分析结果图。
图2为黄酒粗多肽C和黄酒多肽LFW的抗氧化活性评价图。
图3为黄酒多肽LFW对细胞存活率的影响图。
图4为黄酒多肽LFW对细胞免疫促炎因子的调节作用图。
具体实施方式
下式实施例中涉及的黄酒购自浙江古越龙山绍兴酒股份有限公司,浙江古越龙山绍兴酒股份有限公司的黄酒酒精度均为15~18%vol,半干型黄酒。
小鼠巨噬细胞RAW264.7细胞系购于中国科学院上海细胞库。
实施例1:黄酒多肽LFW的分离纯化应用和氨基酸结构鉴定
(1)黄酒的预处理除杂:采用循环水式真空泵抽滤黄酒通过0.22μm的微孔滤膜,可去除黄酒中分子量较大的固形物。所用的超滤组件需经过0.1mol/L NaOH溶液洗涤处理,并用蒸馏水洗至流出液为中性方可使用。
(2)超滤分离:通过超滤系统分级超滤酒样,得到小于3kDa的组分,取小于3kDa的组分进一步分离纯化。
(3)大孔树脂吸附:对大孔树脂进行预处理,使用95%乙醇浸泡DA201-C(比表面积为550-600m2/g,平均孔径为100-110A°,粒径范围为0.3~1.26mm,含水量为65-76%)大孔树脂24h后,使用蒸馏水清洗大孔树脂,直至流出液无明显乙醇气味并清澈透明。分别用1mol/L的HCl和4%的NaOH浸泡24h,以去除树脂在合成过程中的杂质,树脂洗至中性后滤去多余水分备用;取10g(干重)处理后的树脂置于锥形瓶中,加入步骤(2)得到的小于3kDa的组分100mL,于25℃、150r/min条件下吸附24h,收集吸附黄酒多肽后的DA201-C大孔树脂,抽滤去除滤液,用适量蒸馏水冲洗滤干后加入60%乙醇溶液,抽滤后测定多肽含量。随后进行洗脱,洗脱液经旋蒸去除乙醇后冻干,得到黄酒粗多肽组分,即为C组分。
(4)凝胶色谱层析:使用Sephadex G-15葡聚糖凝胶预处理及装柱,装柱后用蒸馏水平衡2个柱体积至检测器基线平稳。用蒸馏水将黄酒粗多肽组分配制成浓度为16mg/mL的溶液,经0.22μm微孔膜过滤后经过凝胶过滤分离层析系统,以蒸馏水(pH 7.0)为流动相,流速为3mL/min,收集洗脱液,合并同一分离峰的洗脱液进行冷冻干燥后得黄酒多肽分离纯化组分,经氨基酸结构鉴定(氨基酸结构鉴定使用电喷雾离子阱质谱仪将多肽进行还原烷基化后经反相色谱分离,鉴定出相应的氨基酸序列),其氨基酸序列为亮氨酸-苯丙氨酸-色氨酸(Leu-Phe-Trp,LFW),质谱图如图1所示。其疏水氨基酸占比为2/3,分子量为464.24Da。
实施例2:黄酒粗多肽C和黄酒多肽LFW的抗氧化活性评价
应用体外DPPH和ABTS自由基清除实验评价两种黄酒多肽的抗氧化活性。
其中黄酒多肽和抗坏血酸(Vc)的配制:取冷冻干燥后样品,以去离子水溶解后以2mg/mL进行浓度梯度稀释;准确称量20mg抗坏血酸溶于10mL去离子水中,按照浓度梯度2.0mg/mL、1.5mg/mL、1.0mg/mL、0.8mg/mL、0.5mg/mL、0.2mg/mL制备样液。
(1)DPPH自由基清除实验
准确称取DPPH试剂5.52mg溶解于无水乙醇中,并定量转入100mL容量瓶中,用无水乙醇定容至100mL棕色容量瓶,得到浓度为0.14mM DPPH贮备液,置于冰箱中冷藏备用。检测方法为,分别取2Ml黄酒多肽样品加入试管,加入2mL DPPH溶液,室温完全避光保持30min,混匀后使用1cm比色皿在OD 517nm下检测吸光值,吸光值记为A1。空白组(A0)为2mL 0.5%DMSO溶液+2mL DPPH溶液,吸光值记为A0。对照组(A2)为2mL样品+2mL无水乙醇溶液,其吸光值记为A2。
自由基清除率(K):K(%)=[1-(A1-A2)/A0]*100%。
(2)ABTS自由基清除实验
将ABTS溶于甲醇配成14mM ABTS溶液将过硫酸钾溶于蒸馏水配成4.9mM过硫酸钾溶液。等体积混合14mM ABTS溶液和过硫酸钾配成ABTS母液,将母液在室温下避光静置12-16小时,使用前用蒸馏水稀释母液以制备为ABTS工作液,该工作液在734nm处的吸光度为0.7±0.02。测定方法为,称取0.15mL黄酒多肽样品加入试管,加入2.85mL ABTS工作液,室温黑暗保持10min,混匀后使用1cm比色皿在OD 734nm下检测吸光值,吸光值记为A1。空白组(A0)为0.15mL 0.5%DMSO溶液+2.85mL ABTS工作液,吸光值记为A0。对照组(A2)为2mL样品+2mL 0.5%DMSO溶液,其吸光值记为A2同上计算自由基清除率。
