CN112625305B - 一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和应用 - Google Patents
一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和应用。该党参果聚糖复合物主要由一种呋喃型果聚糖和一种吡喃型果聚糖组成。呋喃果聚糖为β‑D‑呋喃果糖基‑（2→1）‑连接而成的果聚糖，吡喃果聚糖为β‑D‑吡喃果糖基‑（2→1）‑连接而成的果聚糖，其分子量分别为2000~2500Da和1000~1500Da。该党参果聚糖复合物制备方法包括如下步骤：（1）党参总多糖的提取；（2）党参总多糖提取液乙醇醇沉；（3）党参多糖的分离。该党参果聚糖复合物在药品、保健品、功能食品及饲料添加剂方面的应用。
Description
技术领域
本发明涉及中药多糖成分的制备方法技术领域，具体为一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和其在药品、保健品、功能食品、饲料添加剂方面的应用。
背景技术
党参（Radix Codonopsis）为桔梗科党参属植物党参、素花党参及川党参的干燥根。党参具有补中益气、健脾益肺的功效，临床用于脾肺虚弱、气短心悸、食少便溏、虚喘咳嗽等症。党参主要含有多糖、三萜、炔苷、生物碱、苯丙素等成分，其中多糖含量最高，为党参重要的药理活性成分。戊糖片球菌（Pediococcus pentosaceus）是一种重要的益生乳酸菌，能够产生有益代谢物，进而具有增强机体免疫力，预防和治疗疾病作用。目前有关党参果聚糖复合物的促戊糖片球菌生长几乎没有相关报道。
发明内容
本发明主要是通过对党参总多糖的分离，然后进行促戊糖片球菌生长筛选，得到一种具有促戊糖片球菌生长的党参果聚糖复合物。
本发明是采用如下技术方案实现的：
一种具有促戊糖片球菌生长的党参果聚糖复合物，包括呋喃型果聚糖A和吡喃型果聚糖B。
所述呋喃型果聚糖A的结构通式如下：
所述吡喃型果聚糖B的结构通式如下：
上述党参果聚糖复合物主要由一种呋喃型果聚糖A和一种吡喃型果聚糖B组成。呋喃型果聚糖A为β-D-呋喃果糖基-（2→1）-连接而成的果聚糖，吡喃型果聚糖B为β-D-吡喃果糖基-（2→1）-连接而成的果聚糖。呋喃型果聚糖A的分子量为2000~2500Da；吡喃型果聚糖B的分子量为1000~1500Da。党参果聚糖复合物中，呋喃型果聚糖A和吡喃型果聚糖B的质量比为10:1~1:10。
上述具有促戊糖片球菌生长的党参果聚糖复合物的制备方法，包括如下步骤：
（1）、党参多糖的提取
党参药材按料液比1:10~1:20浸泡后加热回流提取；
（2）、党参多糖的醇沉
将党参多糖提取液经减压浓缩至1/4~1/3体积，之后采用60%~90%乙醇沉淀，然后1000~1500转/分钟离心15~30分钟，去上清，沉淀用乙醚、乙酸乙酯、丙酮、无水乙醇依次洗涤3次，冷冻干燥，得到党参多糖样品；
（3）、党参多糖的分离
将党参多糖用水溶解后过50000分子量超滤膜，滤过液采用Sephadex G-100柱色谱制备，采用HPGPC分析洗脱液色谱特征，收集33.550分钟和35.614分钟两个主要色谱峰的目标洗脱液，得到党参果聚糖复合物。
上述党参果聚糖复合物的结构鉴定如下：
对本发明获得的党参果聚糖复合物采用高效凝胶渗透色谱法进行分子量测定：
色谱条件：安捷伦1260 HPLC系统-RID检测器，分析柱为TSKgel G4000PWXL色谱柱（7.8mm×30cm，10μm），流动相为超纯水，流速0.3mL/min，示差检测器温度35℃，柱温35℃，进样量20μL。
对本发明获得的党参果聚糖复合物进行多糖组成和结构分析：以1H-NMR、13C-NMR及2D-NMR分析。
