Finding Of Corynebacterium Pyruviciproducens And The Study On Its Biological Characteristics And Application

Food plays an irreplaceable role during the metabolic processes in all creations, in reverse, to take food regularly and selectively is the fundamental activity for living organisms to keep life running. Healthy and scientifically proper amount of food can improve immune system, hence intelligence for human being. By time, the knowledge of human being for the nature has been enriched, meanwhile, to explore and produce healthy food is becoming a necessary component of daily life and industry.Notably, foodborne diseases are the most serious threat for human being's health and life. Therefore, the primary problem that "food safety" needs to solve is how to detect, control and remove all microorganism pathogens efficiently and safely. On the other hand, there are some beneficial microorganisms which can synthesize kinds of useful products as food additive or help to keep food safety against pathogenic ones.Corynebacterium genus was first described in 1896, including a group of Gram-positive and rod-shaped bacteria. To date, there are 106 species within this genus. Corynebacterium widely distributed in the nature and can be isolated from mammalians, plants and a variety of environmental sources. While considering its pathogenicity, the genus comprises 3 groups as pathogens for human, pathogens for animals or plants and soil-derived nonpathogenic bacteria. Corynebacterium diphtheria of the first group is the aetiological agent of diphtheria and the rest is usually regarded as opportunistic pathogens, mainly found from immunocompromised people; the second group often brings troubles for agriculture, hence damage human's food. The last group of Corynebacteria is nonpathogenic, further, some of them, such as Corynebacterium glutamicum, are the versatile producers in food industry, pharmacy and daily life.Apparently, Corynebacterium genus plays a two-side role in food industry. One side is good, referring that they are nonpathogenic engineering bacteria which present more and more characteristics useful for manufactures; another side is destructive, meaning that normal food uptake could incur obstacle when they are the pathogens for animals or plants. Bacteria within this genus are greatly diverse, which is one of the properties of Corynebacterium and the consequences of evolution as well. Therefore, more attention and more researches are putting on the genus globally, however, in China, the relevant work is still limited to explore the pathogenicity of C. diphtheria and the capacity of producing glutamate from C. glutamicum.Currently, there are some bottleneck phases while detecting or manipulating Corynebacteria, such as lipophilic strains, often fail to give an expected growth for research. Since these problems seriously slow down the processes of investigating the pathogenic mechanism and industrial application, it is necessary to find the corresponding resolutions.Thereby, the objectives of this work were:1) To use several methods to identify the novel Corynebacterium species, supplying more data for taxonomy and exploring the efficient and feasible classification means as well.2) To check whether discarded cooking oil can be used as a supplement for the culture of lipophilic bacteria in order to optimize the cultivation medium.3) To investigate the feasibility of applying the novel species in food industry.4) To analyze the possible pathogenicity of the novel species.5) To investigate the function which the novel species exerts on important immune cell in order to unravel its "preexisting immunity".Methods:1) Identification of novel species:searching for optimal conditions for cultivation; ensuring the staining charateristic, generating a profile of enzymatic activities by using commercial detection system together with traditional tests; performing sequencing and blast upon 16S rRNA and partial rpo B genes respectively. 2) Optimization of culture medium:simply disposing discarded cooking oil and adding it into conventional broth medium; comparing obtained growth with those in normal medium or the broth medium with serum added.3) Assessment of value to use the novel species in food industry:applying gas-liquid chromatography to detect end products of the novel species and looking for the component useful for food industry.4) Investigation on pathogenicity of the novel species:designing specific primers and probe for detecting target bacteria in various infections and then real-time PCR being applied; performing countercurrent electrophoresis and specific PCR to detect diphtheria toxin and relevant gene.5) Exploration of possibility to use the novel species in the field of biology medicine:using relatively quantitative real-time PCR to evaluate the transcriptional activities of IL-6 and IL-12 genes in DC2.4 cells; implementing flow cytometry detection to check expression of important membrane molecules.Results:1) The Corynebacterium species was lipophilic,β-glucuronidase production and xylose fermentation were two important tests helpful to distinguish it from other commonly lipophilic Corynebacteira, sequences of 16S rRNA and partial rpoB(2700bp-3130bp) genes both proved the target species was a novel Corynebacterium species.2) The medium with discarded cooking oil added could significantly improve the growth of several lipophilic Corynebacteria.3) The interested species could metabolize glucose and produce pyruvic acid of high concentration.4) The detection rate of target species in infectious samples was very low, additionally, the bacteria didn't express diphtheria toxin neither carry the toxin gene.5) Transcription of IL-6 gene in DC2.4 cells was improved when stimulated by the novel Corynebacterium species but that of IL-12 gene was downregulated; the expression of MHC-Ⅱmolecules on the cell membrane increased but CD80, CD86 and CD40 didn't show significant change.Conclusions:1) A hitherto undescribed Corynebacterium species was identified, a combination of various identification methods to explore the general biological, biochemical and molecular biological characteristics prove to be efficient.2) The novel species could produce pyruvic acid of high concentration. A name for this species is hence proposed as Corynebacterium pyruviciproducens. In addition, discarded cooking oil can dramatically improve the growth of two lipophilic coryneform species. The further study should performed in order to explore the putative applications of this species.3) Corynebacterium pyruviciproducens didn't express diphtheria toxin neither include tuberculostearic acid in cell wall. The result of real-time PCR detection also demonstrated the possibility that the species was not responsible for human diseases, which might build a basis for the future application of this Corynebacterium species in industry.4) The meaning of the change regarding the transcriptional activities of two cytokine genes keeps unclear, however, the improved expression of MHC-Ⅱmolecules could be a trigger to initiate a study on the "preexisting immunity" of the novel species.