CN116120477A - 一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖及其制备方法与应用 - Google Patents
一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖及其制备方法与应用。所述胞外多糖EPS0‑1是由半乳糖胺、鼠李糖、葡糖胺、半乳糖、葡萄糖、甘露糖、半乳糖酸和葡萄糖酸按1.4:9.4:4.9:32:37:6.5:4.1:4.4的摩尔比组成;所述胞外多糖EPS0‑1重均分子量为4350~4360kDa。本发明还提供了该胞外多糖EPS0‑1的制备方法。本发明提供的胞外多糖EPS0‑1具有抗低温的性质,能够明显降低溶液冰点,提高冻融循环中微生物的存活率,可以应用在菌种冷冻保藏和组织器官保藏运输中。并且该胞外多糖EPS0‑1还具有吸湿保湿性能,同时在250~750μg/mL的剂量范围内,对人正常肝细胞LO2无细胞毒性,表明其安全无毒副作用,可以用于制备冷冻食品、化妆品、药物组合物和保健品。
Description
技术领域
本发明涉及一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖及其制备方法与应用,属于医药及保健食品技术领域。
背景技术
独特的地理位置和陆海分布使得南极大陆的海洋和大陆环境具有丰富的多样性。低温、高盐、强紫外、干旱等不利环境的选择压力,使南极微生物已经进化出多种策略来抵抗这些恶劣条件,如以胞外多糖(Exopolysaccharides,EPS)为主成分的大分子胞外聚合物。嗜冷菌EPS具有提高微生物存活率、减少冰晶伤害、缓冲温度、pH或盐浓度变化、进行碳与能源传输等功能,保护与帮助菌体的聚集与粘附,有助于生物膜的形成与吸收营养等作用。
狭义的嗜冷菌是指在0~20℃范围内可以正常生长繁殖,最适生长温度小于或等于15℃的微生物。由于嗜冷菌所处低温环境,其释放的大量的EPS不仅保持细胞附着在表面,也在寒冷的环境中起到低温保护作用,还避免物理性细胞损伤,减缓代谢过程,为嗜冷菌创造了良好的生长环境。EPS有以上的功能归因于其结构和大分子缔合,防止水分子的四面体排列并避免其重结晶或冰晶成核,高分子量的EPS有保水的能力,更有利于非极性残基之间的疏水相互作用及增强粘附作用。EPS链上存在带负电荷的残基,如硫酸盐和羧基,使它们能够形成水化粘性三维网络,从而赋予其抗冰冻的粘合性和阻隔性。此外,一些嗜冷细菌分泌的EPS能够清除超氧阴离子,即具有的抗氧化活性,可以作为细胞低温保护剂。
适冷菌产的大分子多糖具有很好的抗低温功能、较大的扩展性能,可以广泛应用于食品、化妆及药品等领域。因此需要对南极嗜冷菌胞外多糖做进一步的研究。
发明内容
针对现有技术的不足,本发明的目的在于提供一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖及其制备方法与应用。
本发明的技术方案如下:
一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1,所述胞外多糖EPS0-1是由半乳糖胺(GalN)、鼠李糖(Rha)、葡糖胺(GlcN)、半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、半乳糖酸(GalA)和葡萄糖酸(GlcA)组成的α-吡喃杂多糖,其摩尔比具体为GalN:Rha:GlcN:Gal、Glc:Man:GalA:GlcA=1.4:9.4:4.9:32:37:6.5:4.1:4.4;所述胞外多糖EPS0-1重均分子量为4350~4360kDa。
根据本发明优选的,所述胞外多糖EPS0-1含有三螺旋结构、侧链和分支。
上述具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的制备方法,包括步骤如下:
(1)种子液的制备:将假交替单胞菌LP6-12-2按1.0~1.5%的接种量接种至2216E基础培养基中,在8~15℃、150~180r/min下培养30~45h,得到种子液;
