CN115058369B - 一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法 - Google Patents
一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法 Download PDFInfo
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- CN115058369B CN115058369B CN202210852621.7A CN202210852621A CN115058369B CN 115058369 B CN115058369 B CN 115058369B CN 202210852621 A CN202210852621 A CN 202210852621A CN 115058369 B CN115058369 B CN 115058369B
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Abstract
本发明提供一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法,还有合生元用乳酸菌的筛选方法,涉及生物发酵领域。通过酸解岩藻胞外多糖获得岩藻寡糖粗提液,再利用乳酸菌选择性发酵岩藻寡糖粗提液中不良的可消化性单糖,获得含有益生菌乳酸菌和岩藻寡糖益生元的互补型合生元产品。寡糖粗提液中残余高热值的可消化性单糖,不利于其作为益生元发挥作用,但这些单糖可以作为生产益生菌的原料。乳酸菌通过消耗岩藻寡糖粗提液中的可消化性单糖生长增殖,同时纯化岩藻寡糖粗提液,获得仅含岩藻糖和岩藻寡糖的优质寡糖益生元,实现了酸解副产物单糖的高值化利用。发酵过程中产生有益代谢产物短链脂肪酸,提高了合生元产品的附加值。
Description
技术领域
本发明涉及生物发酵领域,具体为一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法。
背景技术
岩藻寡糖具有特殊的益生活性,是一种新型的寡糖益生元。除了动植物来源的岩藻寡糖,微生物胞外多糖中也含有丰富的岩藻糖,是岩藻寡糖的新型来源。近几十年来,通过对不同细菌产生的EPS的组成、结构和功能特性的研究,人们发现了上百种富含岩藻糖的细菌胞外多糖,岩藻糖的含量超过30%以上,是开发功能性岩藻寡糖益生元的良好原料,受到不少研究人员的关注。细菌胞外多糖是典型的杂多糖,岩藻糖通常与葡萄糖、半乳糖等共同形成多糖,与母乳寡糖的结构相似,具有优质的商业潜力。
益生元的制备主要为多糖降解和酶促合成,由于缺少高效通用的岩藻多糖酸解酶,酸解是制备岩藻寡糖益生元最简单有效的方法。但胞外多糖酸解会产生大量游离的葡萄糖和半乳糖,这些可消化性单糖热量高,吸收快,会迅速增加体内血糖指数(GI),对人体健康存在潜在不良风险。益生元中残留过多的可消化性单糖会增加益生元的热量,大幅降低益生元的品质,因此,益生元中可消化性单糖的脱除是益生元制备的重要品控点。
目前存在多种分离单糖和寡糖的技术,如有机试剂沉淀、纳滤、层析、色谱等。但由于单糖和寡糖的性质相似,即使醇沉、纳滤、色谱等分离技术已经广泛工业化应用,但存在试剂昂贵、设备复杂和分离效率低的问题,而且利用物化分离方法会导致岩藻二糖,岩藻三糖等小分子益生元的大量损失。因此,开发简单高效,针对性强的单糖脱除技术,具有广阔的应用前景。
发明内容
本发明目的在于提供一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法,以解决目前的多种分离单糖和寡糖的技术,存在试剂昂贵、设备复杂和分离效率低的问题,同时解决现有方法利用物化分离方式会导致岩藻二糖,岩藻三糖等小分子益生元的大量损失的问题。
