专访浙江工商大学肠道微生物消化课题组，发文经验大揭秘
浙江工商大学朱炫课题组长期专注于维生素B12的生产特性和稳定性以及不同形态和结合态的维生素B12对肠道微生物的影响研究。课题组的多篇文章发表于食品科学研究顶级期刊，近期的4篇维生素B12与微生物相互作用的文章分别发表于：
《Food Chemistry》
《Frontiers in Microbiology》
《Journal of Agricultural and Food Chemistry》
《LWT-Food Science and Technology》
我们非常荣幸地邀请到朱炫博士来为我们分享他的研究工作以及他在开展这些研究中的心得体会。
钴胺素是一种以钴元素为核心的卟啉环结构物质，其中对人体有生物活性的物质我们称之为维生素B12。维生素B12作为重要物质，参与了一系列动物和细菌的大量重要生理活动，如DNA合成，脂肪代谢，能量代谢及神经代谢等。
钴胺素广泛的存在于自然界的动物，植物与微生物中，然而只有一部分原核生物和很小一部分藻类具有合成钴胺素的能力。合成钴胺素是一个极其复杂的过程，微生物需要调动26种已知的酶，占1%的基因来参与钴胺素的合成。维生素B12最早的出现解决了微生物无氧呼吸的电子受体问题。随着千年进化过程，微生物分化成了维生素B12合成型、维生素B12半合成型及维生素B12利用型，无论哪种微生物都在基因中存在着大量与维生素B12互作的单元，实现着B12感知、转运、转化和应用的酶合成的调控。
维生素B12通过结构和形态的变化，成为了一双无形的手，调控菌落社群的变化。如果把维生素B12看作微生物技术的结晶，它就是微生物界的芯片。不同种群的细菌具有完全不同的合成和改装的能力，维生素B12作为菌落社群发展的重要调控器，控制着菌群社会发展动力和遗传特性。为此，我们的研究关注着这个重要的物质的产生、加工中的稳定性、对于肠道微生物消化的调控和在特殊医疗情况下的定点应用与释放研究。
通过我们的研究，我们了解了如何通过确立菌群的发展目标，提高维生素B12的产量；通过自然的蛋白保护，减少维生素B12副产物产生，减少对于菌群的不利影响；最后通过不同类型的维生素B12的肠道调控作用，实现了针对不同肠道特征的消化和形态干预，实现了定点控制的食品营养技术。
本课题由浙江工商大学肠道微生物消化研究团队完成。
主要成员：朱炫、石丽华、胥媛媛、向沙沙、冯潇、包璇、叶昆、沈宇标、应轩宇。
该团队通过与德国人类营养所、德国汉堡大学及明斯特大学合作，构建了人体体外肠道评价系统CDMNs和肠道微生物消化模型，同时建设了维生素B12类似物及F430类似物化学物质库，从而构建了从体外高产发酵到体内消化模拟的全过程控制系统。
课题组合照，第二排右一为朱炫博士
文章1: Effect of Lactobacillus reuteri on vitamin B12 content and microbiota composition of furu fermentation
罗氏乳杆菌对腐乳发酵过程中维生素B12产量及菌群组成的影响
Abstract: In order to solve problems of fluctuant vitamin B12 contents in furu, the effect of L. reuteri inoculation on vitamin B12 content of furu in a large scale experiment was studied in addition to evaluating the microbiomes of furus. Results showed that vitamin B12 content in furu inoculated with L. reuteri was gradually increased up to 141.7 ng/g (wet weight), which is higher than the control group (36.0 ng/g). After principal component analysis, samples for bacterial composition in L. r. group were gathered together, but those for the composition of fungi were dispersed. L. reuteri inoculation obviously increased the relative abundance of Firmicutes and Actinobacteria, but reduced the relative amount of Proteobacteria. The group of L. r. and control exhibited a similar pattern of fungi changes. Based on spearman analysis, Lactobacillus, Enterococcus, Streptococcus, and Corynebacteria were reported to be positively related with vitamin B12 contents and supported each other, vice versa Rhodotorula and Penicillium. Regarding the results of PICRUSt, vitamin B12 related genes pathways were enhanced after L. reuteri was applied. In this study, the novel start culture of L. reuteri was successfully introduced to a traditional soybean fermented food to improve the content of vitamin B12 in furu.