(2)发酵液的制备:将步骤(1)所得的种子液以1.0~1.5%的体积接种于2216E改良培养基中,在8~15℃、150~180r/min下培养60~70h,收集发酵液,在8000~9000r/min下离心15~20min,去除菌体,获得发酵液上清;
(3)多糖沉淀:将步骤(2)所得的发酵液上清在50~60℃下减压浓缩至原体积的20~25%后,冷却至室温,加入浓缩液3~4倍体积的95%乙醇,4℃下静置12h以上,7500~8500r/min离心15~20min,获得沉淀;
(4)脱除蛋白:向步骤(3)所得的沉淀中加入去离子水,搅拌至沉淀溶解,在11500~13000r/min高速离心10~15min,去除沉淀收集上清液,向上清液中加入Sevag试剂,充分混匀后剧烈震荡后,静置分层后回收有机试剂除去蛋白质,重复操作5~8次,在55~65℃下减压浓缩,除去有机试剂,得到胞外多糖溶液;
(5)透析冻干:将步骤(4)所得的胞外多糖溶液装入透析袋中进行透析,每隔8h换去离子水,透析48h,然后经过真空冷冻干燥,得到胞外粗多糖;
(6)粗品分离:将步骤(5)所得的胞外粗多糖溶解于Tris-HCl缓冲液中,配制成浓度为8~10mg/mL的多糖溶液,然后通过DEAE-Sepharose Fast Flow阴离子交换层析分离,使用Tris-HCl缓冲液洗脱,流速为0.5mL/min,每10min收集一管,收集含糖的洗脱液,真空冷冻干燥后,得到胞外多糖EPS-0;
(7)纯化:将步骤(6)所得的胞外多糖EPS-0溶解于去离子水中,,配制成浓度为4~6mg/ml的多糖溶液,透析后浓缩冻干,获得具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1。
根据本发明优选的,步骤(1)中,所述假交替单胞菌LP6-12-2于2021年12月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.24106。
根据本发明优选的,步骤(1)中,所述2216E基础培养基的组成如下:蛋白胨0.1~1%(w/v),酵母粉0.05~0.2%(w/v),人工海水盐0.2~4%(w/v)。
根据本发明优选的,步骤(2)中,所述2216E改良培养基的组成如下:蛋白胨0.1~1%(w/v),酵母粉0.05~0.2%(w/v),人工海水盐0.2~4%(w/v),葡萄糖0.5~2%(w/v),蔗糖0.5~2%(w/v)。
根据本发明优选的,步骤(4)中,所述Sevage试剂为氯仿:正丁醇=4:1~5:1的混合溶液,加入量为上清液体积的18~22%。
本发明还提供上述具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1在制备冷冻食品、化妆品、药物组合物和保健品中的应用。
本发明还提供上述具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1在菌种冷冻保藏和组织器官保藏运输中的应用。
本发明的有益效果在于:
1、本发明提供了一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1,该胞外多糖EPS0-1具有抗低温功能,能够明显降低溶液冰点,提高冻融循环中微生物的存活率,可以应用在菌种冷冻保藏和组织器官保藏运输中。并且该胞外多糖EPS0-1还具有吸湿保湿功能,同时在250~750μg/mL的剂量范围内,对人正常肝细胞LO2无细胞毒性,表明其安全无毒副作用,可以用于制备冷冻食品、化妆品、药物组合物和保健品。
2、本发明提供了胞外多糖EPS0-1的发酵生产制备方法,对胞外多糖EPS0-1结构进行了鉴定,从而为实验室研究以及工业生产提供依据,弥补了国内南极细菌胞外多糖研究的匮乏。并且本发明提供的制备方法过程简单,易于操作,便于大规模生产,产量能够达到153.9mg/L。
附图说明
图1为抗低温的南极嗜冷菌胞外多糖EPS0-1的性质和均一度检测结果图;
图中:A为EPS-0的碘显色反应结果图;B为EPS0-1紫外扫描图谱;C为EPS0-1高效凝胶渗透色谱图。
