Increase Of Carotenoid Output From Rhodotorula Mucilaginosa And Its Impacts On Immune Functions And Intestinal Flora Of Mice

Rhodotorula is a type of ekaryote that is discovered in animals and plants,rivers,lakes and marines.Rhodotorula Mucilaginosa is the dominant species in offshore areas of the South China Sea.Rhodotorula is rich of carotenoid which has oxidation resistance,immunity enhancement,anti-tumor and pro-animal growth.Rhodotorula Mucilaginosa ZTHY2 is a probiotics strain which is separated from offshore areas of the South China Sea and it is characteristic of fast reproduction,easy culture,rich metabolites,zero toxic and side effects,and zero pathogenicity.This study increased output of carotenoid from ZTHY2,established a dynamic model and studied influences of increased carotenoid on immune functions and intestinal flora of mice,aiming to provide test data and references to use ZTHY2 as a probiotics and a carotenoid production strain.Single factor experiment and orthogonal test on selection of carbon source and inorganic salt as well as concentration optimization of glucose,soy peptone and yeast extract of ZTHY2 were carried out by using fermentation medium of Rhodotorula as the basis.Bacterial biomass and carotenoid output were used as the evaluation indexes.Results showed that glucose was the best carbon source and Mg SO₄ was the best inorganic salt to increase carotenoid output from ZTHY2.When the fermentation medium was composed of1.5%glucose,0.5%soy peptone,1.5%yeast extract,1%Na Cl and 0.05%Mg SO₄,the biomass of ZTHY2 was 7.5702 g/L,which was 45.9%higher compared to that before optimization.When the fermentation medium was composed of 1.5%glucose,1%soy peptone,1%yeast extract,1%Na Cl and 0.05%Mg SO₄,carotenoid output of ZTHY2 was1.3142 mg/L,which was increased by 78.8%compared to that before optimization.The second generation of culture of ZTHY2 was inoculated into the optimized fermentation medium of ZTHY2.Different culture temperatures,culture times,p H values of medium,inoculum sizes of medium,shake cultivation speeds and liquid amounts in the medium were set.Bacterial biomass and carotenoid output were used as evaluation indexes.Results showed that the carotenoid output and biomass were 1.3658 mg/L and 8.6530 g/L when culture temperature,culture time,p H value of medium,inoculum size of medium,shake cultivation speed and liquid amount in the medium are 30?,60 h,p H6,6%,180 r/min and 60 m L/250 m L.These were 85.8%and 66.8%higher compared to those before optimization,respectively.ZTHY2 was cultured under the optimized fermentation medium and culture conditions.Number of bacterial cells,substrate consumption and carotenoid content in the fermentation process were tested every 3 h.All test data were fitted by Origin 2019b software.Dynamic models of relations among number of bacterial cells of ZTHY2,substrate consumption and carotenoid content were constructed.Parameters of relevant dynamic equations were determined and dynamic curves were fitted.Results showed that all constructed dynamic models of ZTHY2 growth,substrate consumption and carotenoid synthesis could fit with experimental values and they could reflect dynamic process of carotenoid production from ZTHY2 well.A total of 200 KM rats were chosen and divided into five groups.Group 1,Group 2,Group 3 and Group 4 were testing groups and offered intragastric administration of ZTHY2suspensions with different numbers of bacterial cells?2�10⁶CFU/m L,2�10⁷CFU/m L,2�10⁸CFU/m L and 2�10⁹CFU/m L?.Group 5 was the control group and it was offered intragastric administration of sterilized normal saline.Intragastric administration was performed once every day,0.5 m L per mouse and 30 d successively.Each test index was measured by 3 mice,with samples collected at 15d and 30 d.Mice samples were weighted and spleens,thymus glands,livers and kidneys were separated to calculate organ index.Delayed hypersensitivity?DTH?was tested by dinitrofluorobenzene?DNFB?and auricle weighting method.Phagocytosis of macrophages in mice was tested through a carbon clearance experiment.Contents of serum Ig G,Ig A,IL-2,TNF-?,IFN-?,CD4 and CD8molecules of mice were tested by the enzyme-linked immunosorbent assay?ELISA?.Microbial community structure in intestinal tract contents of mice was tested by the high-throughput sequencing technique.According to test results,ZTHY2 could increase thymus and spleen indexes of mice.Long-term intragastric administration influences the thymus index more significantly?P<0.05?,while short-term intragastric administration could not only strengthen DTH of mice,but also increased contents of serum Ig G and Ig A in mice by65.64%and 64.93%,increased contents of serum CD4 and CD8 molecules in mice evidently,and increased serum IL-2 content significantly?P<0.05?.Under long-term?30 d?intragastric administration,phagocytosis of macrophages could be strengthened significantly?P<0.05?,accompanied with significant increases of serum TNF-?and INF-?contents in mice?P<0.05?.ZTHY2 adjusted the family level and category level of intestinal flora structure of mice to increase the relative abundance of firmicutes and lactobacillus,but decreased the relative abundance of bacteroidetes.As a result,beneficial bacteria in intestinal tract of mice were increased,while harmful bacteria were decreased.In this study,the fermentation culture medium of ZTHY2 and culture conditions were optimized,which improved carotenoid content significantly.The 3 dynamic models can reflect the dynamic process that ZTHY2 generates carotenoid.ZTHY2 can strengthen immune functions of mice and enrich and improve the intestinal flora structure of mice.