如图2所示,随着样品在体系中的浓度由200mg/L提高至1800mg/L,两种多肽对疏水性自由基DPPH均增加,其中黄酒多肽LFW的DPPH清除率由55.7±0.9%提高至93.1±1.6%,与阳性对照的清除率趋近。对亲水性自由基ABTS的清除中,与DPPH清除率的趋势相似,其中多肽LFW效果优于粗多肽C,在黄酒多肽LFW浓度为1800mg/L时清除率达到88.1±2.2%。因此,通过评价两种黄酒多肽的自由基清除能力,表明同浓度下黄酒多肽LFW具有更好的抗氧化活性。
表1 不同样品DPPH自由基清除率(%)
表2 不同样品ABTS自由基清除率(%)
实施例3:黄酒多肽在调节免疫因子中的应用
在实施例2的基础上,进一步对分离纯化的黄酒多肽LFW组分使用LPS刺激巨噬细胞RAW264.7的细胞存活率和免疫因子评价其功能性。
利用MTT法即3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法检测黄酒多肽对LPS刺激细胞24h后的细胞存活率。使用小鼠巨噬细胞RAW264.7培养于含10%胎牛血清(v/v)、1%(v/v)双抗(青霉素104μg/mL,链霉素104μg/mL)的DMEM高糖细胞培养基,于T25细胞培养瓶中接种,放置于温度为37℃、CO2浓度为5%的恒温培养箱中培养,取对数生长期细胞用于实验。细胞培养好后,将方瓶中的贴壁细胞经细胞刮刮下并吹散后,利用台盼蓝染料在血球计数板上对细胞密度计数,并调整细胞密度为1×104个/mL在96孔板中铺入细胞。过夜培养细胞贴壁后,除去上清液并加入含有不同浓度黄酒多肽LFW(50,100,200,400μg/mL)的RPMI-1640培养基,培养24h后利用MTT法对细胞存活率进行检测。MTT法检测中,将10μL MTT(5mg/mL)加入96孔板中,在37℃培养4h后去除上清液,加入150μL DMSO,培养30min后在490nm下检测样品读数,细胞存活率依据未加样品的对照组计算。
黄酒多肽对细胞免疫因子的调节作用测定。将培养好的细胞以1.0×104个/mL密度铺板于96孔板中并培养24h,使用不同浓度黄酒多肽LWF(在反应体系中的浓度分别为50,100,200μg/mL)对细胞进行预处理,4h后除去上清液并加入含有0.1μg/mL脂多糖LPS的细胞培养基培养24h,设置未加多肽样品和LPS的对照组,以及LPS阳性组。取细胞上清液用于促炎因子TNF-α、IL-6和IL-1β的浓度。使用Sigma公司的ELISA试剂盒根据说明书进行操作,利用酶标仪检测并根据标品绘制标准曲线,最终计算相关促炎因子的浓度。
应用上述方法进行细胞免疫因子调节评价,如图3所示,黄酒多肽LFW在浓度为50-200μg/mL范围内的细胞存活率均不具有显著差异,因此黄酒多肽LFW的细胞毒性非常低。免疫因子调节方面(图4),使用LPS刺激巨噬细胞后,促炎因子TNF-α、IL-6和IL-1β均显著增加(P<0.0001),而黄酒多肽LFW可抑制促炎因子含量,且随黄酒多肽组分浓度增加而抑制力增强。当黄酒多肽浓度为200μg/mL,可显著降低LPS导致IL-1β升高到正常水平(P<0.0001)。说明该黄酒多肽组分能有效调节由LPS诱导的小鼠巨噬细胞中促炎因子含量。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (6)
1.氨基酸序列为亮氨酸-苯丙氨酸-色氨酸的黄酒多肽在制备清除自由基、辅助抗氧化和/或辅助抗衰老的产品中的应用;所述产品为药物组合物。
2.根据权利要求1所述的应用,其特征在于,所述自由基包括超氧阴离子自由基、羟自由基、羧自由基、脂氧自由基、一氧化氮自由基、硝基自由基或超氧化氢自由基中的至少一种。
3.根据权利要求1所述的应用,其特征在于,所述多肽在所述产品中的浓度为200~1800μg/mL或200~1800 μg/mg。
4.氨基酸序列为亮氨酸-苯丙氨酸-色氨酸的黄酒多肽在制备降低促炎症因子含量、或减轻炎症的产品中的应用;所述产品为药物组合物。
5.根据权利要求4所述的应用,其特征在于,所述促炎因子包括TNF-α、IL-6、IL-1β中的至少一种。
6.根据权利要求4所述的应用,其特征在于,所述多肽在产品中的浓度为50~200 μg/mL或50~200 μg/mg。
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