代糖又称替代糖，甜味剂，被广泛应用于各种食品，饮料，药物中提高它们的甜味。近年来随着对代糖研究的加深，部分研究表明代糖可辅助控制人体的血糖，而另一研究表明代糖损害人体糖耐受性。目前代糖对糖耐受性的影响研究结果存在争议。同时发现代糖对糖代谢损伤的作用可能是在碳水化合物同时存在下才导致的。而且过量摄入代糖有可能会导致心血管疾病，癌症以及代谢综合征等疾病。因此多个国家设置了每日最大摄入量来确保代糖的使用安全。由于代糖的种类繁多，物理和化学性质各异，难以用单一的检测方法对代糖进行检测，尤其是极性与物理性质相差悬殊的天然代糖与人工代糖。因此，亟需开发一种同时检测多种人工和天然代糖的方法，从而有效地监测代糖相关的食品、药品的质量安全及体内代谢。另一方面需要进一步验证长期摄入代糖对糖耐受性的影响。

（1）通过柱切换系统将Hypersil GOLD™C18柱（250×4.6 mm，5.0 mm）与Xtimate sugar-Ca柱（7.8×300 mm，5 mm）结合起来，并通过柱切换节点的调整，构建了柱切换UHPLC-CAD系统，并成功同时分离了12种天然[赤藓糖醇（Erythritol，ERY）、甘露醇（Mannitol，MNT）、木糖醇（Xylitol，XYL）、山梨醇（Sorbitol，SBT）、甜菊糖苷（Steviol，STV）]和人工代糖[安赛蜜（Acesulfame Potassium，ACS-K）、糖精（Saccharin，SAC）、甜蜜素（Cyclamate，CYC）、三氯蔗糖（Sucralose，SCL）、阿斯巴甜（Aspartame，ASP）、阿力甜（Alitame，ALI）、纽甜（Neotame，NEO）]。结果表明该方法可以在60 min内同时分离物理化学性质差异悬殊的12种代糖，并成功应用于在线检测和定量15种从中国当地超市购买的无糖饮料中的代糖定量。该方法的线性良好(R² ≥ 0.9990)，LOD和LOQ分别为0.932-6.25 μg/mL和3.10-20.83 μg/mL。日内和日间精密度的RSD值分别为0.59%-6.88%，平均回收率为85.16%-108.64%。在饮料中共检测到10种代糖（ERY、XYL、MNT、SBT、ACS-K、CYC、SAC、SCL、ASP和STV）。其中天然代糖ERY、XYL、SBT及STV的含量范围为：46.880 ± 5.520 mg/L到81259 ± 2068 mg/L；人工代糖ACS-K、CYC、SCL及ASP的含量范围为：33.610 ± 1.560 mg/L到11105 ± 411.4 mg/L。结果表明天然代糖的添加量明显高于人工代糖，同时还发现，ERY和ACS-K被过量添加于饮料中。该方法也被应用于监测饮用无糖饮料后尿液中代糖的代谢变化，ERY在尿液中的含量随着时间变化，呈现出先增加后减少的趋势。ERY在尿液中的浓度在摄入代糖饮料30-45分钟后达到峰值，之后迅速下降。在摄入代糖饮料60分钟后，尿液中的代糖ERY的浓度保持至少在10 mg/mL的浓度下温和趋势。因此，该方法解决了同时分离和检测高极性天然代糖（包括同分异构体代糖）和人工代糖的难点。此外，该方法为监测代糖在体内的代谢变化提供了新方法。

