|Alternative Title||Process Optimization of Lactobacillus Plantarum Fermented Herbal Compound and Evaluation of its Effect on Intestinal Flora|
|Place of Conferral||兰州|
|Keyword||植物乳杆菌 药食同源类中药 发酵 肠道菌群|
本研究从4株分离自天然发酵乳的植物乳杆菌中筛选发酵中药复方的最佳菌株，以发酵液中的活菌数为指标，通过单因素和正交试验得到益生菌发酵中药复方的最佳工艺参数。依照《保健食品检验与评价技术规范（2003版）》中的相关规定，使用平板计数法评价发酵产物调节肠道菌群的功能，并通过16s rRNA测序技术分别比较了发酵产物、植物乳杆菌和中药复方对小鼠肠道菌群结构组成和多样性的调节作用，为开发具有调节肠道菌群作用的功能性产品提供理论依据。研究结果如下：（1）植物乳杆菌C3发酵中药复方的效果最好，最佳发酵参数为：发酵温度35℃，发酵时间24 h，菌体接种率6%，中药复方提取液浓度0.2 mg/mL。发酵液中的植物乳杆菌在模拟胃液中处理3 h后，存活率为97.5%，在模拟肠液中处理6 h后的存活率为70.21%；发酵液中多糖和芦丁含量较发酵前均显著增加。（2）平板计数的实验结果显示，实验前后自身及组间比较，各组小鼠粪便中乳杆菌和双歧杆菌的数量显著上升，产气荚膜梭菌的数量显著下降；肠杆菌的数量在9个实验组中均无显著变化。实验前后自身比较，肠球菌的含量在中药提取液中剂量和高剂量组中显著下降，其余组中无明显变化；与对照组相比，肠球菌的数量在植物乳杆菌低剂量组显著升高，在中药提取液中剂量和高剂量组显著下降，在剩余6组中无明显变化。依据《保健食品检验与评价技术规范（2003版）》中的相关规定可得，植物乳杆菌和发酵后的中药提取液具有调节肠道菌群的功能。（3）16s rRNA测序结果表明，20天的实验干预对正常小鼠肠道菌群的结构和组成产生明显的影响。3个实验组厚壁菌门与拟杆菌门的比值均显著降低，其中以发酵组效果最为明显， 3组小鼠有益菌属Lactobacillus和Bifidobacterium的含量都显著增加。除此之外，发酵组S24-7, Bacteroides和Odoribacter菌属的相对丰度显著升高，Clostridiales和Erysipelotrichaceae菌属的相对丰度显著降低。通过对16s rRNA测序数据进行PICRUSt功能预测分析发现，发酵产物的干预不同程度地增加了氨基酸代谢、能量代谢、次生代谢物的合成、多聚糖的合成与代谢、萜类和聚酮类化合物的代谢及核苷酸代谢相关的菌属的相对丰度，这说明这些干预可能在促进宿主相关代谢方面发挥有益作用。本研究表明在最佳工艺条件下，植物乳杆菌发酵中药复方不仅可以保证发酵液中的活菌数在7 log10cfu/mL以上，能够达到发酵产品要求，还通过对中药材的生物转化，显著增加了中药提取液中多糖和芦丁的含量；而且发酵产物对小鼠肠道菌群具有明显的有益调节作用，为开发具有调节肠道菌群功能的新型微生态制品奠定基础。
|Other Abstract||Intestinal flora is the most vital microbial community in the human gastrointestinal tract, which plays an important role in maintaining host health and intestinal homeostasis. It will cause various chronic diseases and intestinal metabolic disorders when intestinal flora imbalance happens. Therefore, using the regulation of intestinal flora to improve the human health would become a new method with the intestinal flora as a target. Low oral bioavailability is a common limited factor for Chinese medicine treatment. Microbial fermentation is a long-term biotransformation process that applied to traditional Chinese medicine as the number of viable bacteria is the key affecting factor for conversion effect and quality evaluation of fermented products. In recent years, “Multiple components for multiple targets” has been proposed as the treatment principle of traditional Chinese medicine. On the one hand, Chinese medicine can promote the growth of beneficial bacteria and inhibit the proliferation of certain pathogens simultaneously; on the other hand, the intestinal flora can metabolize the active ingredients of traditional Chinese medicine, while the metabolites can be absorbed easily and exert different biological effects. At the same time, traditional Chinese medicine can also regulate the composition of intestinal flora, protect the intestinal mucosal barrier, and restore intestinal homeostasis.|
In this study, the best strains of fermented traditional Chinese medicine compound were screened from 4 strains of Lactobacillus plantarum, which were isolated from the natural fermented milk. Later, the best process parametersof the probiotics fermentation Chinese medicine compound were obtained by single factor and orthogonal tests with a number of viable bacteria in the fermentation broth as indicators. According to the relevant regulations of “Technical Standards for Testing & Assessment of Health Food (2003 Edition)”, the Colony Forming Unit (CFU) method was used to evaluate the intestinal flora regulation function of fermentation products. The 16S rRNA sequencing technology was used to compare the regulatory effects of fermentation products, Lactobacillus