Isolation And Identification Of A Strain Of Lactobacillus Reuteri With High Expression Of Linoleic Acid Isomerase

BackgroundA large number and variety of microorganisms reside in the intestine of the human body.These microorganisms are collectively referred to as intestinal flora.Among the intestinal flora,there are probiotics that are beneficial to human health.Probiotics are beneficial to the host by colonizing it in the human body and changing the flora of a part of the host.Lactobacillus reuteri is a probiotic that has been studied in depth and can be colonized in a large number of mammals.Conjugated linoleic acids(CLA)is a natural fatty acid with many physiological activities.It has many functions such as anti-cancer,anti-atherosclerosis,lipid-lowering,weight-loss regulation,immune function,and growth.This study involved a strain of Lactobacillus reuteri that highly expresses linoleate isomerase(LAI).The Lactobacillus reuteri is isolated from feces of a healthy infant.The expression level of linoleic acid isomerase is 6-10 times as many as that of Lactobacillus reuteri DSM20016(ATCC 23272)and other probiotics.It can convert linoleic acid(LA)into conjugated linoleic acid.Objective1.Isolate and identify the strain,construct a phylogenetic tree based on 16S rRNA sequencing results,and determine the species relationship of the strain.2.To study the morphological characteristics,culture characteristics,physiological and biochemical characteristics,adhesion ability,drug resistance,resistance to artificial gastric and intestinal fluids and bile salts,animal safety and other biological characteristics of lactobacillus reuteri.3.The efficiency of conversion of linoleic acid to conjugated linoleic acid by this Lactobacillus reuteri was determined and analyze the chemical structure of the product.4.Explore the preliminary production of fermented milk with Lactobacillus reuteri as a mixed or single strain of fermentation.Methods1.Lactobacillus reuteri HI120 was isolated with MRS solid medium,and high expression linoleic acid isomerase strain was screened.The 16S rRNA sequencing method was compared with the NCBI database to identify bacterial species,and the linoleic acid isomerase gene was amplified and analyzed by gene sequencing.2.Study on the biological characteristics of Lactobacillus reuteri HI120,including morphological observation by gram staining.The OD600 value of spectrophotometer was recorded to describe the growth curve.The bacteria were cultured with the cells for 2 h to determine their ability to adhere to human cells.Lactobacillus biochemical identification tube was used for sugar fermentation experiment.Antibiotic resistance was tested.The artificial gastric intestinal fluid and the bile salt solution of different concentrations were used to test resistance to Acid-Base reaction.The safety and acute toxicity of the bacteria were evaluated by intragastric administration in C57 mice.3.Linoleic acid was cultured with Lactobacillus reuteri HI120 for 24 hours.The fatty acid was extracted,and the conversion rate of conjugated linoleic acid was detected.The main chemical structure of conjugated linoleic acid was determined by gas chromatography-mass spectrometry(GC-MS).4.Preliminarily investigate the usage of HI120 to fermented milk and tested the amplification ability of LAI genes in fermented milk.Results1.Lactobacillus reuteri was smeared on MRS solid medium for 24 hours under anaerobic culture,showing milky white,middle bulge,round and shiny colonies.They were positive for Gram staining.In the NCBI database,the BLAST software tool was used to compare the 16S rRNA gene sequence of the strain with other Lactobacillus reuteri 16S rRNA gene sequences to establish a phylogenetic tree of the strain.The results showed that the strain 16S rRNA sequence is 99%homologous to the 16S rRNA gene sequence of the standard strain of Lactobacillus reuteri DSM20016 and other Lactobacillus reuteri.The sequence of LAI gene was 79%similar to that of DSM20016 in NCBI database,and the similarity of amino acid sequence was 87%.2.The growth curve shows HI120 reached its maximum growth rate at about 12 hours and reached a plateau at about 22 hours,and the plateau phase began to decline steadily after maintaining about 6 hours.This strain of lactobacillus reuteri infected human colon cancer cell HCT116 in vitro under aerobic condition.The experimental results showed that the bacterial adhesion was 3.8±2.2 bacteria after 2 hours of culture.The sugar fermentation results of the Lactobacillus reuteri HI120 and the standard strain DSM20016 are consistent,and they can ferment lactose,sucrose,raffinose,and maltose,and the rest of esculin,cellobiose,mannitol.,Salicin,sorbitol,and inulin were negative.This Lactobacillus reuteri HI120 is resistant to compound sulfa and norfloxacin,and resistant to gentamicin,doxycycline,penicillin G,ampicillin/sulbactam,cefazolin,erythromycin,and chlorine.More than 90%of the bacteria survived after 6 hours of treatment with artificial gastric juice,artificial intestinal juice and artificial intestinal juice,and more than 80%survived after 6 hours of treatment with 0.6%bile salt.Mice were given gastric gavage for 3 consecutive days.There were no statistically significant differences in weight gain percentage,WBC count,RBC count and hemoglobin concentration between the medium or high-dose HI120 groups and the PBS control group.Histopathological sections showed that no organ or cytotoxic damage was present in the middle and high dose groups.3.The results showed that the average efficiency of this strain to produce conjugated linoleic acid was 6.1 and 9.1 times that of the standard strain DSM20016 and Bifidobacterium longum NCC2705,respectively.This bacterium converts linoleic acid to conjugated linoleic acid,whose main chemical structure is cis-9,trans-11 conjugated linoleic acid.4.The strain was successfully mixed into a standard yogurt starter culture(Angel)and fermented milk was successfully fermented.The amplification of the linoleate isomerase in fermented milk added to the strain was 528 and 579 times as that of without adding the bacteria or inactivating the bacteria.Conclusion1.In this experimental study,a strain of Lactobacillus reuteri was successfully isolated and identified,characterized by high expression of linoleic isomerase,which can convert linoleic acid into conjugated linoleic acid through co-culture.2.Lactobacillus reuteri HI120 has the potential of probiotics and is expected to be a new biological agent and be used in the production of fermented milk.