图2为胞外多糖EPS0-1的红外光谱图。
图3为胞外多糖EPS0-1的单糖组成分析图。
图中:A为16种单糖标准品离子色谱图;B为EPS0-1离子色谱图;
图中,峰1-16依次代表单糖D-Fuc、D-GalN、D-Rha、D-Ara、D-GlcN、D-Gal、D-Glc、D-GlcNAc、D-Xyl、D-Man、D-Fru、D-Rib、D-GalA、L-GulA、D-GlcA和D-ManA。
图4为EPS0-1高级结构分析图。
图中:A为多糖三螺旋结构检测,刚果红-EPS0-1复合物的λmax随NaOH浓度的变化图;B为多糖的分支情况检测,碘试剂-EPS0-1的紫外-可见扫描光谱图;C为EPS0-1的Cumulant自相关函数拟合曲线;D为EPS0-1的Regularization自相关函数拟合图。
图5为EPS0-1的微观形貌和显微结构。
图中:A为EPS0-1的AFM 2D和3D图;B为EPS0-1的SEM图(从左到右放大倍数分别为500×、2000×和5000×)
图6为EPS0-1对E.coli DH5α的低温保护作用结果图。
图中:A胞外多糖EPS0-1降低冰点的作用;B为不同浓度EPS0-1对大肠杆菌的低温保护作用(*p<0.05,**p<0.01,vs对照组)。
具体实施方式
下面结合实施例对本发明的具体实施过程作进一步详述,所描述的实施例仅是本发明的一部分实施例,并不是全部的实施例。
下述的实施例中所使用的实验技术方法和科学术语如无特殊说明均与常规技术人员通常理解的含义相同。所涉及的实验耗材和试剂如无特殊备注均为一般商业途径可获取。
实施例中所述高效生产胞外多糖的假交替单胞菌(Pseudoalteromonas sp.)LP6-12-2,于2021年12月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.24106。
2216E基础培养基的组成如下:蛋白胨0.1~1%(w/v),酵母粉0.05~0.2%(w/v),人工海水盐0.2~4%(w/v)。
2216E改良培养基的组成如下:蛋白胨0.1~1%(w/v),酵母粉0.05~0.2%(w/v),人工海水盐0.2~4%(w/v),葡萄糖0.5~2%(w/v),蔗糖0.5~2%(w/v)。
实施例1、抗低温的南极嗜冷菌胞外多糖EPS0-1的制备
一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的制备方法,包括步骤如下:
(1)种子液的制备:从-80℃超低温冰箱中取出冻存的假交替单胞菌LP6-12-2,按1%接种量接种至100mL的2216E基础培养基中,在10℃、150r/min下培养50h,得到种子液;
(2)发酵液的制备:将步骤(1)所得的种子液按照1%的体积比接种于100mL的2216E改良培养基中,在10℃、150r/min下培养60h,收集发酵液,在8000r/min下离心15min,去除菌体,获得发酵液上清;
(3)多糖沉淀:将步骤(2)所得的发酵液上清在60℃下减压浓缩至原体积的25%,冷却后向浓缩液中加入其3-4倍体积的95%乙醇,4℃过夜,然后在8000r/min下离心15min,收集沉淀;
(4)脱除蛋白:向步骤(3)所得的沉淀中加入足量去离子水,搅拌2h至沉淀充分溶解,在12000r/min下离心10min,去除沉淀,收集上清液;向上清液中加入其1/4体积的Sevage试剂(氯仿:正丁醇=5:1,V/V),剧烈震荡,重复操作7次,至无明显蛋白出现,在60℃下减压浓缩除去有机试剂,得到胞外多糖溶液;
(5)透析冻干:将步骤(4)所得的胞外多糖溶液装入至2000Da的透析袋中进行透析,每隔8h换去离子水,透析48h,透析后的多糖溶液装入冻干皿中,于-80℃冷冻,之后放入真空冷冻干燥机中干燥,得到胞外粗多糖;
(6)粗品分离:将步骤(5)所得的胞外粗多糖溶解于上样缓冲液(0.02M、pH为7.8的Tris-HCl缓冲液)中,配制成浓度为10mg/mL的多糖溶液,进行DEAE-Sepharose Fast Flow阴离子交换层析,使用Tris-HCl缓冲液洗脱,流速为0.5mL/min,每10min收集一管,收集含糖的洗脱液,真空冷冻干燥后,得到胞外多糖EPS-0;