为达成上述目的,本发明提出如下技术方案:一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法,包括如下步骤:
第一步,酸解岩藻胞外多糖,其中,通过薄层色谱(TLC)对酸解寡糖产物进行半定量分析,选取酸解的最佳条件;
第二步,将第一步中获得的酸解液通过电渗析脱酸,使得酸解液的pH大于3,再加入缓冲盐调pH中性,制得岩藻寡糖粗提液;
第三步,向第二步中获得岩藻寡糖粗提液中接种活化后的乳酸菌,岩藻寡糖粗提物中的可消化性单糖被乳酸菌发酵并代谢脱除,得到含有岩藻寡糖益生元和乳酸菌益生菌的发酵液,再向发酵液中加入灭菌后脱脂乳粉和甘油作为冻干保护剂,真空冷冻干燥后得到固体合生元制剂。
进一步的,在本发明中,第一步中先对岩藻胞外多糖进行单糖组成测定,岩藻胞外多糖包括岩藻糖、葡萄糖,半乳糖组成的胞外多糖,岩藻糖含量大于30%。
进一步的,在本发明中,岩藻寡糖粗提液的制备过程如下,向质量分数为0.5-5%的岩藻胞外多糖溶液中加入0.01-0.3 M盐酸,50-100 ℃酸解0.5-8 h,酸解液电渗析脱酸至pH 3.0~4.0,加入K2HPO4调pH为6.0-7.0,岩藻寡糖粗提液是含有可消化性单糖和岩藻寡糖的混合物。
进一步的,在本发明中,电渗析脱酸包括如下过程,岩藻胞外多糖溶液的电导值低于100-500 mS/cm时停止电渗析操作,岩藻寡糖粗提液终pH为3.0-4.0。
进一步的,在本发明中,乳酸菌发酵过程如下,乳酸菌使用MRS培养基于35-37 ℃活化6-24 h后,4-10 ℃离心1-10 min获得菌体沉淀。按照0.1-1%的质量比向岩藻寡糖粗提液中接种活化后的乳酸菌,发酵条件为:发酵温度35-37 ℃,转速100-300 rpm,发酵时间6-36 h。
进一步的,在本发明中,乳酸菌包括植物乳杆菌、干酪乳杆菌、戊糖片球菌和乳酸乳球菌。
进一步的,在本发明中,所述岩藻寡糖益生元为充分脱除可消化性单糖的功能性岩藻寡糖,分子量小于5000 Da。
一种胞外多糖来源岩藻寡糖发酵型合生元用乳酸菌的筛选方法,步骤一,对岩藻胞外多糖进行酸解,获得岩藻寡糖粗提液;
步骤二,选择戊糖片球菌、干酪乳杆菌、乳酸乳球菌和植物乳杆菌分别进行发酵处理,发酵处理包括配制MRS肉汤培养基,接种上述菌种,37 ℃活化24 h后,4 ℃离心获得菌体沉淀,发酵时按质量比设置接菌量为1%,1%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为37 ℃,转速为180 rpm,时间为24 h;
步骤三,选择合适的菌种,具体通过分析发酵过程中乳酸菌的生长存活情况,分析乳酸菌脱除岩藻寡糖粗提液中可消化性单糖的情况,分析合生元发酵液中有益代谢产物的生成情况,分析合生元产品中乳酸菌的活菌计数,分析合生元产品中岩藻寡糖的分子量测定,进行选定菌种。
有益效果,本申请的技术方案具备如下技术效果:
1、利用某些乳酸菌优先利用可消化性单糖的原则,发酵去除岩藻寡糖粗提液中不良的可消化性单糖,降低益生菌生产成本的同时纯化寡糖益生元,产生有益代谢产物短链脂肪酸,提高产品的附加值。
2、胞外多糖来源的岩藻寡糖作为益生元,寡糖组成与母乳岩藻寡糖相似,是婴儿益生元的潜在选择,具有人群定向性。另外,岩藻糖作为稀有糖,具有抗炎、抗癌、抗凝血和抗病毒等特殊活性,辅助增益合生元的功能效果。
3、利用电渗析脱酸工艺,破除岩藻寡糖粗提液中强酸对益生菌菌株生长的绝对抑制,是发酵型合生元制备的必要前提,电渗析技术简单高效,更适用于工业化操作推广。
应当理解,前述构思以及在下面更加详细地描述的额外构思的所有组合只要在这样的构思不相互矛盾的情况下都可以被视为本发明公开的发明主题的一部分。