plantarum and Chinese medicine compound on the structure and diversity of mice intestinal flora.Further,it provides a theoretical basis for the development of functional products having the function of regulating intestinal flora. The research results are as follows: (1) Lactobacillus plantarum C3 fermented traditional Chinese medicine compound showed the best effect. The optimal fermentation parameters were: fermentation temperature 35°C, fermentation time 24 h, bacterial cell inoculation rate 6%, Chinese herbal compound extract concentration 0.2 mg/mL. Under the conditions of fermentation, the number of viable bacteria in the fermentation broth reached 9 log10cfu/mL. The survival rate of the cells treated in simulated gastric juice for 3h was 97.5%, and the survival rate after simulated intestinal fluid treatment for 6h was 70.21% in the fermentation broth. The content of polysaccharide and rutin increased significantly compared with that before fermentation. (2) The results of plate counting showed that the number of Lactobacillus, Bifidobacterium and Clostridium perfringens in of mice in each group changed significantly, while the number of Enterobacter did not change significantly in experimental groups (self-comparison and intergroup comparison). The content of Enterococcus decreased significantly in HM and HH groups , but there was no significant change in the other groups by self-comparison. The number of Enterococcus was significantly increased in PL group, decreased significantly in HH groups, and there was no significant change in other groups by intergroup comparison. According to Technical Standards for Testing and Assessment of Health Food (2003 Edition), Lactobacillus plantarum and the traditional Chinese medicine extract after fermentation could regulate intestinal flora. (3) The results of 16s rRNA sequencing showed that experimental intervention had a significant effect on the structure and composition of the intestinal flora on normal mice. The ratios of the Firmicutes and the Bacteroides in the three experimental groups were significantly reduced, and the effect of the fermentation group was the most obvious. The contents of the beneficial bacteria Lactobacillus and Bifidobacterium in the three groups were significantly increased. In addition, the relative abundances of the fermentation groups S24-7, Bacteroides and Odoribacterwere significantly increased, whereas the relative abundances of Clostridiales and Erysipelotrichaceae were significantly reduced. And the intervention of fermentation products increased the relative abundance of bacteria related to the metabolism of amino acids, energy metabolism, synthesis of secondary metabolites, synthesis and metabolism of polysaccharides, metabolism of steroids and polyketides, and nucleotide metabolism in different degrees, which played a beneficial role in promoting host-related metabolism.
This study shows that,Lactobacillus plantarum fermented traditional Chinese medicine compound under suitable conditions ensures the number of viable bacteria in the fermentation broth to be above 7 log10cfu/mL.Also, it significantly increases the polysaccharide and rutin content in the fermentation broth.The compound also greatlyregulated the intestinal flora.This compound thus could be substantial in laying the foundation for the development of new micro-ecological products with functions to regulate intestinal flora.
|赵唐. 植物乳杆菌发酵中药复方工艺优化及对小鼠肠道菌群的作用研究[D]. 兰州. 兰州大学,2019.|
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