(7)纯化:将步骤(6)所得的胞外多糖EPS-0溶解于去离子水中,,配制成浓度为5mg/ml的多糖溶液,然后将胞外多糖溶液装入至300kDa的透析袋中充分透析,透析后的多糖溶液装入冻干皿中,于-80℃冷冻,之后放入真空冷冻干燥机中干燥,获得具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1。
本实施例制备的具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的产量可达150.0~170.0mg/L,适合大规模工业化生产。
实施例2、抗低温的南极嗜冷菌胞外多糖EPS0-1的性质分析
本发明对具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的性质鉴定过程如下:
1、碘显色反应、BCA法检测蛋白、紫外光谱扫描及糖含量分析
a、碘显色反应:分别向1mL阳性对照(2mg/mL可溶性淀粉溶液)、EPS0-1(0.2mg/mL)和阴性对照(dH2O)中加入5μL碘液,混匀后观察,结果如图1A所示。
由图1A可知,阳性对照淀粉溶液变为蓝色,而胞外多糖EPS0-1和阴性对照dH2O均无明显颜色变化,仅有稀释碘液的淡黄色。这表明胞外多糖EPS0-1中不含淀粉。
b、BCA法检测蛋白:以牛血清蛋白为阳性对照,通过BCA法检测胞外多糖EPS0-1(1mg/mL)中是否含有蛋白。结果发现,胞外多糖EPS0-1样品未见明显颜色反应。这表明,胞外多糖EPS0-1中不含蛋白质。
将盛有1mg/mL胞外多糖EPS0-1溶液的比色杯置于紫外分光光度计中,设置波长范围为200~400nm,进行紫外扫描,结果如图1B所示。
由图1B可知,扫描图谱中在260nm和280nm处均无明显吸收峰,表明样品中不含核酸、多肽和蛋白质等物质。苯酚硫酸法检测多糖含量,根据葡萄糖标准曲线的公式计算胞外多糖EPS0-1的糖含量,结果显示胞外多糖EPS0-1的糖含量为91.5%±1.78%。
c、胞外多糖EPS0-1均一度检测:将胞外多糖EPS0-1配制成5mg/mL溶液,12000rpm离心10min,取上清液,用0.22μm的微孔滤膜过滤,然后将滤液转置于进样瓶中进行高效凝胶渗透色谱。色谱条件为示差检测器;流动相为水和50%甲醇溶液;RID-20A色谱柱:SB-804HQ凝胶柱(8×300mm);流速为0.5mL/min,40℃柱温;5μL进样量,结果如图1C所示。
由图1C可知,胞外多糖EPS0-1仅有一个组分,即在6.73min处的单一较对称的洗脱峰,并且胞外多糖EPS0-1无味,无臭,易溶于水,但溶解度较小(<10mg/mL),饱和糖溶液粘度较高;均不溶于甲醇、二甲基亚砜、苯、乙醇、氯仿、丙酮等有机试剂。
2、胞外多糖EPS0-1红外光谱分析
胞外多糖EPS0-1采用ATR法进行红外光谱扫描分析:取50mg胞外多糖EPS0-1放在傅里叶变换红外光谱仪Nicolet iS50的样品池中,采用ATR模式,在4000~400cm-1波长下进行扫描记录,扫描条件为:扫描次数32,分辨率为4,自动大气背景扣除,结果如图2所示。
由图2可知,胞外多糖EPS0-1具有糖类特征峰3304和2926cm-1处,分别是OH和亚甲基中的C-H伸缩振动的特征谱带;在1724和1245cm-1处吸收峰,说明存在糖醛酸和硫酸基团;1648、1544和1375cm-1处存在吸收峰,说明胞外多糖EPS0-1存在氨基基团;1130、1037和1003cm-1存在吸收峰,说明胞外多糖EPS0-1由吡喃糖苷组成;847cm-1处的吸收峰是由α吡喃糖C-H剪刀式振动引起,是典型的α构型吸收峰,即胞外多糖EPS0-1为α构型多糖。
以上结果表明,胞外多糖EPS0-1是含有糖醛酸、氨基和硫酸基的α构型多糖。
实施例3、具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的单糖组成分析
单糖组成测定:利用离子色谱仪测定胞外多糖EPS0-1的单糖组成,分为以下步骤进行:
