Isolation And Identification Of Oral Lactobacillus And The Study Of Its Probiotic Function

lactic acid bacteria is recognized probiotics that has been widely distributed around us and applied in food processing,health care and other fields.As the normal microflora of human body,lactobacillus plays a crucial role to the health of people.Particular in recent years,studies have consistently shown that it helps control weight,can protect the cardiovascular system,regulate immune response and inhibit tumor,etc.With the development of society and the improvement of medical technology,people have attached increasing importance to oral health care.However,dental caries,periodontal disease and other oral diseases have occurred frequently,while the traditional treatments have had limited effect.Over the past few years,probiotic therapy has achieved remarkable results in the treatment of intestinal diseases,and the application of probiotics in the oral cavity has been rare.The main purpose of the paper is to separate and screen the high quality lactobacilli,evaluate the probiotic effect and clarify its probiotic function.Carring out the preliminary groups experiment and making it into a kind of tablet that is easy to store and transport.It will be useful to improve the oral environment of soldiers and helpful to enhance the combat capability of the army.Firstly,we collected saliva samples from the oral cavity of healthy volunteers and isolated a series of lactobacillus.By measuring their inhibiting ability for pathogenic bacteria,antibiotic resistance,biofilm formation ability,acid production capacity and H₂O₂ production capacity,a potential high quality lactobacillus was selected.Through sequencing and biochemical identification of its 16s rRNA,it was confirmed to be a saliva lactobacillus.Then,it was preserved in the China General Microbiological Culture Collection Center and obtained the number of CGMCC 12857.In order to understand the information of the genome of 12857,the whole genome was sequenced and compared the annotation results to multiple databases.In addition to the basic information of the genome,multiple other genes functions were also compared such as metabolic pathways and transporters,so as to explore the probiotic function of12857 from the nucleic acid level and explain its probiotic mechanism.As a kind of active strain which will be applied to human body,it’s necessary to evaluate its toxicological characteristics.The SD rats were used for acute toxicity test and repeated dose toxicity study,accordance to the Technical Guidelines for Drug Toxicity Study for Single Dose and Technical Guidelines for Drug Toxicity Study for Repeated Doses,issued by Food and Drug Administration of China.Whether the 12857would result in acute death or chronic effects in experimental animals was observed.Through the observation and determination of general behavioral characteristics,body weight,viscera index,hematology and biochemistry index of experimental animals,no significant or rational difference was found between the experimental group and the control group animals.Finally,it is believed that 12857 has no acute or chronic toxicity to animals and can be applied as safe food which is non-toxic and harmless.In the process of vitro probiotics evaluation of 12857,the oral normal cells were simulated with Hela cells cultured in vitro to evaluate the immunoregulation function of12857 at the cellular level.Easy to colonize the oral environment is the precondition for the function of probiotics.The adhesion ability of 12857 to cells was determined to assess whether they were easy to colonize the oral environment.Meanwhile,the effects of 12857 on cytotoxicity and growth activity were measured by LDH and MTT respectively.In addition,the proteins of autophagy and apoptotic were tested with the method of western blot to evaluate whether 12857 could affect the normal autophagy and apoptosis of cells.LPS is a kind of common cell inflammatory factor.In order to explore the regulation of 12857 over cell inflammatory reaction,the study observed whether it can cause the expression quantity change of several common cell inflammatory factors,such as TNF-α,IL-1βand IFN-βunder high inflammatory state and normal state of cells respectively.Results show that compared with the control strain,12857 demonstrates strong adhesion and easy colonization in oral environment.At the same time,it is free from the destruction of the cell membrane that could cause the cells apoptosis and necrosis.With less effect on cell activity,it is conducive to the growth and reproduction of cells.No abnormal apoptosis and necrosis of cells incurred by 12857 was detected with WB.Apart from that,12857 can cause the rise of proinflammatory cytokine of TNF-αand IFN-β,and suppress the rise of IL-1βcaused by LPS.The experimental results show that 12857 can adjust the release of cytokines and control the inflammatory reaction of cells.For the purpose to store and transport easily and improve the oral health of the soldiers,the 12857 should be made into tables.At the Pilot Scale Production Base of Bio-engineering Research Institute,Academy of Military Medical Sciences,large-scale fermentation,centrifugal and lyophilization of 12857 were performed.The lyophilized bacteria powder was delivered to Xinfu Ruikang Biological Pharmaceutical Technology Co.,Ltd for tabletting and packaging to make the 12857 lactic acid bacteria lozenges.The lactic acid bacteria lozenges were distributed to the enlisted military volunteers for a month-long population experiment.After that,the plaque and saliva samples weretaken respectively before and after the experiment,and the total nucleic acid of samples were extracted for high-throughput sequencing of 16s second generation.Based on the results of OTU clustering analysis,the Rarefaction curve,Beta diversity analysis,and multi-sample species classification,we explored the oral microbial composition andchange of the volunteers.The results revealed that the oral microflora of the soldiers was mainly composed of Proteobacteria,Firmicutes,Bcteroides and Actinobacteria.With the intervention of 12857 lactic acid bacteria,the structure change of salivasamples were relatively significant,indicating it is susceptible to the interference from the outside environment.The Metastats analysis results show that the content of lactic acid bacteria was significantly increased after the intervention.Meanwhile,the content of a series of oral pathogenic bacteria such as Actinomyces,Porphyromonsginglivlis,Prevotre and streptococcus,etc were significantly reduced.The results of theexperiment showed that not only in vitro but in vivo environment,lactobacillus 12857can inhibit the growth of harmful bacteria and protect the oral environment.At the same time,a rapid,sensitive and convenient LAPM detection method was established to measure the common pathogenic bacteria of periodontal disease,Fusobacterium nucleatum.This technology can detect the bacteria at the level of"species"to supplement the results of 16s rRNA second-generation high-throughput sequencing.Additionally,compared with the traditional PCR technology,LAMP is convenient,simple,sensitive and specificity.The detection results of clinical samples display that the detection rate and sensitivity were higher than that of conventional PCR.To sum up,the study isolated a high-quality strain of lactic acid bacteria,which is safe to consume and capable of oral health.It could be applied to the soldiers on a special mission or under limited sanitary conditions to protect their oral environment and maintain oral health.It also lays a foundation for the development and application of lactobacillus products in the oral cavity.