（2）选取了70只C57BL/6J雄性小鼠考察了长期摄入天然或人工代糖以及结合碳水化合物能量后对糖代谢影响。随机将小鼠分为10组。依据分组对各组小鼠进行长达13周的长期代糖的灌胃实验，并对各组小鼠进行长期的体重、体长、空腹血糖等生化指标的监测，同时进行了OGTT、ITT等实验来评价长期摄入代糖对小鼠糖代谢的影响。结果显示ACS-K组和SCL组各项指标和对照组无异。而纯ERY溶液灌胃组在空腹血糖值，糖化血红蛋白值上与对照组无明显差异，但是OGTT曲线与AUC值又存在统计学差异（p < 0.01）。且空腹胰岛素比对照组高（p < 0.05），ERY组与正常对照组相对比HOMA-IR指数明显更高，且有显著性差异（p < 0.05）。因此ERY组对胰岛素敏感性可能有一定影响。最后长期摄入纯ASP溶液灌胃组的各项生化指标显示小鼠糖耐受能力显著降低，产生糖尿病的症状。例如空腹高血糖：7.6 ± 1.03 mmol/L、高糖化血红蛋白：5.8 ± 0.19%、与正常组对比空腹胰岛素过高（p < 0.01）、OGTT曲线的AUC值比正常对照组要高（p < 0.05），ITT曲线下降恢复缓慢，HOMA-IR指数为2.4 ± 1.22比正常对照组要高且具有显著性差异（p < 0.01），表现胰岛素抵抗。其次单独摄入ASP和与碳水化合物一起摄入的ASP组相比，与碳水化合物一起摄入的ASP组表现的糖代谢损伤现象更严重，如高空腹血糖：9.2 ± 0.95 mmol/L，高糖化血红蛋白：6.6 ± 0.76%，OGTT曲线的AUC值更高，且有显著差异（p < 0.05），HOMA-IR指数为4.0 ± 2.38等。这说明代糖与碳水化合物同时摄入后会导致加深糖耐受性损伤，进而促进糖代谢损伤。

（3）通过对小鼠粪便的菌群进行16S rRNA分析，进一步探究了小鼠肠道菌群与代糖和糖代谢之间的关系。发现小鼠肠道菌群在α多样性分析上各组间无显著性差异。在小鼠肠道菌群β多样性分析上W与G组，ASP组，ERY-A组与W组物种组成相似度较高，PCoA分析无明显组别差异，物种组成无明显差异。因此说明长期摄入ASP或单独摄入ERY不会对肠道菌群造成明显的改变。代糖SCL组，ACS-K组，ERY-B组分别与正常饮水组W组相比较各组间PCoA分析有明显组别距离差异，说明长期单独摄入代糖SCL或ACS-K会导致肠道菌群结构明显发生改变。而从肠道菌群物种组成角度分析其中SCL 组与 ACS-K组 Verrucomicrobiota大幅度减少，而ERY-B组相对G组Verrucomicrobiota门大幅度增加。该结果表明小鼠长期摄入代糖实验中，SCL组，ACS-K组，还有ERY-B组在门水平上显著改变了肠道菌群的种类，且该门肠道菌群与糖代谢密切相关。从属水平，各组与对照组W、G组相对比Muribaculum属的肠道菌群各组皆有减少，其中ACS-B组相对增加。其次CAG-485属菌各组皆有增加，最后SCL组和ACS-K组中的AKK菌大幅度减少。说明肠道菌群在摄入SCL组和ACS-K组的代糖后，肠道菌群发生了明显的改变。因此实验结果表明小鼠肠道菌群在小鼠摄入各种代糖后，其中摄入SCL与ACS-K组的小鼠肠道菌群发生了明显的改变，小鼠的糖代谢相关的有益菌明显减少。