结合附图从下面的描述中可以更加全面地理解本发明教导的前述和其他方面、实施例和特征。本发明的其他附加方面例如示例性实施方式的特征和/或有益效果将在下面的描述中显见,或通过根据本发明教导的具体实施方式的实践中得知。
附图说明
附图不意在按比例绘制。在附图中,在各个图中示出的每个相同或近似相同的组成部分可以用相同的标号表示。为了清晰起见,在每个图中,并非每个组成部分均被标记。现在,将通过例子并参考附图来描述本发明的各个方面的实施例,其中:
图1为不同盐酸浓度、不同温度、不同酸解时间下获得的结果图。
图2为实施例3岩藻寡糖粗提液电渗析过程中电导率和pH值的变化图,图2中A为酸解条件1的电导率和pH值;图2中B为酸解条件2的电导率和pH值。
图3为实施例6不同发酵条件下各组益生菌菌株生长增殖情况,图3中A为合生元1、合生元2、合生元3和合生元4中益生菌菌株OD600值变化图;图3中B为合生元1、合生元2、合生元3和合生元4中益生菌菌株pH值变化图;图3中C为合生元A、合生元B、合生元C和合生元D中益生菌菌株OD600值变化图;图3中D为C为合生元A、合生元B、合生元C和合生元D中益生菌菌株pH值变化图。
图4为实施例7不同发酵条件下岩藻寡糖粗提液中TLC组成变化图。图4中A为合生元A、合生元B、合生元C和合生元D中寡糖粗提液组成变化图;图4中B为合生元1、合生元2、合生元3和合生元4中寡糖粗提液组成变化图。
图5为实施例10合生元中岩藻寡糖益生元的分子量图。
具体实施方式
为了更了解本发明的技术内容,特举具体实施例并配合所附图式说明如下。
在本公开中参照附图来描述本发明的各方面,附图中示出了许多说明的实施例。本公开的实施例不必定意在包括本发明的所有方面。应当理解,上面介绍的多种构思和实施例,以及下面更加详细地描述的那些构思和实施方式可以以很多方式中任意一种来实施,这是因为本发明所公开的构思和实施例并不限于任何实施方式。另外,本发明公开的一些方面可以单独使用,或者与本发明公开的其他方面的任何适当组合来使用。
实施例1、岩藻胞外多糖的制备和单糖组成测定。
选取肠杆菌F-CE2,密执安棍状杆菌M1和肠杆菌M1发酵提取岩藻胞外多糖,并确定岩藻胞外多糖的主要单糖组成。
肠杆菌F-CE2和肠杆菌M1使用LB液体培养基活化,密执安棍状杆菌M1使用YPG培养基(酵母膏5 g/L,蛋白胨10 g/L,葡萄糖5 g/L,pH=6.5)活化。肠杆菌F-CE2和肠杆菌M1的液体发酵培养基为TGN液体培养基(胰蛋白胨10 g/L,葡萄糖20 g/L,磷酸氢二钠10 g/L,pH=6.5)。密执安棍状杆菌M1的液体发酵培养基为YPC培养基(蛋白胨10 g/L,葡萄糖20 g/L,碳酸钙5 g/L,pH=6.5)。菌株于活化培养基中活化三代后,接入液体发酵培养基中,接菌量为0.5%(V/V),发酵温度为30 ℃,转速为150 rpm,发酵时间为72 h。发酵结束后,将发酵液4000 rpm离心15 min,去除菌体沉淀;将上清液于55 ℃旋转蒸发仪中旋蒸至原体积的1/4,随后加入3倍体积的95%乙醇过夜醇沉;6000 rpm离心10 min收集醇沉沉淀。将沉淀用超纯水复溶,用10 kDa透析袋透析三天后冻干,获得三种不同菌株来源的岩藻胞外多糖。使用PMP柱前衍生化法测定岩藻寡糖粗提液中游离单糖的种类和含量。检测操作条件如下:
PMP柱前衍生化法具体为:称取冻干后的岩藻胞外多糖配制为10 g/L岩藻胞外多糖溶液,岩藻胞外多糖溶液和各单糖标准品溶液(1 g/L)与两倍体积NaOH(0.3 mol/L)和PMP(1-苯基-3-甲基-5-吡唑啉酮)甲醇溶液(0.5 mol/L)混合,70 ℃反应60 min后,冷却至室温,HCl(0.3 mol/L)中和NaOH,二氯甲烷反复萃取除去PMP试剂,过0.22 µm微孔滤膜后进行HPLC分析。HPLC分析使用Agilent 1260高效液相色谱仪,紫外检测器和C18色谱柱分析单糖组成。系统温度设置为30 ℃;流动相为0.05 mol/L KH2PO4(pH 6.7):CH3CN=83:17(V:V);流速为1 mL/min;进样量为10 µL;检测波长245 nm。
