【作者】 李雄；

【导师】 游丽君；

【作者基本信息】 华南理工大学 ， 食品科学与工程， 2021， 博士

【摘要】 功能性低聚糖是不被消化的选择性发酵膳食纤维,可以促进益生菌的生长并产生短链脂肪酸,有益于宿主健康。目前,市场上对于功能性低聚糖的需求迅速增长,市场潜力巨大。脆江蓠(Gracilaria chouae)是我国特有的大型经济红藻,富含碳水化合物,可以作为功能性低聚糖的来源。本论文以脆江蓠为原料,采用高温水热降解法制备不同分子量的脆江蓠多糖,比较其流变特性、体外消化酵解特性的差异;优化高温水热法降解制备脆江蓠低聚糖的工艺条件,并探讨其降解机制;评价所得低聚糖的益生活性并通过转录组学分析其益生活性机制。具体研究内容及结果如下:(1)比较不同产地脆江蓠水提粗多糖化学组成和流变性质,选择荣成产地的脆江蓠原料;采用高温水热处理对该原料多糖进行降解,并通过超滤分离得到不同分子量的多糖组分,分别为水提粗多糖、分子量>10 k Da、5-10 k Da、<5 k Da。研究不同分子量脆江蓠多糖的化学组成、流变性质、体外消化和酵解特性,发现高温水热处理后,分子量<5 k Da的多糖总糖含量有所增加,硫酸基含量减少,单糖组成变化较小;高温降解后,多糖的表观粘度、储存模量G’和损失模量G"都迅速降低,多糖溶液趋于刚性,流动性增加;不同分子量的多糖在模拟胃肠道消化液中消化一段时间后,多糖的分子量没有明显变化,但降解后的脆江蓠多糖在体外发酵过程中,pH值更低,且短链脂肪酸的含量更高;降解后的多糖可以减缓参与蛋白质发酵相关细菌如Bacteroides、Parabacteroides、Lachnoclostridium和Phascolarctobacterium等的生长,分子量<5 k Da的脆江蓠多糖还能促进产丁酸、乙酸等短链脂肪酸的有益细菌如Anaerostipes、Eubacterium、Roseburia、Ruminococcus和Lachnospira等的生长,更好地调节肠道菌群。(2)以低聚糖得率为指标,对高温水热法制备脆江蓠低聚糖的工艺条件进行优化。结合单因素实验和响应面优化结果得出最优工艺条件为;水解温度180℃,反应时间49 min,液料比为7。此条件下制备所得的脆江蓠低聚糖(GCO)得率达到72.35%。低聚糖分子量分布为3.14 k Da(占42.14%)、1.99 k Da(占40.23%)和1.01 k Da(占17.63%),远低于粗多糖(平均分子量为1.79×10~3k Da)。通过体外酵解实验研究,发现添加低聚糖GCO的发酵培养基中pH值更低,乳酸产量更高。结果表明脆江蓠粗多糖被降解成低聚糖以后,微生物可及性得到有效提高,乳杆菌属Lactobacillus的相对丰度显著增加。(3)通过离子交换纤维素DE-52对脆江蓠水提粗多糖进行分离纯化得到2个主要组分并命名为GCP-0.2和GCP-0.4,测得总糖含量分别为61.15±2.27%和54.81±0.96%,硫酸基含量分别为25.68±1.68%和32.04±0.23%。纯化多糖GCP-0.4的单糖主要由半乳糖(96.38%)、葡萄糖(0.37%)和木糖(3.25%)组成。GCP-0.2和GCP-0.4的平均分子量分别为1.19×10~3 k Da和9.89×10~2 k Da。一维和二维核磁共振波谱解析GCP-0.4的主要结构为→3)-4-O-sulfate-β-Galp(1→4)-2-O-sulfate-3,6-anhydro-α-D-Galp-(1→,是一种iota-卡拉胶;GCP-0.2核磁共振波谱显示其同样具有iota-卡拉胶的结构信息。在脆江蓠多糖高温降解中发现,延长高温降解时间会显著降低多糖水解液的pH值,增加还原糖及游离硫酸基含量;自由基抑制剂能显著抑制高温处理对脆江蓠多糖的降解,酸可以显著缩短多糖的降解时间,无硫酸基的葡聚糖被高温降解的反应效率明显较弱。推测其降解机制为:高温下溶液中生成的自由基攻击多糖分子,断裂部分糖苷键并降低溶液的pH值,弱酸环境催化脆江蓠多糖的部分硫酸酯键水解,进一步降低溶液中的pH值;多糖的糖苷键在酸性环境下迅速断裂,导致分子量迅速降低。(4)采用葡聚糖凝胶G-25柱层析法对脆江蓠低聚糖GCO进行分离纯化,选择其中电导率较低的组分命名为GCO1;采用分级醇沉法对GCO进行纯化,选择65-92%乙醇醇沉组分,命名为GCO2。以上两种低聚糖的总糖含量分别为72.63±0.95%和54.63±0.42%,硫酸基含量分别为22.87±1.90%和37.08±1.92%。GCO1的单糖主要由半乳糖(90.45%)、葡萄糖(5.15%)、木糖(3.43%)和葡萄糖醛酸(0.97%)组成;GCO2的单糖主要由半乳糖(97.50%)、葡萄糖(0.77%)、木糖(1.59%)、半乳糖醛酸(0.01%)和葡萄糖醛酸(0.13%)组成。MALDI-Tof MS/MS的结果显示,低聚糖GCO1主要是由不同聚合度(DP=2-12)的半乳糖组成,且部分连有硫酸基,与低聚糖GCO2有较大差异。两种低聚糖均能显著促进保加利亚乳杆菌、德氏乳杆菌、嗜酸乳杆菌和乳双歧杆菌的生长和增殖。(5)通过转录组学比较分析添加脆江蓠低聚糖GCO1和GCO2后,保加利亚乳杆菌和双歧杆菌基因在转录水平上的差异,结果表明,GCO1可以显著上调保加利亚乳杆菌247个基因的转录,下调295个基因的转录;显著上调乳双歧杆菌93个基因的转录,下调128个基因的转录。GCO2可以显著上调保加利亚乳杆菌138个基因的转录,下调221个基因的转录;显著上调乳双歧杆菌74个基因的转录,下调68个基因的转录。脆江蓠低聚糖主要通过影响保加利亚乳杆菌碳水化合物的代谢通路,并上调fab Z、fab F、acc B、acc C等基因以促进脂肪酸的合成,减少脂肪酸代谢,积累更多的脂肪酸;脆江蓠低聚糖通过提高乳双歧杆菌物质转运及其相关转运蛋白通路基因的转录水平,促进碳水化合物的代谢以及脂肪酸的合成,促进乳双歧杆菌的增殖。更多还原

【Abstract】 Functional oligosaccharides are non-digested dietary fiber,which can promote the growth of probiotics and produce short-chain fatty acids.Functional oligosaccharides are beneficial to the health of the host.The demand for functional oligosaccharides is currently showing a rapid growth trend.The domestic production capacity is far from being able to meet the market,and the huge market potential is urgently needed to be developed.Gracilaria chouae,an economic red algae in China,is rich in carbohydrates,and can be used as a source of functional oligosaccharides.G.chouae