（4）基于对小鼠血浆和肠道内容物进行非靶向代谢组学分析，进一步探究了代糖对糖耐受性的影响，以及碳水化合物能量在代糖对糖代谢中的影响。结果显示血浆样品共筛选147种显著差异代谢物，肠道内容物样品中共筛选了137个显著差异代谢物。并对显著差异代谢物进行了代谢通路的富集分析。对表现出糖尿病症状的ASP组进行代谢途径分析。富集结果显示ASP的代谢与苯丙氨酸、酪氨酸和色氨酸的生物合成，苯丙氨酸代谢密切相关。对ASP的代谢途径进行分析，发现在ASP摄入到体内后，经分解产生苯丙氨酸，苯丙氨酸转化为苯丙酮酸，苯丙酮酸含量过高增加炎症反应，进而导致胰岛β细胞受损，产生胰岛素抵抗，进而诱发高血糖。另一方面苯丙酮酸转化为预苯甲酸盐进而转化为对羟基丙酮酸，最后转化为L-酪氨酸，同时苯丙氨酸也会转化为L-酪氨酸，当L-酪氨酸含量过高时，可能会进而诱发糖尿病肾病。

综上所述，本论文建立了一种柱切换UHPLC-CAD同时分离检测12种天然和人工代糖的新方法，该方法准确、有效，并且成功解决了多种代糖混合同时检测的难点，为饮用代糖饮料后的体内代谢研究提供了一种新方法。同时本研究通过小鼠长期摄入代糖的实验，并结合血浆与肠道内容物非靶向代谢组学和小鼠粪便16S rRNA分析发现，而长期的代糖ASP的摄入不会显著改变肠道菌群的种类，但会显著影响血糖水平，造成糖耐受性损伤，并发现ASP对糖代谢的影响可能与苯丙氨酸代谢密切相关。同时发现ASP与碳水化合物的共同摄入会加剧对机体糖代谢的损伤。本研究为阐明代糖对糖耐受性影响提供数据支撑，为后续深入研究代糖对糖代谢的影响奠定了基础，最终为更安全，更合理地使用代糖提供保障。

Sweeteners, also known as sugar substitute, are widely used in various foods, beverages and drugs to improve their sweetness. In recent years, with the deepening of the research on sweeteners, some studies have shown that sweeteners can assist in the control of blood glucose. Another study showed that sweeteners impair human glucose tolerance. Therefore, the current research results of the effect of sweeteners on glucose tolerance are controversial. At the same time, it was found that the effect of sweeteners on glucose metabolism damage may be caused by the simultaneous presence of carbohydrates. And excessive intake of sweeteners may lead to cardiovascular disease, cancer, metabolic syndrome and other diseases. Therefore, many countries have set the maximum daily intake to ensure the safe use of sweeteners. Due to the wide variety of sweeteners and different physical and chemical properties, it is difficult to use a single detection method to detect sweeteners, especially natural and artificial sweeteners with great differences in polarity and physical properties. Therefore, it is urgent to develop a method to detect a variety of artificial and natural sweeteners at the same time, so as to better monitor the quality and safety of food and medicine related to sweeteners and the metabolism in vivo. On the other hand, the effect of long-term intake of sweeteners on glucose tolerance needs to be verified.