结果如表1所示,两种肠杆菌胞外多糖主要由岩藻糖(~40%)、葡萄糖(~20%)、半乳糖(~20%)组成,含有少量的鼠李糖,葡萄糖醛酸和半乳糖醛酸。密执安棍状杆菌胞外多糖主要由岩藻糖(~35%)、葡萄糖(~30%)、半乳糖(~20%)组成,含有少量甘露糖和鼠李糖。
三种不同菌株来源的岩藻胞外多糖的单糖组成表明,岩藻胞外多糖中岩藻糖的含量大于35%,普遍含有大量葡萄糖和/或半乳糖。除岩藻糖外,岩藻胞外多糖降解后,必定以葡萄糖和/或半乳糖为主要单糖副产物。葡萄糖和半乳糖作为典型的可消化性单糖,严重影响岩藻寡糖作为益生元的功能活性。
表1为实施例1中三种不同菌株来源的岩藻胞外多糖的单糖组成。
实施例2、岩藻寡糖粗提液的制备
选取密执安棍状杆菌胞外多糖(主要含有岩藻糖、葡萄糖和半乳糖)酸解制备岩藻寡糖粗提液。1 g 岩藻胞外多糖用100 mL不同浓度的HCl(0.01、0.05、0.1、0.15、0.2、0.3M)溶解,并在不同温度条件(50°C、 70 °C 、80 °C和100 °C)下搅拌,并在不同酸解时间条件下0.5-8 h,进行多组不同条件下的实验。
每隔一段时间(1、2、4、6 h)取样(2 mL),通过薄层色谱(TLC)对酸解寡糖产物进行半定量分析,选取酸解的最佳条件。薄层层析色谱操作条件如下:
使用Silica gel 60 F254 薄层层析板,规格为20 cm×20 cm,点样量为1.5 µL,丁醇:乙酸:水(6:3:1,v/v)的溶液作为流动相,层析展开时间为4 h,苯胺-二苯胺试剂(4 mL苯胺、4 g 二苯胺、200 mL 丙酮和 30 mL 85% 磷酸)作为显色剂,于110 ℃干热2 min显色。
通过不同盐酸浓度,不同温度和不同酸解时间获得的酸解寡糖产物组成如图1所示,岩藻胞外多糖酸解产生大量岩藻糖,葡萄糖和半乳糖单糖。单糖副产物的含量随着酸解的加深持续增加,寡糖的含量初始由于多糖的酸解而增加,随单糖副产物的产生相应减少。通过不同温度,不同酸浓度,不同酸解时间对岩藻胞外多糖的酸解结果确定岩藻寡糖粗提液中的可消化性单糖是葡萄糖和半乳糖。
实施例3、电渗析脱酸工艺
选择实施例2中确定的酸解条件,定义为酸解条件1(0.2 M HCl,80 ℃,2 h)和酸解条件2(0.15 M HCl,70 ℃,6 h),进而对岩藻胞外多糖进行酸解。然后,将岩藻寡糖酸解液冷却至室温并通过电渗析系统去除其中的盐酸。在溶液的电导值低于300 mS/cm时停止电渗析操作,并监测电渗析过程中的电导率和pH值变化(15 min测定一次)。电渗析后的岩藻寡糖粗提液在55 °C下通过旋转蒸发浓缩,加入三倍95%的乙醇(v/v)过夜醇沉。醇沉后通过离心(4000 rpm,20 min)收集乙醇上清液并冷冻干燥以获得pH约为3.5的岩藻寡糖粗提物。电渗析操作条件如下:
电极室、浓缩室和进料室分别为H2SO4水溶液(2 L)、Na2SO4水溶液(2 L)和酸解溶液(1 L)。电源输出电压保持在20 V,工作电流范围为0.2~1.0 A。离子交换膜的单膜有效面积为120×250 mm2。
电渗析过程中电导率和pH的变化情况如图2所示,盐酸解后,即电渗析初始时,酸解条件1和酸解条件2获得的岩藻寡糖酸解液的pH均小于1.5电导率大于20000 us/cm,基本没有菌株能够耐受强酸环境生长,因此需要脱酸,解除强酸对菌株的生长抑制。通过70/120min电渗析后,岩藻寡糖粗提液的pH达到3.0-4.0,电导率低于300 us/cm,脱酸率达到90%以上,再通过适量缓冲盐的加入,加入K2HPO4调pH为6.0-7.0,保证后续发酵的正常进行。
实施例4、岩藻寡糖发酵型乳酸菌合生元的制备