polysaccharides were prepared with different molecular weights by hydrothermal treatment,and its rheology,digestion and fermentation characteristics were compared in vitro.The hydrothermal process was optimized to obtain G.chouae oligosaccharides,and the degradation mechanism was explored.The prebiotic effect of G.chouae oligosaccharides was evaluated and the mechanisms was explored through transcriptome.The main results are as follows:(1)The chemical composition and rheological properties of the crude G.chouae polysaccharides which extracted by hot-water from different areas were compared,and the G.chouae from Rongcheng,Shandong,China was used in the following experiment.G.chouae polysaccharide was degraded by hydrothermal treatment and separated by ultrafiltration to obtain polysaccharide fractions with different molecular weights(named GCPs).The chemical compositions,rheological properties,digestion and fermentation characteristics of GCPs were studied in vitro.Results showed that the total carbohydrate content of<5 k Da fraction was increased but the content of sulfate group was decreased after hydrothermal treatment.Hydrothermal could rapidly decrease the apparent viscosity,storage modulus G’,and loss modulus G"of G.chouae polysaccharide.The molecular weight of the GCPs did not change rapidly after simulated gastrointestinal digestion in vitro.However,the degraded polysaccharide had a lower pH value and a higher content of short-chain fatty acids during the fermentation in vitro.The growth of the protein fermentation related bacteria such as Bacteroides,Parabacteroides,Lachnoclostridium,and Phascolarctobacterium were remitted in the degraded polysaccharides.Furthermore,the growth of beneficial bacteria that produce short-chain fatty acids such as Anaerostipes,Eubacterium,Roseburia,Ruminococcus,and Lachnospira were promoted in<5 k Da fraction.(2)The hydrothermal degradation process of G.chouae polysaccharide was optimized based on the yield of oligosaccharides(GCO).The results showed that the optimal conditions were:temperature at 180℃,7 of liquid-to-material ratio and 49min of the reaction time.Under these conditions,the yield of oligosaccharides was72.35%.The molecular weight distribution of GCO was 3.14 k Da(accounting42.14%),1.99 k Da(accounting 40.23%),and 1.01 k Da(accounting 17.63%),which was lower than that of the crude polysaccharide(1.79×10~3 k Da).In the in vitro fermentation,the pH value of the fermentation medium of GCO was lower and the production of lactic acid was higher than those of G.chouae polysaccharide.The results suggested that hydrothermal treatment could greatly improve the accessibility of microorganisms and significantly increase the relative abundance of Lactobacillus.