上述分析证明：党参果聚糖复合物主要由一种呋喃型果聚糖和一种吡喃型果聚糖组成。呋喃果聚糖为β-D-呋喃果糖基-（2→1）-连接而成的果聚糖，吡喃果聚糖为β-D-吡喃果糖基-（2→1）-连接而成的果聚糖，其分子量分别为2000~2500Da和1000~1500Da。
本发明获得的党参果聚糖复合物对体外粪菌培养中戊糖片球菌生长具有显著促进作用，实验具体步骤如下：
（1）、粪菌体外培养：将党参果聚糖复合物按照2%的比例与1%的MRS琼脂高压灭菌后制成平板，将粪便（取自一名年轻健康女性）按0.1g/mL PBS稀释，纱布过滤，将滤液平铺于上述平板上厌氧培养，待生长出菌落后，将菌落再次接种于该平板，三次接种培养后接入乳酸细菌培养基扩大培养；
（2）、优势细菌培养：再次接种于党参复合物为唯一碳源的液体内进行培养，检测其生长；
（3）、16SrRNA序列分析：从步骤（2）中培养获得的细菌进行16SrRNA序列分析，鉴定菌种类型。
本发明获得的党参果聚糖复合物能够显著促进戊糖片球菌生长，则具有在药品、保健品、功能食品及饲料添加剂中的应用。
附图说明
图1表示党参果聚糖复合物凝胶渗透色谱图。
图2A表示党参果聚糖复合物酸水解产物HPLC图谱。
图2B表示党参果聚糖复合物标准D-果糖HPLC图谱。
图2C表示党参果聚糖复合物标准D-葡萄糖HPLC图谱。
图3A表示党参果聚糖复合物1H-NMR谱图。
图3B表示党参果聚糖复合物13C-NMR谱图。
图3C表示党参果聚糖复合物DEPT135谱图。
图4表示党参果聚糖复合物H-H COSY图谱。
图5表示党参果聚糖复合物HSQC图谱。
图6表示党参果聚糖复合物HMBC图谱。
图7表示戊糖片球菌在党参果聚糖复合物中的生长曲线图。
具体实施方式
下面对本发明的具体实施例进行详细说明。
一、具有促戊糖片球菌生长的党参果聚糖复合物的制备方法，如下：
（1）、党参多糖提取
党参药材按料液比1:10~1:20浸泡30分钟，加热回流提取3次，每次1.5小时，合并三次提取液。
（2）党参多糖的制备
党参多糖提取液经减压浓缩至1/4~1/3体积、60%~80%乙醇沉淀、1000转/分钟离心15分钟，去上清，沉淀用乙醚、乙酸乙酯、丙酮、无水乙醇依次洗涤3次，冷冻干燥，得到党参多糖样品。
（3）、党参多糖的分离
将党参多糖用水溶解后过50000分子量超滤膜，滤过液采用Sephadex G-100柱色谱制备，采用HPGPC分析洗脱液色谱特征，收集目标洗脱液得到党参果聚糖复合物。
二、党参多糖HPGPC分析
色谱条件为安捷伦1260 HPLC系统-RID检测器，分析柱为TSKgel G4000PWXL色谱柱（7.8mm×30cm，10μm），流动相为超纯水，流速0.3mL/min，示差检测器温度30℃，柱温30℃，进样量20μL，获得HPGPC图谱。谱图呈现如下特点：在 33.55min处有一个明显色谱峰（呋喃型果聚糖）和35.614min处有一色谱峰（吡喃型果聚糖），其所对应多糖分子量大小分别为2000~2500Da和1000~1500Da。
图1的结果表明党参果聚糖复合物主要由33.550分钟和35.614分钟两个主要色谱峰，根据HPGPC分子量和时间（分钟）线性曲线可知33.550分钟和35.614分钟两个主要色谱峰代表的多糖分子量大小分别为2000~2500Da和1000~1500Da。
图2A、2B、2C的结果表明党参果聚糖复合物酸水解产物主要为D-果糖和D-葡萄糖，说明组成党参果聚糖复合物多糖的单糖为D-果糖和D-葡萄糖。
二、党参多糖NMR分析
党参多糖样品（50mg）溶解于1mL氘代水（D2O）中，400MHz核磁共振仪进行1H-NMR、13C-NMR、13C-DEPT135及2D-NMR分析，获得分析该果聚糖复合物NMR谱。
图3A、3B、3C的结果表明党参果聚糖复合物的糖信号主要为果糖信号和微弱的葡萄糖信号，与酸水解结果一致。根据碳信号强弱可知33.550分钟和35.614分钟两个主要色谱峰代表的多糖分别为β-D-呋喃果糖基-（2→1）-连接而成的果聚糖和β-D-吡喃果糖基-（2→1）-连接而成的果聚糖。