岩藻寡糖粗提液的制备:使用实施例1确定的酸解条件1(0.2 M HCl,80 ℃,2 h)对岩藻胞外多糖进行酸解。将pH=1的岩藻寡糖酸解液冷却至室温并通过120 min电渗析脱酸至pH=3.0,然后用NaHCO3将岩藻寡糖液的pH调为6.7,115 ℃灭菌30 min,获得岩藻寡糖粗提液。
合生元1:
戊糖片球菌发酵:配制MRS肉汤培养基,接种戊糖片球菌,37 ℃活化24 h后,4 ℃离心2 min获得菌体沉淀。发酵时按质量比设置接菌量为1%,1%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为37 ℃,转速为180 rpm,时间为24 h。
合生元2:
干酪乳杆菌发酵:配制MRS肉汤培养基,接种干酪乳杆菌,37 ℃活化24 h后,4 ℃离心2 min获得菌体沉淀。发酵时按质量比设置接菌量为1%,1%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为37 ℃,转速为180 rpm,时间为24 h。
合生元3:
乳酸乳球菌发酵:配制MRS肉汤培养基,接种乳酸乳球菌,37 ℃活化24 h后,4 ℃离心2 min获得菌体沉淀。发酵时按质量比设置接菌量为1%,1%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为37 ℃,转速为180 rpm,时间为24 h。
合生元4:
植物乳杆菌发酵:配制MRS肉汤培养基,接种植物乳杆菌,37 ℃活化24 h后,4 ℃离心2 min获得菌体沉淀。发酵时按质量比设置接菌量为1%,1%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为35 ℃,转速为180 rpm,时间为24 h。
合生元的包埋冻干:发酵液加入8.5 %(W/V)灭菌后脱脂乳粉、2.0 %(V/V)甘油作为冻干保护剂。真空冷冻干燥后得到合生元。
实施例5、岩藻寡糖发酵型乳酸菌合生元的制备
岩藻寡糖粗提液的制备:使用实施例1确定的酸解条件2(0.15 M HCl,70 ℃,6 h)对岩藻胞外多糖进行酸解。将pH=1.5的岩藻寡糖酸解液冷却至室温并通过70 min电渗析脱酸至pH=3.5,然后用NaHCO3将岩藻寡酸解液的pH调为7.2,115 ℃灭菌30 min,获得岩藻寡糖粗提液。
合生元A:
戊糖片球菌发酵:配制MRS肉汤培养基,接种戊糖片球菌,35 ℃活化12 h后,10 ℃离心10 min获得菌体沉淀。发酵时按质量比设置接菌量为0.5%,0.5%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为35 ℃,转速为120 rpm,时间为18 h。
合生元B:
干酪乳杆菌发酵:配制MRS肉汤培养基,接种干酪乳杆菌,37 ℃活化12 h后,10 ℃离心10 min获得菌体沉淀。发酵时按质量比设置接菌量为0.5%,0.5%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为35 ℃,转速为120 rpm,时间为18 h。
合生元C:
乳酸乳球菌发酵:配制MRS肉汤培养基,接种乳酸乳球菌,35 ℃活化12 h后,10 ℃离心10 min获得菌体沉淀。发酵时按质量比设置接菌量为0.5%,0.5%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为35 ℃,转速为120 rpm,时间为18 h。
合生元D:
植物乳杆菌发酵:配制MRS肉汤培养基,接种植物乳杆菌,37 ℃活化12 h后,10 ℃离心10 min获得菌体沉淀。发酵时按质量比设置接菌量为0.5%,0.5%菌体由无菌PBS重悬后接种于岩藻寡糖粗提液中,发酵温度为35 ℃,转速为120 rpm,时间为18 h。
