| Lactobacillus is a kind of microorganism widely existing in nature,including Lactobacillus,Bifidobacterium,Streptococcus,etc;It has significant value in the fields of food,biotechnology,and healthcare.Especially as probiotics,it has important physiological functions such as regulating intestinal microbiota balance,regulating immune response,alleviating intestinal diseases,and reducing serum cholesterol.Due to the change of dietary habits and the lack of theoretical knowledge related to dietary structure,more and more people are facing the problem of overweight and obesity in recent years.The proportion of obese people is gradually increasing and more and more people are inclined to minors,which eventually leads to the occurrence of many diseases,such as cardiovascular disease,type Ⅱ diabetes,polycystic ovary syndrome,infertility,and many cancers.In recent years,probiotics have gradually entered the public’s vision and played an important role in the food,health care products and medical industry.Therefore,the development of new functional probiotics is still a research hotspot.In this study,we isolated,screened and identified Lactobacillus canadensis from the fecal samples of healthy infants,and evaluated its biochemical characteristics,safety and functional characteristics.Finally,we screened a strain with better comprehensive performance to carry out the animal experiment of high-fat diet mice,and evaluated its lipid-lowering performance.(1)We took 125 fecal samples from healthy infants as the research object,and finally isolated and identified 15 strains of Lactobacillus gasseri.These 15 strains were all positive for Gram staining.LGI 6-5,6-9,and 6-15 were short bacilli,while the remaining strains were long bacilliform;The 16S rRNA sequence length is around 1500 bp,and the sequence information is highly homologous to that of Lactobacillus gasseri;The results of biochemical characteristics showed that 6 strains could ferment raffinose;Seven strains can ferment sorbitol;The results of contact enzyme test and gelatin liquefaction test were negative;Both can ferment glucose to produce acid,but do not produce gas;The results of starch hydrolysis experiments were all positive;The production of hydrogen sulfide showed that 10 strains were positive and the rest were negative;The results of the experiment on producing acetylmethyl methanol by fermenting glucose showed that 9 strains were positive,while the others were negative without discoloration;The results of aerobic type determination showed that 9 strains were facultative anaerobic type,and the rest were microaerobic type;The experimental results of litmus milk showed that 3 strains were neutral,and the remaining 12 strains made litmus pink,indicating that during the fermentation process,acid was produced and the acidity value was high,which could make the milk coagulate.Through a series of isolation,identification,and biochemical analysis methods,we have a clear understanding of the morphological,physiological,and biochemical characteristics of 15 strains of Lactobacillus gasseri.(2)We evaluated the safety and functional characteristics of 15 strains of Lactobacillus gasseri,and found that except for LGI 6-9,which had a small area of hemolytic circles on the blood plate,other strains showed no hemolysis.In antibiotic experiments,it was found that the sensitivity rates to tetracycline,vancomycin,linezolid,rifampicin,and imipenem were 86.7%,86.7%,80%,80%,and 100%,respectively.It showed inherent resistance to trimethoprim.Therefore,through safety evaluation,we excluded LGI 6-9 strains and conducted functional evaluation on the remaining 14 strains.In the bacteriostatic experiment,we found that 11 strains had the ability to inhibit the growth of Escherichia coli,and 7 strains had the ability to inhibit Staphylococcus aureus.Among them,LGI 6-1,LGI 6-2,LGI 6-3,LGI 6-4,LGI 6-6,and LGI 6-14 strains showed different degrees of inhibition on both Staphylococcus aureus and Escherichia coli.In the acid and bile salt tolerance experiments,it was found that LGI 6-1 and LGI 6-6 strains had good acid and bile salt resistance characteristics,reaching a survival rate of over 90%after 2 hours at pH=2,and still reaching a survival rate of 20%after 4 hours.Adhesion experiments showed that strains LGI 6-1,LGI 6-2,LGI 6-4,and LGI 6-8 simultaneously had certain self aggregation,co aggregation,and hydrophobic abilities.The results of cholesterol lowering experiments in vitro showed that the degradation rates of LGI 6-1 and 6-3 were 43.03%and 40.83%,respectively.The results of the in vitro triglyceride reduction experiment showed that the degradation rates of LGI 6-10 and 6-11 were 42.62%and 39.31%,respectively,and the degradation rate of LGI 6-1 was 27.3%.Based on the above safety and functional characteristics experiments,we finally selected LGI 6-1 as the test strain for in vivo experiments.(3)We used 3 week old mice as the experimental object,established a hyperlipidemic mouse model after 8 weeks,and divided into five groups to design the experiment:blank group(Ctrl),hyperlipidemic injury group(HFD),Lactobacillus gasseri LGI 6-1 treatment group(HFLR),Lactobacillus acidophilus ATCC 43121 treatment group(HFLA),and the combination of two bacteria treatment group(HFM)for animal experiments.The results showed that the Lee’s index in the HFM group was significantly lower than that in the HFD group;The weight of adipose tissue decreased significantly;In the HE staining results,there were also significant changes in the status of liver cells and adipocytes;The contents of T-CHO(total cholesterol)in liver and HDL-C(high density lipoprotein cholesterol)in serum increased significantly;The content of free fatty acids in the liver decreased significantly;The content of GSH(glutathione)increased significantly,while the content of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)decreased significantly;TNF-α、IL-1β(Inflammatory factors)levels significantly decreased;PPARγ、The expression of FAT/CD36(related lipogenic gene)was significantly reduced.Based on the above results,we isolated and identified 223 strains of Lactobacillus and Enterococcus from 125 infant fecal samples,providing more bacterial resources for the development of probiotics;After evaluating the safety and functionality of 15 strains of Lactobacillus gasseri in vitro,we selected LGI 6-1 to explore the mechanism of lipid lowering in vivo.We found that the combination of LGI 6-1 and the strain of Lactobacillus acidophilus ATCC 43121 had a significant alleviative effect on various indicators of the body in high-fat diet mice. |
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