(1) First, in order to solve the difficulty of simultaneously detecting a variety of sweeteners with widely different physical and chemical properties, Hypersil GOLD™C18 column (250 × 4.6 mm, 5.0 mm) was combined with Xtimate sugar-Ca column (7.8 × 300 mm, 5 mm), and through the adjustment of switching nodes, the column switching uhplc-cad system was finally constructed, and 12 natural [erythritol (ERY), mannitol (MNT), xylitol (XYL), sorbitol (SBT), stevioside (STV)] and artificial sweeteners [acesulfame (ACS-K), saccharin (SAC), cyclamate (CYC), sucralose (SCL) aspartame (ASP), alitame (ALI), neotame (NEO)] were successfully separated at the same time. The results showed that the method could simultaneously separate 12 kinds of sweeteners mixtures with great differences in physical and chemical properties within 60 min, and was successfully applied to online detection and quantification of 15 kinds of sugar-free beverages purchased from local supermarkets in China. The linearity of the method was good (R² ≥ 0.9990), and the LOD and LOQ were 0.932-6.25 μg/mL and 3.10-20.83 μg/mL, respectively. The RSD values of intra-day and inter-day precision were 0.59%-6.88%, and the average recovery was 85.16%-108.64%. A total of 10 kinds of sweeteners (ERY, XYL, MNT, SBT, ACS-K, CYC, SAC, SCL, ASP and STV) were detected in beverages. The contents of ERY, XYL, SBT and STV of natural sweeteners ranged from 46.880 ± 5.520 mg/L to 81259 ± 2068 mg/L; the contents of ACS-K, CYC, SCL and ASP in artificial sweeteners ranged from 33.610 ± 1.560 mg/L to 11105 ± 411.4 mg/L. The results showed that the amount of natural sweeteners was significantly higher than that of artificial sweeteners, and it was also found that ERY and ACS-K were added in excess in beverages. At the same time, in order to further explore the metabolic excretion of sweeteners in the body, this method has also been applied to monitor the dynamic metabolic changes of sweeteners in urine after drinking sugar-free beverages. ERY showed a trend of first increasing and then decreasing in urine. The content of ERY peaked at 30-45 minutes, and then rapidly decreased to 60 minutes, maintaining a mild curve trend at a concentration of at least 10 mg/mL. Therefore, this method solves the difficulties in the simultaneous separation and detection of highly polar natural and artificial sweeteners, including isomeric sweeteners. In addition, this method provides a new method for monitoring the dynamic metabolic changes of sweeteners in vivo.

(2) In order to further explore the effect of carbohydrate energy on glucose tolerance and carbohydrate metabolism, 70 C57BL/6J male mice were selected to investigate the effect of long-term intake of natural or artificial sweeteners on glucose metabolism. The mice were randomly divided into 10 groups. According to the grouping, the mice in each group were subjected to a 13 week long-term gavage experiment, and the long-term body weight, body length, fasting blood glucose and other biochemical indicators of the mice in each group were monitored. At the same time, OGTT, ITT and other experiments were carried out to evaluate the effect of long-term intake of sweeteners on the glucose metabolism of mice. The results showed that the indicators of ACS-K group and SCL group were the same as those of the control group. However, there was no significant difference in fasting blood glucose and glycated hemoglobin between the pure ERY solution gavage group and the control group, but there was statistical difference in OGTT curve and AUC value (p < 0.01). And fasting insulin was higher than that of the control group (p < 0.05). The HOMA-IR was significantly higher in the ERY group compared to the normal control group, and there was a significant difference (p < 0.05). So the ERY group may have some influence on insulin sensitivity. Finally, the biochemical indicators of the long-term intake of ASP showed that the glucose tolerance of mice was significantly reduced, resulting in the symptoms of diabetes. For example, fasting hyperglycemia: 7.6 ± 1.03 mmol/L, hyperglycated hemoglobin: 5.8 ± 0.19%, compared with the normal group, fasting insulin was too high (p < 0.01), AUC value of OGTT curve was higher than that of the normal control group (p < 0.05), ITT curve decreased and recovered slowly, HOMA-IR is 2.4 ± 1.22, which is higher than the normal control group and has a significant difference (p < 0.01), showing insulin resistance, etc. Secondly, compared with the ASP group with carbohydrate intake alone, the ASP group with carbohydrate intake showed more serious glucose metabolism damage, such as high fasting blood glucose: 9.2 ± 0.95 mmol/L, high glycated hemoglobin: 6.6 ± 0.76%, AUC value of OGTT curve was higher, and there was a significant difference (p < 0.05), HOMA-IR is 4.0 ± 2.38. This indicates that the intake of sweeteners together with carbohydrates will lead to the deepening of glucose tolerance injury, and then deepen the damage of glucose metabolism.