图4结果能够清晰归属β-D-呋喃果糖基3位、4位、5位的氢信号和β-D-吡喃果糖基3位、4位、5位的氢信号。
图5结果能够清晰归属β-D-呋喃果糖基1位、3位、4位、5位、6位的氢信号和碳信号以及β-D-吡喃果糖基1位、3位、4位、5位、6位的氢信号和碳信号。
图6结果能够清晰确认两种果糖基的-（2→1）-连接方式。
表1 党参果聚糖复合物H和C核磁数据归属。
三、获得的党参果聚糖复合物对体外粪菌培养中戊糖片球菌生长促进作用。
（1）、粪菌体外培养：将党参果聚糖复合物按照2%的比例与1%的MRS琼脂高压灭菌后制成平板，将粪便（取自一名年轻健康女性）按0.1g/mL PBS稀释，纱布过滤，将滤液平铺于上述平板上厌氧培养，待生长出菌落后，将菌落再次接种于该平板，三次接种培养后接入乳酸细菌培养基扩大培养；
（2）、优势细菌培养：再次接种于党参复合物为唯一碳源的液体内进行培养，检测其生长；
（3）、16SrRNA序列分析：对从步骤（2）中培养获得的细菌进行16SrRNA序列分析，鉴定菌种类型。
根据党参果聚糖复合物为唯一碳源所促生长的戊糖片球菌16SrRNA测序结果，通过与数据库比对确认为戊糖片球菌。
图7结果表明，党参果聚糖复合物在48小时内能够显著促进戊糖片球菌生长。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照本发明实施例进行了详细说明，本领域的普通技术人员应当理解，对本发明的技术方案进行修改或者等同替换，都不脱离本发明的技术方案的精神和范围，其均应涵盖权利要求保护范围中。
<110>山西医科大学
<120>一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和应用
<160>1
<210>1
<211>1479
<212>RNA
<213>戊糖片球菌（Pediococcus pentosaceus）
<220>
<223>益生乳酸菌
<400>1
GGCGTGCGGG TGCTATACAT GCAAGTCGAA CGAACTTCCG TTAATTGATT ATGACGTACT 60
TGTACTGATT GAGATTTTAA CACGAAGTGA GTGGCGAACG GGTGAGTAAC ACGTGGGTAA 120
CCTGCCCAGA AGTAGGGGAT AACACCTGGA AACAGATGCT AATACCGTAT AACAGAGAAA 180
ACCGCATGGT TTTCTTTTAA AAGATGGCTC TGCTATCACT TCTGGATGGA CCCGCGGCGT 240
ATTAGCTAGT TGGTGAGGTA AAGGCTCACC AAGGCAGTGA TACGTAGCCG ACCTGAGAGG 300
GTAATCGGCC ACATTGGGAC TGAGACACGG CCCAGACTCC TACGGGAGGC AGCAGTAGGG 360
AATCTTCCAC AATGGACGCA AGTCTGATGG AGCAACGCCG CGTGAGTGAA GAAGGGTTTC 420
GGCTCGTAAA GCTCTGTTGT TAAAGAAGAA CGTGGGTAAG AGTAACTGTT TACCCAGTGA 480
CGGTATTTAA CCAGAAAGCC ACGGCTAACT ACGTGCCAGC AGCCGCGGTA ATACGTAGGT 540
GGCAAGCGTT ATCCGGATTT ATTGGGCGTA AAGCGAGCGC AGGCGGTCTT TTAAGTCTAA 600
TGTGAAAGCC TTCGGCTCAA CCGAAGAAGT GCATTGGAAA CTGGGAGACT TGAGTGCAGA 660