(1)单糖标准品及配制:将16种单糖标准品被配成10mg/mL的标准溶液。各单糖标准溶液又被配置5mg/mL标准品作为标准。不同单糖质量可根据绝对定量方法测定,再根据单糖摩尔质量计算其摩尔比;所述16种单糖为D-Fuc、D-GalN、D-Rha、D-Ara、D-GlcN、D-Gal、D-Glc、D-GlcNAc、D-Xyl、D-Man、D-Fru、D-Rib、D-GalA、L-GulA、D-GlcA和D-ManA。
(2)样品配制:称胞外多糖EPS0-1和各单糖样品(10mg)置于安瓿瓶,加三氟乙酸(3M,10mL),120℃水解3h,将酸水解的样品溶液转移至管中,吹干(氮吹仪),加入5mL ddH2O涡旋混匀,稀释10倍,12000rpm/min离心5min,取上清IC分析。
(3)将称胞外多糖EPS0-1和各单糖样品进行离子色谱分析。离子色谱条件:电化学检测器,色谱柱为DionexCarbopacTMPA20(3*150mm);流动相A为ddH2O,B为15mM NaOH,C为15mM NaOH&100mM NaOAC;0.3mL/min流速;5μL进样量;30℃柱温,结果如图3所示。
在离子色谱图中将胞外多糖EPS0-1中每种单糖吸收峰的保留时间与16种标准品峰的保留时间进行比对,确定胞外多糖EPS0-1的单糖组成;根据单糖标准品和内标的摩尔浓度及峰面积,计算出样品中不同单糖的摩尔质量,进而计算出单糖的摩尔比。由图3可知,胞外多糖EPS0-1的单糖峰2、3、5、6、7、10、13、15分别为盐酸氨基半乳糖(GalN)、鼠李糖(Rha)、盐酸氨基葡萄糖(GlcN)、半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、半乳糖醛酸(GalA)、葡萄糖醛酸(GlcA),摩尔比为1.4:9.4:4.9:32:37:6.5:4.1:4.4。这表明,胞外多糖EPS0-1是由半乳糖和葡萄糖等八种单糖组成的酸性杂多糖。此外,胞外多糖EPS0-1中含有少量氨基糖和糖醛酸,这与其红外光谱分析结果一致。
实施例4:具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的高级结构分析
1、多糖三螺旋结构利用刚果红实验进行测定:将2mg/mL的胞外多糖EPS0-1溶液和刚果红试剂(100μmol/L)按1:1比例混合,再加入NaOH溶液(1mol/L)使样品的NaOH最终浓度维持在0、0.05、0.10、0.15、0.20、0.25、0.30、0.35、0.40、0.45、0.50mol/L。在该过程中用紫外分光光度计记录每个NaOH浓度下的最大吸收波长λmax(400~700nm),以不含胞外多糖EPS0-1的刚果红试剂作为对照。当多糖具有三螺旋结构构象时,能与刚果红结合形成特殊络合物,络合物的最大吸收波长同刚果红相比在一定的NaOH浓度范围内产生红移,即表现为最大吸收波长变大。当NaOH浓度大于一定浓度后,碱性条件破坏了分子间的氢键,最大吸收波长急剧下降,多糖三股螺旋结构解体变为单股螺旋成为无规则的线团式结构,结果如图4A所示。
由图4A可知,在0~0.5M NaOH浓度范围内,刚果红-EPS0-1复合物的λmax随NaOH浓度的增加而降低。在初始条件下,向胞外多糖EPS0-1溶液中加入的刚果红,形成刚果红-多糖复合物溶液,此过程中溶液颜色由红色变为紫色,其λmax显著大于刚果红的λmax,表明红移现象发生,红移值为15nm;当加入NaOH后,λmax急剧下降,表明低浓度NaOH破坏了胞外多糖EPS0-1分子间的氢键,即多糖三股螺旋解聚;当NaOH浓度大于0.1mol/L,λmax趋于稳定,表明三螺旋结构已解体,解体的胞外多糖EPS0-1多糖链不能再与刚果红结合形成络合物。这表明,EPS0-1多糖链中存在三螺旋结构,且该螺旋结构的内部结合较弱,低浓度的碱就可破坏该结构。
2、多糖分支程度利用碘-碘化钾实验进行测定:将多糖溶液与碘试剂(0.02%I2和0.2%KI,w/v)混匀,在300~600nm范围内进行光谱扫描,若在565nm处有最大吸收则说明多糖有较少的分支和/或较短的侧链;反之,则说明多糖含有较长的侧链和/或较多的分支,结果如图4B所示。