(3)The crude G.chouae polysaccharide was separated and purified by DEAE Cellulose DE-52 to obtain two main components which named GCP-0.2 and GCP-0.4.The total carbohydrate contents of GCP-0.2 and GCP-0.4 were 61.15±2.27%and54.81±0.96%,respectively,and the sulfate contents were 25.68±1.68%and32.04±0.23%,respectively.GCP-0.4 was mainly composed of galactose,glucose and xylose,with a molar percentage of 96.38:0.37:3.25.The average molecular weights of GCP-0.2 and GCP-0.4 were 1.19×10~3 k Da and 9.89×10~2 k Da,respectively.The backbone of GCP-0.4 were→3)-4-O-sulfate-β-Galp(1→4)-2-O-sulfate-3,6-anhydro-α-D-Galp-(1→.The results of hydrothermal degradation showed that the pH value of the hydrolysate was decreased and contents of reducing sugars and sulfate ion were increased,with the increase of the degradation time.The degradation process was significantly inhibited by free radical inhibitors,promoted by acid.The degradation efficiency of dextran(without sulfate group)was obviously slower than that of G.chouae polysaccharide(with sulfate group).The degradation mechanism might be:the free radicals generated in the solution at high temperature attacked polysaccharide molecules to break some glycosidic bonds and lowered the pH value of the solution.The part of sulfate bond was hydrolyzed at low pH environment and further reduced the pH value.Then the glycosidic bond of the polysaccharide was rapidly broken to obtain small molecular weight polysaccharides.(4)Sephadex G-25 column chromatography was used to separate and purify GCO.The component with lower conductivity was selected as GCO1;The graded alcohol precipitation method was also used to purify the GCO,and the component which was precipitated by 65-92%of ethanol concentration was selected as GCO2.The total sugar contents of GCO1 and GCO2 were 72.63±0.95%and 54.63±0.42%,respectively,and the sulfate contents were 22.87±1.90%and 37.08±1.92%,respectively.GCO1 was composed of galactose(90.45%),glucose(5.15%),xylose(3.43%)and glucuronic acid(0.97%),while GCO2 was composed of galactose(97.50%),glucose(0.77%),xylose(1.59%),galacturonic acid(0.01%)and glucuronic acid(0.13%).The results of MALDI-Tof MS/MS showed that GCO1 was mainly composed of galactose with different polymerization degrees(DP=2-12),which is quite different from GCO2.Both GCO1 and GCO2 could significantly promote the growth of Lactobacillus bulgaricus,Lactobacillus delbrueckii,Lactobacillus acidophilus and Bifidobacterium lactis.(5)Transcriptome analysis was carried out to explore the effects of GCO1 and GCO2 on the transcriptional differences of Lactobacillus bulgaricus and Bifidobacterium lactis,respectively.The results showed that GCO1 could significantly up-regulate 247 genes in Lactobacillus bulgaricus and 93 genes in Bifidobacterium lactis,and down-regulate 295 and 128 genes,respectively.GCO2could significantly up-regulate 138 genes in Lactobacillus bulgaricus and 74 genes in Bifidobacterium lactis,and down-regulate 221 and 68 genes,respectively.G.chouae oligosaccharides might mainly affect the carbohydrate metabolic pathway of Lactobacillus bulgaricus,and up-regulate fab Z,fab F,acc B and acc C genes to promote fatty acid synthesis,reduce fatty acid metabolism and accumulate more fatty acids.G.chouae could also promote the carbohydrate metabolism and fatty acid synthesis of Bifidobacterium lactis by increasing the gene transcription levels of material transport and related transporter pathway.更多还原