文章2: Stability of vitamin B12 with the protection of whey proteins and their effects on the gut microbiome
维生素B12对乳清蛋白保护作用的稳定性及其对肠道微生物的影响
Abstract: Cobalamin degrades in the presence of light and heat, which causes spectral changes and loss of coenzyme activity. In the presence of beta-lactoglobulin or alpha-lactalbumin, the thermal- and photostabilities of adenosylcobalamin (ADCBL) and cyanocobalamin (CNCBL) are increased by 10–30%. Similarly, the stabilities of ADCBL and CNCBL are increased in the presence of whey proteins by 19.7% and 2.2%, respectively, when tested in gastric juice for 2 h. Due to the limited absorption of cobalamin during digestion, excess cobalamin can enter the colon and modulate the gut microbiome. In a colonic model in vitro, supplementation with cobalamin and whey enhanced the proportions of Firmicutes and Bacteroidetes spp. and reduced those of Proteobacteria spp., which includes pathogens such as Escherichia and Shigella spp., and Pseudomonas spp. Thus, while complex formation could improve the stability and bioavailability of cobalamin, these complexes might also mediate gut microecology to influence human nutrition and health.
文章解读：VB12对乳清蛋白保护作用的稳定性及其对肠道微生物的影响
文章3: Cobalamin (Vitamin B12) Induced a Shift in Microbial Composition and Metabolic Activity in an in vitro Colon Simulation
维生素B12在体外结肠模拟中诱导微生物组成和代谢活动的转变
Abstract: Cobalamin deficiency is believed to be related to disturbances in cell division, neuropathy, nervous system disease and pernicious anemia. Elderly people are usually advised to supplement their diets with cobalamin. As cobalamin has several forms, the effects of different forms of cobalamin on gut microbiota were investigated in this study. After 7 days of supplementation, methylcobalamin had reduced the diversity of gut microbiota compared to that in the control and cyanocobalamin groups. After supplementation with methylcobalamin, the percentage of Acinetobacter spp. Had increased to 45.54%, while the percentages of Bacteroides spp., Enterobacteriaceae spp. and Ruminococcaceae spp. had declined to 11.15, 9.34, and 2.69%, respectively. However, cyanocobalamin had different influences on these bacteria. Both cobalamins increased the amount of short-chain fatty acids, particularly butyrate and propionic acid. The cyanocobalamin group showed increased activity of cellulase compared with that in the other two groups. According to CCA and PICRUSt analysis, methylcobalamin had a positive correlation with Pseudomonas bacteria, propionic acid, and butyrate. Methylcobalamin promoted lipid, terpenoid, and polyketide metabolism by gut bacteria, promoted the degradation of exogenous substances, and inhibited the synthesis of transcription factors and secondary metabolites. Our results indicate that the various forms of cobalamin in the food industry should be monitored and regulated, because the two types of cobalamin had different effects on the gut microbiome and on microbial metabolism, although they have equal bio-activity in humans. Given the effects of methylcobalamin on gut microbiota and microbial metabolism, methylcobalamin supplementation should be suggested as the first option.
文章解读：VB12在体外结肠模拟中诱导微生物组成和代谢活动的转变
文章4: Impact of Cyanocobalamin and Methylcobalamin on Inflammatory Bowel Disease and the Intestinal Microbiota Composition.
氰钴胺和甲钴胺对炎症性肠病和肠道微生物组成的影响
Abstract: Patients with inflammatory bowel disease (IBD) are usually advised to supplement various types of vitamin B12, since vitamin B12 is generally absorbed in colon. Thus, in the current study, the influence of cyanocobalamin (CNCBL) or methylcobalamin (MECBL) ingestion on IBD symptoms will be investigated. Then, whether and how the application of various cobalamins would modify the taxonomic and functional composition of the gut microbiome in mice will be examined carefully. Dextran sulfate sodium-induced IBD mice were treated with MECBL or CNCBL; disease activity index (DAI) scores and intestinal inflammatory condition of mice were evaluated. Faecal samples were collected; microbiota composition was determined with a 16s rRNA analysis, functional profiles were predicted by PICRUSt and short-chain fatty acids were measured. The consequence of higher relative abundances of Enterobacteriaceae and isomeric short chain fatty acids by cobalamins treatment revealed that a high concentration of CNCBL, but not MECBL, supplementation obviously aggravated IBD. A microbial ecosystem rich in Escherichia/Shigella and low in Lactobacillus, Blautia, and Clostridium XVIII was observed in IBD mice after a high concentration of CNCBL supplementation. In cobalamin-dependent enzymes, CNCBL was more efficient in adenosyl-cobalamin system than MECBL, vice versa in MECBL system. The distinct effects of various cobalamins were associated with the distribution and efficiency of vitamin B12-dependent riboswitches. CNCBL had a strong inhibitory effect on all riboswitches, especially on btuB and pocR riboswitches from Enterobacteriaceae. CNCBL aggravated IBD via enhancing the proportion of Enterobacteriaceae organisms through riboswitch and enzyme systems. The present study provides a critical reference for offering a suitable amount and type of cobalamin during a symbiotic condition.
