Screening Of Excellent Lactobacillus Casei Strains By Heavy Ion Mutagenesis And Its Application In Anti-colon Cancer

Colon cancer is a malignant tumor that presents in the digestive tract.In recent years,the morbidity and mortality of the whole world remain high,which seriously threatens human life and health.In 2019,the national cancer center ranked colon cancer as the third most common cancer,at 38.8%.At present,the treatment of colon cancer includes surgical resection,chemical treatment and traditional Chinese medicine treatment.They are of great significance to the treatment and rehabilitation of colon cancer patients,but there are some deficiencies.Surgical treatment is harmful to the intestine;chemical treatment drugs are easy to produce drug resistance,toxic side effects;the individual difference of traditional Chinese medicine treatment is great;and there is no effect of prevention of colon cancer.In recent years,it has been found that probiotics can adhere to the intestinal tract surface,forming a dense barrier to prevent the colonization of pathogenic bacteria,and can combine with carcinogens to expel from the body,which has the effect of preventing cancer.In addition,the cell wall peptidoglycan of probiotic ingredients and secondary metabolites of extracellular polysaccharide can activate the cells in the immune system,enhance the phagocytosis of macrophage,strengthen the killing activity of NK cells and enhance T cell-mediated immune response,etc,so as to enhance the body's immune function,inducing cancer cell apoptosis,relief on the occurrence and development of cancer.Therefore,it is of great clinical significance and application value to develop a functional probiotic system with little side effects,good therapeutic effect and low cost for the prevention and treatment of colon cancer.A good strain is the key prerequisite for the preparation of probiotics.How to quickly and efficiently screen the excellent probiotic strains has become the current research hotspot.The acid environment of gastric juice and bile salt environment in small intestine can inhibit the growth of probiotics.Therefore,the primary prerequisite for probiotics to play a role in the intestinal tract is its acid tolerance,bile salt tolerance and high adhesion.However,the wild-type strains isolated from the nature usually have the disadvantages of small number of living bacteria,low adhesiveness and poor tolerance,which can not meet the requirements of probiotic preparation.Therefore,it is very important to mutate and domesticate the wild-type strains.In this study,a strain of Lactobacillus casei JY?NCBI number:MN400655?was isolated and identified from fresh corn stalk.JY was irradiated with different doses of¹²C⁶⁺heavy ion beam.Acid resistance?pH=3?and bile salt tolerance?0.3%?were used as the main screening indexes,combined with the survival rate of the strains in artificial gastrointestinal fluid and the adhesion rate of Caco-2 cells,three mutant strains with excellent properties were screened.MTT method was used to study the inhibition of proliferation of Caco-2 cells in vitro by three mutant strains.Finally,a mutant strain JY300-8 with good inhibition effect on Caco-2 cells was obtained,and the mechanism of inhibition was studied.The preliminary evaluation of the effect of the mutant strain JY300-8 on the prevention of colon cancer in mice was completed,which provided an excellent candidate strain and experimental basis for the development of probiotics with the effect of prevention and treatment of colon cancer.Main research contents and results are given as followed:1.Isolation and identification of JY strain and study on its biological characteristics A lactobacillus JY was isolated from corn stalk and identified as Lactobacillus casei by 16S r DNA sequence?NCBI registration number:MN400655?.JY strain grew rapidly,the number of viable bacteria reached 10¹⁰ CFU/mL after cultured for 24 h.It had66.12%and 37.01%survival rate in the artificial gastric and intestinal fluid,The bacteriostatic zone of JY strain against Escherichia coli,Staphylococcus aureus and Salmonella reached 19.40±1.44 mm,15.80±1.75 mm and 14.80±1.45 mm.JY strain showing certain probiotics potential.2.Screening of excellent mutant strains by heavy ion mutagenesisJY strain was irradiated with different doses of ¹²C⁶⁺heavy ion beam.Acid resistance?pH=3?and bile salt tolerance?0.3%?were used as the main screening indexes.In combination with the survival rate of strains in artificial gastric juice and intestinal juice and the adhesion of intestinal epithelial cells,the good mutant strains were screened.The results showed that the strains JY300-8,JY300-10 and JY160-15 showed good probiotic potential.The yield of lactic acid was 41.6-83.3%higher than that of the original strain.After the strains were cultured 24 h under acidic environment?pH=3?,the od600 value was 200%higher than the original strain.The survival rate of artificial gastric juice for 3 h and artificial intestinal juice for 6 h were higher than 100%,which was the highest level reported at present.The adhesion rates of intestinal epithelial cells were higher than 85%.These three strains can be used as candidate strains for preparation of probiotics.3.Effects of excellent mutants act on proliferative activity and apoptosis of Caco-2 cells in vitroThe inhibition of three mutants JY300-8,JY300-10 and JY160-15 act on Caco-2 cells under different time and action concentration was studied by MTT method.The study found that Lactobacillus casei JY300-8 has the best inhibitory effect on colon cancer cells.When JY300-8 acted on colon cancer cells at 100:1?cells:cells?for 72 hours,the inhibition rate of colon cancer cells reached 65.27%,which was 8 times higher than that of the original strain and 0.47 times higher than that of the commercial probiotics.Annexin V/PI double staining method was used to detect the effect of JY300-8 bacteria on the apoptosis of Caco-2 cells.The results showed that the probiotics inhibited the proliferation of Caco-2 cells by inducing apoptosis,and the late apoptosis rate reached43.5%.4.Effects of different components of JY300-8 on proliferation of CT-26 cellsThe effects of JY300-8 extracellular polysaccharides and bacterial cells on the proliferation of CT-26 cells at different concentrations and time were detected by MTT method.The results showed that both the bacterial somatic cells and extracellular polysaccharides of Lactobacillus casei could inhibit the proliferation of CT-26 cells.The inhibitory effect of bacterial cells was stronger than that of exopolysaccharide.When JY300-8 bacterial cells acted on CT-26 cells at 100:1?cells:cells?for 72 h,the inhibition rate of CT-26 cells reached 84.40±1.05%,and the inhibition rate of 500ug/mL extracellular polysaccharide acted on CT-26 cells reached 55.5±3.76%for 72h.We determined that the cells of Lactobacillus casei JY300-8 were selected as the choice for subsequent intervention in colon cancer mice.5.Study on intervention of JY300-8 bacterial cells in colon cancer of miceMale BALB/C SPF six-week-old mice were used as experimental subjects.After two weeks of gavage treatment with live and inactivated JY300-8 bacterial cells,CT-26 cells were injected subcutaneously to establish a colon cancer model and monitored for 25days to evaluate the antagonistic ability of probiotics against colon cancer.The results showed that both viable bacteria and inactivated bacteria had tumor inhibition effect.The tumor inhibition effect of inactivated bacteria was better than that of live bacteria,with the tumor inhibition rate of 60.33%and the tumor inhibition rate of live bacteria being 42.96%.The tumor inhibition rate of commercial probiotics was 53.9%?P<0.05?and 9.59%higher than that of commercial probiotics.In a word,heavy ion irradiation mutagenesis is of great significance to the breeding of probiotics.JY300-8 strain can be used as an excellent candidate for the development of functional probiotic preparation,and has a good application prospect in the prevention and treatment of colon cancer.