AGAGGACAGT GGAACTCCAT GTGTAGCGGT GAAATGCGTA GATATATGGA AGAACACCAG 720
TGGCGAAGGC GGCTGTCTGG TCTGCAACTG ACGCTGAGGC TCGAAAGCAT GGGTAGCGAA 780
CAGGATTAGA TACCCTGGTA GTCCATGCCG TAAACGATGA TTACTAAGTG TTGGAGGGTT 840
TCCGCCCTTC AGTGCTGCAG CTAACGCATT AAGTAATCCG CCTGGGGAGT ACGACCGCAA 900
GGTTGAAACT CAAAAGAATT GACGGGGGCC CGCACAAGCG GTGGAGCATG TGGTTTAATT 960
CGAAGCTACG CGAAGAACCT TACCAGGTCT TGACATCTTC TGACAGTCTA AGAGATTAGA 1020
GGTTCCCTTC GGGGACAGAA TGACAGGTGG TGCATGGTTG TCGTCAGCTC GTGTCGTGAG 1080
ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCTTATTAC TAGTTGCCAG CATTAAGTTG 1140
GGCACTCTAG TGAGACTGCC GGTGACAAAC CGGAGGAAGG TGGGGACGAC GTCAAATCAT 1200
CATGCCCCTT ATGACCTGGG CTACACACGT GCTACAATGG ATGGTACAAC GAGTCGCGAG 1260
ACCGCGAGGT TAAGCTAATC TCTTAAAACC ATTCTCAGTT CGGACTGTAG GCTGCAACTC 1320
GCCTACACGA AGTCGGAATC GCTAGTAATC GCGGATCAGC ATGCCGCGGT GAATACGTTC 1380
CCGGGCCTTG TACACACCGC CCGTCACACC ATGAGAGTTT GTAACACCCA AAGCCGGTGG 1440
GGTAACCTTT TAGGAGCTAG CCGTCTAAGT GACAGATTG 1479
Claims (4)
1.一种具有促戊糖片球菌生长的党参果聚糖复合物，其特征在于：包括呋喃型果聚糖A和吡喃型果聚糖B；
所述呋喃型果聚糖A的结构通式如下：
所述吡喃型果聚糖B的结构通式如下：
所述呋喃型果聚糖A的分子量为2000~2500Da；所述吡喃型果聚糖B的分子量为1000~1500Da；
呋喃型果聚糖A和吡喃型果聚糖B的质量比为10:1~1:10。
2.一种权利要求1所述的具有促戊糖片球菌生长的党参果聚糖复合物的制备方法，其特征在于：包括如下步骤：
（1）、党参多糖的提取
党参药材按料液比1:10~1:20浸泡后加热回流提取；
（2）、党参多糖的醇沉
将党参多糖提取液经减压浓缩至1/4~1/3体积，之后采用60%~90%乙醇沉淀，然后1000~1500转/分钟离心15~30分钟，去上清，沉淀用乙醚、乙酸乙酯、丙酮、无水乙醇依次洗涤3次，冷冻干燥，得到党参多糖样品；
（3）、党参多糖的分离
将党参多糖用水溶解后过50000分子量超滤膜，滤过液采用Sephadex G-100柱色谱制备，采用HPGPC分析洗脱液色谱特征，收集33.550分钟和35.614分钟两个主要色谱峰的目标洗脱液，得到党参果聚糖复合物。
3.一种权利要求1所述的党参果聚糖复合物在培养促戊糖片球菌生长方面的应用。
4.一种权利要求1所述的党参果聚糖复合物在制备保健功能食品及饲料添加剂方面的应用。
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