黄小编：从以上四篇文章内容上来看，您的研究思路非常连贯，从上游维生素B12的生产、加工到下游其在肠道营养及微生物消化代谢中的研究，您的思路非常严谨、清晰，请问您最开始是如何想到做这样一个课题的呢，可以给我们分享一下您的经验吗？
朱老师：从我博士开始就研究着维生素B12和它的类似物。对于我们来说一直好奇，维生素B12如此复杂的一个分子，分子量大于1000，存在于生物体内，并且经过几万年的遗传还如此稳定和重要。同时维生素B12的工业合成存在着环保和能耗等问题。所以我们首先通过传统发酵食品思路，找到切实解决的方法，通过传统结合工业的手段提高了产量。在此过程中我们发现，维生素B12稳定性变成了我们的首要问题。结合后面研究我们发现氰钴胺，这类非自然的维生素B12，存在很大弊端，所以我们思考如何通过食品手段提高B12稳定性，最后我们发现乳清蛋白是一个很好的手段。而后我们就要研究，不同的形态和结合态的维生素B12对于肠道微生物的影响。对于我们而言肠道无非是另外一个发酵系统，而维生素B12在微生物体系中的存在和发酵是息息相关的。为此，我们引入了我们发酵思路，来分析和解决肠道微生物菌落和消化能力的变化，从而了解B12的角色和作用。我们觉得做研究最重要的是，首先是要以上帝视角观察事物的发展，没有预设目标和概念。通过切实的逻辑证明，了解事物发展的规律，再使用我们的工程师手段，来使得事物至少表面上，向着有利于人类的方向发展，我们就可以扮演好“上帝工程师”的角色。
黄小编：您上面4篇文章发表的期刊，在食品学研究杂志中的影响力都是数一数二的，您的研究无论是在质量还是数量上都非常高，您觉得您的研究成果能发表在顶级期刊上主要取决于哪些因素呢？在前期实验设计和后期文章编写上您有什么心得跟大家分享一下吗？
朱老师：此问题同上，寻找问题的过程中让自己变成一个“上帝工程师”的角色。
黄小编：在整个实验设计构思、数据分析及文章写作、投稿上您有遇到过什么难题吗？您又是如何攻克和解决的呢？这个可能是我们广大科研工作者最关心的一个问题呢~
朱老师：最大的问题，就是无知，总觉得理所当然，往往实验结果和预想不一样。第一，放弃所谓的执着，尊重逻辑证明的结果；第二，扩大阅读，很多思路往往是在其他学科研究中发现的。
黄小编：在您的研究论文中有提到联川生物参与提供了技术支持，您最开始为什么会选择联川生物呢？您怎么评价您的这位合作伙伴呢？
朱老师：我首先是认识了郎女士（也就是小编的老板郎秋蕾女士~），郎女士让我感觉是个很可靠、很稳重的人，接下来的实验数据也是可信，后期的好多人，比如小朱，比如小黄都给人可靠的感觉。当然，我不是学计算机出身的，好多问题处理都是联川给我解决的，而且数据质量也相当好。
1.Xuan,B., Xiang, S., Chen, J., Shi, Y., Chen, Y., Wang, H., & Zhu, X. (2018).Effect of Lactobacillus reuteri on vitamin B12 content and microbiotacomposition of furu fermentation. LWT. doi:10.1016/j.lwt.2018.10.045
2.HuanhuanWang,Shou, Y., Zhu, X., Xu, Y., Shi, L., Xiang, S., Feng, X., Han, J., Stability ofVitamin B12 with the Protection of Whey Proteins and Their Effects on the GutMicrobiome, Food Chemistry(2018).
3.XuY, et al. (2018) Cobalamin (Vitamin B12) Induced a Shift in MicrobialComposition and Metabolic Activity in an in vitro Colon Simulation. Front.Microbiol. 9:2780. doi:10.3389/fmicb.2018.02780
4.Xuan Zhu, Shasha Xiang, et al. (2018) Impact of Cyanocobalamin andMethylcobalamin on Inflammatory Bowel Disease and the Intestinal MicrobiotaComposition. J. Agric. Food Chem. DOI: 10.1021/acs.jafc.8b05730