合生元的包埋冻干:发酵液加入6.0 %(W/V)灭菌后脱脂乳粉、1.0 %(V/V)甘油作为冻干保护剂。真空冷冻干燥后得到合生元。
实施例6、合生元发酵过程中乳酸菌的生长存活情况
按照实施例4和5的工艺条件进行操作,并定义为合生元1、合生元2、合生元3、合生元4、合生元A、合生元B、合生元C和合生元D,测定每组发酵过程中0 h、6 h、12 h、18 h和24h发酵糖液的OD600值和pH值,评价8种合生元中乳酸菌的生长增殖情况。
OD600值和pH值的测定分别使用酶标仪和pH计,结果如图3所示。图3中A和图3中B为合生元1、合生元2、合生元3、合生元4的OD600值和pH值,合生元4中植物乳杆菌生长情况最佳,最大OD600值达0.84,pH降至3.70;合生元2和3中的干酪乳杆菌和乳酸乳球菌也能够在发酵过程中迅速增殖,24 h时OD600值分别为0.62和0.66,pH值为4.32和4.13。合生元1中戊糖片球菌增殖略缓慢,最大OD600值为0.48,最低pH值为5.07。
图3中C和图3中D为合生元A、合生元B、合生元C和合生元D的OD600值和pH值,与合生元A、合生元B、合生元C和合生元D中乳酸菌生长趋势类似,合生元D中植物乳杆菌生长情况最佳,之后依次为合生元C和B,合生元A中戊糖片球菌增殖较为缓慢。
8种合生元发酵液的OD600值和pH值结果表明,4株乳酸菌均能够在两种不同的岩藻寡糖粗提液中生长增殖,并代谢碳水化合物产酸,证明利用无氮源岩藻寡糖粗提液制备益生菌的可行性。
实施例7、合生元发酵过程中岩藻寡糖粗提液的纯化效果
利用薄层层析色谱法(TLC法)测定每组发酵过程中0 h、6 h、12 h、18 h和24 h岩藻寡糖粗提液组分变化,评价乳酸菌脱除岩藻寡糖粗提液中可消化性单糖的情况。TLC操作方法如实施例2所述,合生元1、合生元2、合生元3、合生元4、合生元A、合生元B、合生元C和合生元D的TLC组成如图4中A和图4中B所示,合生元1和合生元A中,发酵结束后,葡萄糖被戊糖片球菌消耗利用,但没有完全脱除半乳糖单糖;合生元2和合生元B中,干酪乳杆菌在发酵终点完全去除葡萄糖和半乳糖,而没有消耗岩藻寡糖组分;合生元3和合生元C中,乳酸乳球菌能够快速利用葡萄糖和半乳糖。合生元4和合生元D中,植物乳杆菌完全去除葡萄糖和半乳糖。
利用高效液相色谱法(HPLC法)测定每组发酵过程中0 h、6 h、12 h、18 h和24 h时岩藻寡糖粗提液中可消化性单糖和岩藻寡糖的含量,确定乳酸菌对岩藻寡糖粗提液中可消化性单糖的脱除率和对岩藻寡糖的保留率。HPLC测定方法如下:
取1 mL合生元发酵液,过0.22 µm微孔滤膜后上机分析。使用Agilent 1260高效液相色谱仪,示差折光检测器和OHpak SB-802.5色谱柱,系统温度设置为30 ℃;流动相为100% H2O;流速1 mL/min;上样量为10 µL。使用不同分子量的寡糖标品(岩藻糖(164 Da)、甘露糖(180 Da),甘露二糖(342 Da),甘露三糖(504 Da),甘露四糖(666 Da),甘露五糖(828 Da),3650 Da,5000 Da)绘制标准曲线。按照峰面积比计算可消化性单糖的脱除率和岩藻寡糖的保留率,结果如表2所示。
合生元1和合生元A部分去除岩藻寡糖粗提液中的可消化性单糖,脱除率大于50%,并基本没有损失岩藻寡糖组分;合生元2、合生元3、合生元B和合生元C能够完全脱除岩藻寡糖粗提液中的可消化性单糖,脱除率大于97%,并保留岩藻寡糖组分,保留率大于90%;合生元4和合生元D完全去除岩藻寡糖粗提液中的可消化性单糖,并保留大部分岩藻寡糖组分,寡糖保留率大于60%。
可消化性单糖的脱除率和岩藻寡糖的保留率测定结果表明,4株乳酸菌均能够对岩藻寡糖粗提液进行纯化,消耗可消化性单糖并保留岩藻寡糖组分,将岩藻寡糖粗提液转化为脱除葡萄糖和半乳糖的优质岩藻寡糖益生元。
