| D-psicose is a differential isomer of the C-3 position of D-fructose,a crucial rare sugar and a new low-calorie sweetener.In 2011,D-psicose was approved as Generally Regarded as Safe(GRAS)by the US Food and Drug Administration(FDA)for use in various foods and dietary supplements.It has been found that D-psicose is an excellent alternative to sucrose in foods and can be used not only as a low-calorie sweetener in beverages and dietary supplements but also has a variety of physiological functions that are important in lowering blood sugar,anti-obesity,and anti-atherosclerosis.Probiotics are live microorganisms that provide health benefits to the host when given in adequate amounts.Probiotics are generally considered safe and well-tolerated and have now been significantly effective in preventing and treating various diseases,particularly those involving the gastrointestinal tract.In recent years,there has been a rising trend to explore whether synbiotics(a combination of probiotics and prebiotics)have good bioactivity to prevent metabolic diseases.In this study,D-allulose and probiotics were used as the entry point for the combined application of the two,with the following main research aspects:1.Screening study of probiotic strains capable of utilizing D-psicose.Thirty probiotic strains were screened as starting songs based on the List of Edible Strains issued by the Health and Wellness Commission of the People’s Republic of China and the strains in the laboratory collection.Two probiotic strains that could grow normally in D-psicose MRS medium were screened by measuring the growth curve,viable count,and p H change of the strains,namely Lacticaseibacillus rhamnosus 217-189 and Bacillus coagulans 217-176 were identified as the subsequent experimental strains,taking into account the literature research and the growth of Lc.rhamnosus 217-189 and Bacillus coagulans 217-176 in different MRS media.Lc.rhamnosus 217-189 was identified as the subsequent experimental strain.2.A study of the metabolic mechanism of Lc.rhamnosus 217-189 using D-psicose.The exponential phase of Lc.rhamnosus 217-189 in D-psicose MRS medium and glucose MRS medium was collected,RNA was extracted,and transcriptome sequencing was performed,resulting in a total of 129 genes differentially expressed in both,of which 51 genes were down-regulated,and 78 genes were up-regulated.Analysis of the differentially expressed genes revealed that most of them were related to the bacterial phosphotransferase system(PTS),tagatose 6-phosphate pathway,glycerol pathway,and fructose pathway,and the related genes were significantly up-regulated(P<0.05).Moreover,four significant differential genes,such as the tagatose 1,6-bisphosphate aldolase gene,were randomly screened and verified by quantitative fluorescence PCR,and the results were consistent with the transcriptome results.Then combined with the metabolomic analysis,the mechanism of metabolism of D-psicose utilization by Lc.rhamnosus 217-189 was determined as follows: extracellular D-psicose phosphorylation was transported to intracellular via ABC transporter and PTS system,converted to Dtagatose,D-fructose and a small number of other sugars,and finally catabolized by tagatose-6-phosphate pathway and fructose pathway,and then used by bacterial growth.3.Study of Lc.rhamnosus 217-189 combined with D-psicose in a mouse model of type II diabetes mellitus.A mouse model of type II diabetes was established by feeding a high-sugar,high-fat diet and injecting streptozocin(STZ)to induce the administration of Lc.rhamnosus 217-189 and D-psicose sugar alone or in combination,and the experimental results showed that Lc.rhamnosus 217-189 and D-psicose had a remitting effect on the type II diabetes model.Two,the Combined administration’s remission effect was better than that alone.The combination of Lc.rhamnosus 217-189 and D-psicose restored the body weight of type II diabetic mice by 4.36±1.70 g.They reduced the fasting blood glucose(FBG)to 16.18±0.54 mmol/L and the oral glucose tolerance test(OGTT)to 18.18±0.54 mmol/L.The concentrations of serum inflammatory factors were also reduced: TNF-α decreased by 34.41±2.21 pg/m L,IL-1β by 39.55±3.18 pg/m L,IL-6 by105.98±12.14 pg/m L,and the concentration of the anti-inflammatory factor IL-10 was increased by 30.45±0.5 pg/m L.The effects on serum insulin(INS),catalase(CAT),glutathione peroxidase(GSH-PX),malondialdehyde(MDA),and Superoxide Dismutase(SOD),and hepatic glycogen content or activity in the liver were elevated to different degrees.The pathological histological observation of mouse pancreatic tissue and the analysis of intestinal flora diversity revealed that Lc.rhamnosus 217-189 combined with D-psicose could restore islet atrophy in type II diabetic mice.They could regulate the balance of mouse intestinal flora to a certain extent and restore the normal state of mouse intestinal flora.The remission effect of the combination of the two drugs was better than that of using them alone.4.Optimization of fermentation conditions for the transformation of D-psicose ketose by Escherichia coli.The inducible p ET22b-psi and the constitutive p LP3804-psi containing D-psicose 3-epimerase(DPEase)were constructed and transferred to E.coli DE3/XL1-Blue for inducible expression.The fermentation conditions(induction conditions: medium type,induction temperature,inducer type,inducer addition time,metal ions,induction time and incubation conditions: incubation temperature,substrate p H,substrate concentration,bacterial volume,incubation time)were optimized,and the yield of D-psicose transformation by E.coli containing p LP3804-psi plasmid under the optimal reaction conditions was determined to be 33.91%,and the yield of D-psicose transformation by E.coli containing p ET22b-psi plasmid was 17.81%.After the system was stabilized,Lactobacillus plantarum 217-8 was inoculated at 2%,and the purity of Dpsicose in the E.coli system containing p LP3804-psi plasmid was 89.67% after incubation at 37°C for 48 h.The purity of D-psicose in the E.coli system containing p ET22b-psi plasmid was 27.30%,thus achieving high purity production of D-psicose and providing the next step for The transformation of D-psicose ketose using probiotic bacteria provided a basis for the next step. |
PDF Full Text Request