由图4B可知,碘试剂-EPS0-1的扫描光谱显示其最大吸收峰并不在565nm处,而是在350nm处,说明胞外多糖EPS0-1存在较多的侧链和/或较长的分支。
3、胞外多糖EPS0-1粒度大小和分子均一度利用动态光散射DLS分析:利用动态光散射检测颗粒因布朗运动产生的散射光干涉增强或干涉衰减(宏观表现为光的波动),自相关函数可描述光的波动特性这一原理,比较样品颗粒大小和变化趋势以及判断样品均一性。将1mg/mL的胞外多糖EPS0-1溶液加入到清洁的比色杯中,利用动态光散射仪检测胞外多糖EPS0-1粒度大小和样品分子均一度,每个样品重复三次,每个重复扫描10次,结果如图4C所示。
由图4C可知,胞外多糖EPS0-1的Cumulant自相关函数拟合曲线平滑且顺利回落到基线,表明数据可靠;该曲线呈单一衰减态势且衰减时间较长,说明胞外多糖EPS0-1组分中粒子尺寸较大,组分相对单一,几乎没有其他小颗粒成分的影响。
4、通过Regularization自相关函数计算胞外多糖EPS0-1不同组分或尺寸间的值如图4D所示。
由图4D可知,胞外多糖EPS0-1颗粒中存在2种尺寸的组分,在粒径在1~10nm之间存在一种颗粒较小的组分1,在10~1000nm之间存在大颗粒组分2,但组分1和组分2各自尺寸相同,且存在一定形式结合。这表明胞外多糖EPS0-1多糖存在不同程度的结合,导致胞外多糖EPS0-1溶液中存在较大聚集体。
5、胞外多糖EPS0-1的超微结构利用原子力显微镜AFM和扫描电子显微镜分析
用ddH2O溶解胞外多糖,制成1mg/mL胞外多糖EPS0-1溶液,将溶液连续稀释至终浓度为0.1mg/mL,通过0.22μm过滤器过滤后,将10μl样品滴到新鲜切割的云母玻片上并在室温下干燥,胞外多糖EPS0-1的AFM图像由配有Z型扫描仪的Bruker TESPA-V2扫描探针显微镜在轻敲模式下扫描获得。设置参数分别为:弹簧常数为42N/m,共振频率约为320kHz,扫描区域设置为4μm2,分辨率为512×512点。使用JPKSPM Data Processing软件(6.1.111版,德国)处理获得胞外多糖EPS0-1的AFM图像,结果如图5A所示。
将冷冻干燥制得的胞外多糖EPS0-1样品用双面胶带固定在SEM样品柱上,并在真空下使用溅射镀膜机喷金,喷金后将样品柱放入SEM中。在5kV加速电压和500×、2000×、5000×放大倍率的条件下获得胞外多糖的微观图像,结果如图5B所示。
由图5A可知,胞外多糖EPS0-1组分在溶液中没有呈现明显的链形,但呈现出较大的多糖颗粒。胞外多糖EPS0-1的高度为2.0~5.5nm,高于单个多糖链0.1~1.0nm的高度,表明它们产生了分子间和分子内聚集。
由图5B可知,胞外多糖EPS0-1为片状和棒状结构(500×和2000×),其表面光滑致密(5000×),表明胞外多糖EPS0-1交联程度较高,结构紧实。动态光散射分析、原子力显微镜和扫面电子显微镜观察结果均显示胞外多糖EPS0-1多糖链在溶液中发生不同程度的交联,佐证了刚果红实验的结果,即胞外多糖EPS0-1存在三螺旋构象。
实施例5:具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的抗低温功能
1、溶液冰点降低有助于阻止冰晶形成,进而起到低温保护作用,根据此性质对胞外多糖EPS0-1进行抗低温测试,具体方法为:向15mmol/L NaCl溶液中分别加入终浓度为10μg/mL、100μg/mL和1000μg/mL的胞外多糖EPS0-1,以15mmol/L NaCl溶液为对照组,然后将其置于冰盐浴中,测定溶液冰点,结果如图6A所示。
由图6A可知,低浓度的胞外多糖EPS0-1(10μg/mL)对溶液的冰点没有影响,且降温速度与对照组相比,有所加快,到达冰点时间的比对照组提前了20s。随EPS0-1浓度的增加,溶液的冰点也在不断下降,当胞外多糖EPS0-1浓度达到100μg/mL和1000μg/mL时,冰点相比对照组分别下降2℃和4℃。这表明,胞外多糖EPS0-1可以起到降低溶液冰点的作用。