表2为合生元发酵过程中可消化性单糖的脱除率和岩藻寡糖的保留率。
合生元类型 | 可消化性单糖脱除率(%) | 岩藻寡糖保留率(%) |
合生元1 | 55.3 | 96.5 |
合生元2 | 99.4 | 95.4 |
合生元3 | 99.2 | 95.7 |
合生元4 | 100 | 60.3 |
合生元A | 67.7 | 97.6 |
合生元B | 97.7 | 95.8 |
合生元C | 98.5 | 92.4 |
合生元D | 100 | 82.7 |
实施例8、合生元发酵液中合生元短链脂肪酸的产量
利用液相色谱法(HPLC法)测定每组合生元发酵液中合生元短链脂肪酸的产量,评价8组合生元发酵液中有益代谢产物的生成情况。检测操作条件如下:
取1 mL合生元发酵液,加入50 µL磺基水杨酸水溶液(20 g/L)剧烈涡旋2 min,4℃静置30 min脱除发酵液中杂质蛋白,4 ℃,10000 rpm离心3 min,取上清液进样。使用配备紫外检测器(G1314F,210 nm)的安捷伦1260高效液相色谱系统测定纯培养和粪便发酵过程中短链脂肪酸的产量。色谱柱Shodex RSpak KC-811色谱柱(6 μm, 8.0 mm × 300 mm)Shodex RSpak KC-G 68 保护柱(10 μm, 6.0 mm × 50 mm)。流动相为 0.1% H3PO4,流速为 0.8 mL/min。乳酸(10.96 mmol/L)、甲酸(15.83 mmol/L)、乙酸(17.25 mmol/L)、丙酸(13.24 mmol/L)和丁酸(10.76 mmol/L)作为标准品,异己酸(7.82 mmol/L)作为内标。
表3为8组合生元发酵液中各种短链脂肪酸的产量,8组合生元中的短链脂肪酸主要由乳酸,甲酸,乙酸,丙酸组成,合生元4的总短链脂肪酸含量最高,达到48.54 mmol/L,乳酸,甲酸和乙酸的含量均大于10 mmol/L;合生元2和合生元3的短链脂肪酸含量均超过30mmol/L,甲酸和乳酸的产量低于合生元4;合生元1的短链脂肪酸产量最少为21.99 mmol/L,且没有丙酸生成。合生元A、合生元B、合生元C和合生元D的短链脂肪酸产生情况类似合生元1、合生元2、合生元3和合生元4,由于合生元A、合生元B、合生元C和合生元D的酸解条件较为温和,游离的葡萄糖和半乳糖含量较少,因此短链脂肪酸产量总体略低于合生元1、合生元2、合生元3和合生元4。
短链脂肪酸供给肠道营养,调节肠道pH值,有效抑制致病菌生长定植。8组合生元发酵液的短链脂肪酸测定结果表明,8组合生元均生成有益代谢产物短链脂肪酸,提高了合生元的营养性和功能性。
表3为8组合生元发酵液中各种短链脂肪酸的产量。
实施例9、合生元产品中乳酸菌的活菌计数
通过平板计数法(GB 4789.35—2016)计8组合生元中的乳酸菌活菌数(cfu)。在无菌操作台下,称取0.1 g待测合生元溶解于1 mL无菌生理盐水中,漩涡振荡10min待活菌充分释放后,进行活菌数测定:取0.5 mL菌液,放入装有4.5 mL无菌生理盐水的试管中稀释,振荡混匀,再从中取出0.5 mL稀释液放入4.5 mL无菌生理盐水中混匀,每个稀释度取1 mL于灭菌培养皿中,冷却至室温的MRS固体培养基倾注混匀,每个梯度设置三个重复,36 ± 1℃厌氧培养72 ± 2 h。从样品稀释到平板倾注要求在15 min内完成。
四组合生元中的乳酸菌数目如表4所示,合生元4中植物乳杆菌活菌数最高,活菌数为2.1×108 cfu/g;合生元2和3中的植物乳杆菌和干酪乳杆菌活菌数相差不大;合生元1中戊糖片球菌活菌最低,为2.2×107 cfu/g。合生元A、合生元B、合生元C和合生元D的活菌数趋势类似合生元1、合生元2、合生元3和合生元4,由于合生元A、合生元B、合生元C和合生元D的岩藻寡糖粗提液中作为碳源的游离葡萄糖和半乳糖含量较少,且冻干时冻干保护剂的加入量偏低,因此益生菌增殖情况弱于合生元1、合生元2、合生元3和合生元4,且活菌数总体略低于合生元1、合生元2、合生元3和合生元4。