(3) In order to further explore the relationship between the intestinal flora of mice and sweeteners, the 16S rRNA analysis of mouse fecal flora showed that there was no significant difference among the groups in the α diversity analysis of mouse intestinal flora. In the analysis of β diversity of intestinal flora in mice, the species composition of W and G groups, ASP group, ERY-A group and W group were similar, and there was no significant group difference and species composition difference in PCoA analysis. Therefore, long-term intake of ASP or intake of ERY alone will not cause significant changes in intestinal flora. Compared with the normal drinking water group W, there were significant differences in PCoA analysis between the sweeteners SCL group, ACS-K group, ERY-B group and the normal drinking water group W group, indicating that long-term intake of sweeteners SCL or ACS-K alone will lead to significant changes in the structure of intestinal flora. From the perspective of species composition of intestinal flora, verrucomicrobiota decreased significantly in SCL group and ACS-K group, while verrucomicrobiota increased significantly in ERY-B group compared with group G. The results showed that the SCL group, ACS-K group, and ERY-B group significantly changed the species of intestinal flora at the phylum level in the long-term intake of glucose metabolism experiment in mice, and the intestinal flora of this phylum was closely related to glucose metabolism. At the subordinate level, the intestinal flora of each group and the control groups W and G were relatively reduced compared with the Muribaculum group, and the ACS-B group was relatively increased. Secondly, CAG-485 bacteria increased in all groups, and finally Akk bacteria decreased significantly in SCL group and ACS-K group. It showed that the intestinal flora had changed significantly after the intake of sweeteners in SCL group and ACS-K group. Therefore, the experimental results showed that the intestinal flora of mice in the SCL and ACS-K groups were significantly changed after the mice ingested various sweeteners, and the beneficial bacteria related to glucose metabolism of mice were significantly reduced.

(4) In order to further explore the effect of sweeteners on glucose tolerance and the effect of carbohydrate energy on glucose metabolism, this study conducted a non targeted metabolomic analysis of mouse plasma and intestinal contents. The results showed that 147 significantly different metabolites were screened in plasma samples and 137 significantly different metabolites were screened in intestinal contents samples. And the enrichment analysis of metabolic pathways was carried out for the significantly different metabolites. Metabolic pathway analysis was performed on the ASP group showing diabetic symptoms. The enrichment results showed that the metabolism of ASP was closely related to the biosynthesis of phenylalanine, tyrosine and tryptophan, and phenylalanine metabolism. The metabolic pathway of ASP was analyzed. It was found that after ASP was ingested into the body, it was decomposed to produce phenylalanine, which was converted into Phenylpyruvate. The high content of Phenylpyruvate increased the inflammatory reaction, which led to the damage of pancreatic β cells, insulin resistance, and then induced hyperglycemia. On the other hand, Phenylpyruvate is converted to pre benzoate, then to p-hydroxypyruvate, and finally to L-tyrosine. At the same time, phenylalanine is also converted to L-tyrosine. When the content of L-tyrosine is too high, diabetic nephropathy may be further induced.

To sum up, this paper established a new method of column switching UHPLC-CAD for the simultaneous separation and detection of 12 kinds of natural and artificial sweeteners, which was accurate and effective, and successfully solved the difficulties of simultaneous detection of a variety of sweeteners, providing a new method for the in vivo metabolism research after drinking sweeteners. At the same time, through the experiment of long-term intake of sweeteners in mice, combined with non targeted metabolomics of plasma and intestinal contents and 16S rRNA analysis of mouse feces, this study found that long-term intake of sweeteners ASP would not significantly change the species of intestinal flora, but would significantly affect blood glucose levels, causing damage to glucose tolerance, and found that the effect of ASP on glucose metabolism might be closely related to phenylalanine metabolism. At the same time, it was found that the co intake of ASP and carbohydrate would aggravate the damage to the body's glucose metabolism. This study provided data support for clarifying the impact of sweeteners on glucose tolerance, and laid a foundation for further research on the impact of sweeteners on glucose metabolism, and ultimately provided protection for people to use sweeteners more safely and reasonably.

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