2、胞外多糖EPS0-1能显著提高E.coli DH5α的抗低温能力。
具体为:向大肠杆菌E.coli DH5α中添加0.01%(w/v)胞外多糖EPS0-1,1次冻融后,大肠杆菌E.coli DH5α的存活率为73.0%;4次冻融后,其存活率仍有43.0%;向大肠杆菌E.coli DH5α中添加0.5%(w/v)胞外多糖EPS0-1,大肠杆菌E.coli DH5α经4次冻融后的存活率达到88.4%,其冻融保护效果即与同浓度的EPS和30%甘油的相当;向大肠杆菌E.coli DH5α中添加1%(w/v)胞外多糖EPS0-1经4次冻融后的存活率达到89.5%,略高于0.5%(w/v),与30%甘油抗低温效果的相当,以大肠杆菌E.coli DH5α为对照,结果如图6B所示。
由图6B可知,胞外多糖EPS0-1在低温保护中起主要作用;当胞外多糖EPS0-1浓度达到0.5%(w/v)时即可具有极高的保护效率。
实施例6、具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的保湿功能
测定胞外多糖EPS0-1的吸湿性和保湿性,具体方法如下:
将配制好的饱和碳酸钠溶液和饱和硫酸铵溶液放置于不同的干燥器内,平衡干燥器内的相对湿度分别为43%和81%,然后根据重量法分析胞外多糖EPS0-1、海藻酸钠和透明质酸的吸湿性。再利用硅胶形成干燥的环境,测定胞外多糖EPS0-1、海藻酸钠和透明质酸的保湿性。
结果发现,在湿度为43%的环境中,2h内胞外多糖EPS0-1的吸湿率即达到11.4%,随后逐渐增加,24h时达到最高20.5%;虽然略低于同期透明质酸25.7%的吸湿率,但明显高于海藻酸钠的8.70%吸湿率。在湿度为81%的环境中,2h内胞外多糖EPS0-1与透明质酸的吸湿效果相当,吸湿率为16.5%;36h时,胞外多糖EPS0-1吸湿率达到最高为33.1%。在干燥环境中,胞外多糖EPS0-1、海藻酸钠和透明质酸的保湿率均逐渐下降,胞外多糖EPS0-1的保湿能力介于透明质酸和海藻酸钠。
实施例7、具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的细胞毒性检测
利用人正常肝细胞系LO2研究胞外多糖EPS0-1的细胞毒性,具体方法如下:
取2mg/mL胞外多糖EPS0-1母液,将其配制成250、500、750和1000μg/mL胞外多糖EPS0-1溶液,向已铺LO2细胞的96孔板中,每孔加入200μL胞外多糖EPS0-1溶液,每组浓度设置6个复孔,培养24h。去培养液,按100μL/孔加培养基,10μL/孔加CCK-8增强型溶液,混匀,孵育后(37℃,4h),于450nm下检测吸光度。细胞活力(%)=OD样品-空白/OD阴性对照-空白×100%,以LO2细胞为对照。
结果发现,LO2细胞经250、500、750μg/mL胞外多糖EPS0-1溶液处理24h后,与对照相比LO2细胞存活率虽略有提升,但无显著性差异。LO2细胞经250、500、750μg/mL胞外多糖EPS0-1溶液处理24h后,与对照相比1000μg/mL胞外多糖EPS0-1溶液对LO2细胞有一定促进增殖的作用,增加了21.0%。这表明,胞外多糖EPS0-1对正常肝细胞没有毒性。
综上所述,本发明制备的胞外多糖EPS0-1是具有氨基和糖醛酸的大分子量的杂多糖,具有很好的抗低温、吸湿和保湿功能,并且对体外培养的正常肝细胞无毒性,因此可作为抗冻剂、化妆品和药品添加剂进行开发和应用。
以上所述仅为本发明所用较佳方案的实施例,但本发明的实施方式不受上述实施例限制,凡在本发明原则和精神内所做的任何修改、同等替换和改进,均在本发明的保护范围之内。
Claims (9)
1.一种具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1,其特征在于,所述胞外多糖EPS0-1是由半乳糖胺(GalN)、鼠李糖(Rha)、葡糖胺(GlcN)、半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、半乳糖酸(GalA)和葡萄糖酸(GlcA)组成的α-吡喃杂多糖,其摩尔比具体为GalN:Rha:GlcN:Gal、Glc:Man:GalA:GlcA=1.4:9.4:4.9:32:37:6.5:4.1:4.4;所述胞外多糖EPS0-1重均分子量为4350~4360kDa。