8组合生元中活菌数测定结果表明,8组合生元中的乳酸菌活菌数均达百万级,证明利用岩藻寡糖粗提液中的可消化性单糖增殖益生菌的可行性。
表4为合生元产品中乳酸菌的活菌数。
合生元类型 | 乳酸菌活菌数(×107 cfu/g) |
合生元1 | 2.2±0.15 |
合生元2 | 7.2±0.22 |
合生元3 | 6.4±0.10 |
合生元4 | 20.5±0.14 |
合生元A | 1.5±0.13 |
合生元B | 3.2±0.13 |
合生元C | 4.7±0.27 |
合生元D | 14.8±0.10 |
实施例10、合生元产品中岩藻寡糖的分子量测定
选取单糖脱除率高于98%,寡糖保留率大于90%的合生元B、合生元C、合生元2和合生元3,利用高效液相色谱测定上述合生元中岩藻寡糖的分子量分布。检测操作条件如实施例7。
结果如图5所示,95.6%以上的产物分子量较小,基本分布于5000 Da以下,没有对应于可消化性单糖的峰组分。HPLC液相结果表明岩藻寡糖的分子量低于5000 Da,研究表明肠道中有益菌偏好优先吸收利用小分子寡糖,而岩藻寡糖作为低分子量的寡糖,有利于其作为益生元改善肠道菌群的功效。
虽然本发明已以较佳实施例揭露如上,然其并非用以限定本发明。本发明所属技术领域中具有通常知识者,在不脱离本发明的精神和范围内,当可作各种的更动与润饰。因此,本发明的保护范围当视权利要求书所界定者为准。
Claims (3)
1.一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法,其特征在于,包括如下步骤:
第一步,酸解岩藻胞外多糖,其中,通过薄层色谱(TLC)对酸解产物进行半定量分析,选取酸解的最佳条件;
第二步,将第一步中获得的酸解液通过电渗析脱酸,使得酸解液的pH大于3,再加入缓冲盐调pH中性,制得岩藻寡糖粗提液;
第三步,向第二步中获得岩藻寡糖粗提液中接种活化后的乳酸菌,乳酸菌为植物乳杆菌(Lactobacillusplantarum)、干酪乳杆菌(Lactobacillus casei)和乳酸乳球菌(Lactococcus lactis)其中的一种,岩藻寡糖粗提物中的可消化性单糖被乳酸菌发酵并代谢脱除,得到含有岩藻寡糖益生元和乳酸菌益生菌的发酵液,再向发酵液中加入灭菌后脱脂乳粉和甘油作为冻干保护剂,真空冷冻干燥后得到固体合生元制剂;
电渗析脱酸包括如下过程,岩藻胞外多糖溶液的电导值低于300m S/cm时停止电渗析操作,岩藻寡糖粗提液终pH为3.0-4.0;
乳酸菌发酵过程如下,乳酸菌使用MRS培养基于35-37℃活化6-24h后,4-10℃离心1-10min获得菌体沉淀,按照0.1-1%的质量比向岩藻寡糖粗提液中接种活化后的乳酸菌,发酵条件为:发酵温度35-37℃,转速100-300rpm,发酵时间6-36h。
2.根据权利要求1所述的一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法,其特征在于,第一步中先对岩藻胞外多糖进行单糖组成测定,岩藻胞外多糖包括岩藻糖、葡萄糖,半乳糖组成的胞外多糖,岩藻糖含量大于30%。
3.根据权利要求1所述的一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法,其特征在于,所述岩藻寡糖益生元为充分脱除可消化性单糖的功能性岩藻寡糖,分子量小于5000Da。
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