Study On The Effect Of Fucoidan On The Growth And Antibacterial Ability Of Lactobacillus Rhamnosus (with A Clinical Case Report)

Objective:We compare the effects of two sterilization methods on the fucoidan and the growth of Lactobacillus rhamnosus.The better sterilization method will be selected for the next study.The effects of sterilized fucoidan on the metabolism,morphology,biofilm formation and antibacterial ability of Lactobacillus rhamnosus are studied.Methods:The filtration sterilization(0.22?m)and high-temperature autoclave sterilization(121?,20 min)were used to sterilize the fucoidan.The FT-IR was used to compare the chemical group changes between heater-sterilized fucoidan(HSF)and filter-sterilized fucoidan(FSF).The High-performance liquid chromatography was used to analyze the molecular weight and chemical bonds of the two sterilized fucoidan.The effects of fucoidan on the growth of Lactobacillus rhamnosus were studied by the CFU counting method.According to the results of the above experiments,the better-sterilized method was selected for the next study.Then,we explored the influence of fucoidan on the morphology,metabolism,biofilm formation and antibacterial ability of Lactobacillus rhamnosus.we divided the experiment into 4 groups:0 mg/m L fucoidan(control group),0.8mg/m L fucoidan group,8 mg/m L fucoidan group and 8 mg/m L fucoidan group.Scanning electron microscopy was used to study the effect of fucoidan on the morphology of Lactobacillus rhamnosus and to calculate the change of its elongation.The influence of fucoidan on the metabolism of Lactobacillus rhamnosus was detected by p H meter.The molecular composition of Lactobacillus rhamnosus cultured in MRS with fucoidan was analyzed by FT-IR.The antibacterial effect of fucoidan on Lactobacillus rhamnosus was detected by the agar diffusion test.The effect of fucoidan on the formation of Lactobacillus rhamnosus biofilm was observed by Laser Confocal Microscope.One-way analysis of variance was used to compare the data.Results:The FT-IR results of two sterilization fucoidan showed the same peak positions at 3404(O-H),2937(C-H),1640(C=O),1250(S=O),1053(C-O-C)and 840(C-O-S)cm^-1,and there is no significant difference.The molecular weights of fucoidan after filter sterilization and high-temperature autoclave sterilization were 276 kDa and 77 kDa,respectively,and high-temperature autoclave sterilization treatment caused the molecular weight drop sharply.After filter sterilization and high-temperature autoclave sterilization,the total sugars were 78.61%and 70.22%,the uronic acid was 2.01%and 0.90%,and the sulfate group was 29.65%and 28.42%.The composition of monosaccharides changed with little or even no obvious changes.At the concentration of 0.8 mg/m L of fucoidan,the CFU of Lactobacillus rhamnosus in the filter sterilization group and the high-temperature autoclave group were 4.1×10⁹CFU/m L and 4.5×10⁹CFU/m L,respectively.There was no difference between the two groups(p>0.05).Compared with the 0 mg/m L group(3.0×10⁹CFU/m L),the two groups promoted the proliferation of Lactobacillus rhamnosus(p<0.05).At the concentration of 8 mg/m L,the CFU counts of Lactobacillus rhamnosus in the filter sterilization group and the high-temperature autoclave sterilization groups were 4.4×10⁹CFU/m L and 5.7×10⁹CFU/m L,respectively.The high-temperature autoclave group was significantly better than the filter sterilization group(p<0.01).The CFU of Lactobacillus rhamnosus in the 8 mg/m L group was higher than that in the 0.8 mg/m L group(p<0.05).Compared with 0 mg/m L,there was a significant difference between the groups(p<0.01);in the filter sterilization group,the CFU of Lactobacillus rhamnosus in the 8 mg/m L group was not different from that in the 0.8 mg/m L group(p>0.05).During the co-cultivation process of fucoidan and Lactobacillus rhamnosus which were sterilized by high-temperature autoclave,the p H value of the medium of all groups decreased slightly in the first 8 hours.As the time went from 8h to 16h,the p H value of the culture medium decreased significantly.The p H value of the medium of the 0,0.8 and 8 mg/m L group decreased significantly faster than that of the 80 mg/m L group,and the p H value was lower than the 80 mg/m L group.Under co-cultivation with sterilized fucoidan,it was found that the elongation of Lactobacillus rhamnosus in the 0.8 mg/m L group and the 8 mg/m L group(2.43±0.38,2.66±0.48)was no different when compared with the 0 mg/m L group(2.56±0.48).But in the 80 mg/m L group,the elongation was 3.11±0.69 and the difference was significant(p<0.05).The Lactobacillus rhamnosus biofilm thickness of the 0 mg/m L group,the 0.8 mg/m L group,8 mg/m L group,and 80 mg/m L group measured by the Laser Confocal Microscope were21.85?m,17.03?m,9.48?m,and 6.07?m in respectively;With the concentration of fucoidan increased,the thickness of the biofilm formed by Lactobacillus rhamnosus decreased.The agar diffusion experiment showed that the diameters of the antibacterial rings of the 0 mg/m L group,0.8 mg/m L group,and 8 mg/m L group against Staphylococcus aureus were 18.15,20.27,and 21.44 mm,respectively.The 8 mg/m L group and the 0.8 mg/m L group are different from the 0 mg/m L group(p<0.05),while the80 mg/m L was 15.41 mm,which was smaller than the other three groups in the diameter of the bacteriostatic ring(p<0.05);The diameters of the inhibitory rings of the 0 mg/m L group,0.8 mg/m L group,and 8 mg/m L group against Escherichia.coli were 27.0,27.46,and 28.56 mm,respectively.There was a difference between the 8 mg/m L group and the0 mg/m L group(p<0.01),and there was no difference between the 0.8 mg/m L group and the 0 mg/m L group(p>0.05).While the 80 mg/m L group was 19.09 mm,which was smaller than the other three groups in the diameter of the bacteriostatic ring(p<0.01);The diameters of the inhibitory rings against Enterococcus faecalis in the 0 mg/m L group,0.8mg/m L group,and 8 mg/m L group were 23.4,23.3,and 25.35 mm.There was a difference between the 8 mg/m L group and the 0 mg/m L group(p<0.01),and there was no difference between the 0.8 mg/m L group and the 0 mg/m L group(p>0.05).While the80 mg/m L was 19.87 mm,which was smaller in diameter than the other three groups(p<0.01)Conclusion:The high-temperature autoclave sterilization did not denature the fucoidan,but reduced its molecular weight and changed some of its chemical components.Fucoidan promoted the proliferation of Lactobacillus rhamnosus,and high-temperature autoclave sterilization fucoidan was better than filter-sterilized fucoidan.Fucoidan promoted the growth,metabolism and antibacterial ability of Lactobacillus rhamnosus,but it was closely related to the concentration and may have a threshold effect.