2.如权利要求1所述的具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1,其特征在于,所述胞外多糖EPS0-1含有三螺旋结构、侧链和分支。
3.权利要求1所述的具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1的制备方法,其特征在于,包括步骤如下:
(1)种子液的制备:将假交替单胞菌LP6-12-2按1.0~1.5%的接种量接种至2216E基础培养基中,在8~15℃、150~180r/min下培养30~45h,得到种子液;
(2)发酵液的制备:将步骤(1)所得的种子液以1.0~1.5%的体积接种于2216E改良培养基中,在8~15℃、150~180r/min下培养60~70h,收集发酵液,在8000~9000r/min下离心15~20min,去除菌体,获得发酵液上清;
(3)多糖沉淀:将步骤(2)所得的发酵液上清在50~60℃下减压浓缩至原体积的20~25%后,冷却至室温,加入浓缩液3~4倍体积的95%乙醇,4℃下静置12h以上,7500~8500r/min离心15~20min,获得沉淀;
(4)脱除蛋白:向步骤(3)所得的沉淀中加入去离子水,搅拌至沉淀溶解,在11500~13000r/min高速离心10~15min,去除沉淀收集上清液,向上清液中加入Sevag试剂,充分混匀后剧烈震荡后,静置分层后回收有机试剂除去蛋白质,重复操作5~8次,在55~65℃下减压浓缩,除去有机试剂,得到胞外多糖溶液;
(5)透析冻干:将步骤(4)所得的胞外多糖溶液装入透析袋中进行透析,每隔8h换去离子水,透析48h,然后经过真空冷冻干燥,得到胞外粗多糖;
(6)粗品分离:将步骤(5)所得的胞外粗多糖溶解于Tris-HCl缓冲液中,配制成浓度为8~10mg/mL的多糖溶液,然后通过DEAE-Sepharose Fast Flow阴离子交换层析分离,使用Tris-HCl缓冲液洗脱,流速为0.5mL/min,每10min收集一管,收集含糖的洗脱液,真空冷冻干燥后,得到胞外多糖EPS-0;
(7)纯化:将步骤(6)所得的胞外多糖EPS-0溶解于去离子水中,,配制成浓度为4~6mg/ml的多糖溶液,透析后浓缩冻干,获得具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1。
4.如权利要求3所述的制备方法,其特征在于,步骤(1)中,所述假交替单胞菌LP6-12-2于2021年12月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.24106。
5.如权利要求3所述的制备方法,其特征在于,步骤(1)中,所述2216E基础培养基的组成如下:蛋白胨0.1~1%(w/v),酵母粉0.05~0.2%(w/v),人工海水盐0.2~4%(w/v)。
6.如权利要求3所述的制备方法,其特征在于,步骤(2)中,所述2216E改良培养基的组成如下:蛋白胨0.1~1%(w/v),酵母粉0.05~0.2%(w/v),人工海水盐0.2~4%(w/v),葡萄糖0.5~2%(w/v),蔗糖0.5~2%(w/v)。
7.如权利要求3所述的制备方法,其特征在于,步骤(4)中,所述Sevage试剂为氯仿:正丁醇=4:1~5:1的混合溶液,加入量为上清液体积的18~22%。
8.权利要求1所述的具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1在制备冷冻食品、化妆品、药物组合物和保健品中的应用。
9.权利要求1所述的具有抗低温、保湿功能的南极嗜冷菌胞外多糖EPS0-1在菌种冷冻保藏和组织器官保藏运输中的应用。
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