| Astaxanthin is one of the ketone carotenoids(non-vitamin A source),which is the most potent natural antioxidant in nature with high economic and application value.Free astaxanthin with natural trans structure is easy to be absorbed and utilized by the human body,but the source is small,and production is low.At the same time,astaxanthin has unstable properties,poor solubility in water,and low bioavailability,which limits its full application in functional food.Therefore,it is essential to choose appropriate preparation technology to expand the application of astaxanthin in functional food.Xanthophyllomyces dendrorhous could produce free trans-astaxanthin through fermentation.However,the low astaxanthin production of X dendrorhous,which is susceptible to glucose metabolic suppression,limits its industrial application.Therefore,in this study,nitrogen-doped carbon dots(N-CDs)were used to increase the accumulation of astaxanthin precursors in Spirulina platensis,and S.platensis was used to replace the organic nitrogen source and part of the glucose for the fermentation of X.dendrorhous to improve the accumulation of astaxanthin.The astaxanthin-loading microparticles were prepared to improve the stability and bioavailability of astaxanthin,and its protective mechanism against oxidative stressed Caco-2 cells was explored.The research results are as follows(1)Utilization of N-CDs to promote the growth of S.plate uarantee for the application of N-CDs N-CDs prepared by bovine serum albumin(BSA)through hydrothermal reaction had good water dispersivity,and emitted intense blue light in the visible range.The cytotoxicity test results showed that N-CDs with the concentration range of 1-40 mg/L did not show any toxicity to the mouse mononuclear macrophages RAW 264.7,which provided a safety evaluation guarantee for the application of N-CDs Compared with the control group,N-CDs effectively promoted the growth of S.platensis and the accumulation of bioactive substances.The best cultivation condition for S.platensis was N-CDs 10 mg/L,culture temperature 26?,light intensity 4500 Lux,culture period 10 d,nitrogen-limited stress(NaNO31.25 g/L).Under the best culture condition,the biomass of S.platensis reached 1.48 g/L,?-carotene and zeaxanthin reached 1.20 mg/g and 0.43 mg/g,which were increased by 140.32%,96.72%,and 95.45%,respectively,compared with the original S.platensis culture conditions(2)Based on the astaxanthin anabolic pathway,the fermentation of S.platensis by X.dendrorhous to increase the accumulation of astaxanthin was studied.The results showed that there was a good linear relationship between the biomass of X.dendrorhous and the bacterial cell concentration.After replacing organic nitrogen source and some glucose by S.platensis,the growth stability of X.dendrorhous was delayed,and the biomass increased.Lower pH(pH<5)was not conducive to biomass accumulation but promoted the synthesis of astaxanthin.And the astaxanthin was mainly accumulated during the stable period of X.dendrorhous.The fermentation scheme(3.5 g/L of S.platensis,pH 5.5,and 104 h of the fermentation time)for maximizing the accumulation of astaxanthin was optimized by random centroid optimization(RCO)method.Compared with standard culture,biomass content of X.dendrorhous and astaxanthin content increased by 33.60%and 58.46%,respectively.Compared with standard fermentation,?-cryptoxanthin,zeaxanthin,canthaxanthin and astaxanthin content increased by 27.76%,359.66%,251.18%and 60.14%in the X.dendrorhous fermentation with S.platensis;compared with S.platensis,the content of ?-carotene,?-cryptoxanthin and zeaxanthin was reduced by 76.61%,76.96%and 20.08%,respectively.The results indicated that the S.platensis promoted the development of carotenoids metabolism of X.dendrorhous to the synthesis direction of astaxanthin,thereby promoting the accumulation of astaxanthin.By extraction,separation,purification,and recrystallization from the lyophilized powder of X.dendrorhous fermentation with S.platensis,the purified astaxanthin was obtained,and the purity was 91.32%.(3)The astaxanthin preliposomes(AP)and astaxanthin-polymer-lipid nanocapsules(APLN)with different loadings were prepared by using purified astaxanthin,and their characterization were analysed.FE-SEM and TEM results showed that the two astaxanthin-loading microparticles were spherical structure and had good dispersion in water.The particle size distribution and dispersion degree of AP and APLN were uniform with a large electrostatic repulsive force between the particles.AP and APLN both exhibited higher encapsulation efficiency and effective loading,and selecting this loading particle for subsequent research.Solid-state characterization analysis(FTIR and DSC)indicated that astaxanthin interacted with each other during the dispersion process in the carrier and the embedding effect was excellent.The storage stability experiment results showed that the two astaxanthin-loading microparticles(1%loading)significantly reduced the degradation of astaxanthin.The degradation rate of APLN at different storage temperatures is lower,which decreased by 2.96%(0?),2.09%(25?),and 5.22%(37?)compared with AP,indicating that APLN has a better effect on improving the stability of astaxanthin.In vitro gastrointestinal simulated digestion results showed that the particle size of the two astaxanthin-loading microparticles increased first and then decreased,corresponding to the trend of the measured encapsulation rate.Notably,after the intestinal simulation,the encapsulation rate of AP and APLN decreased to 32.97%and 49.89%,which indicated that astaxanthin was released at a large scale at this stage.The results of chemical antioxidant activity showed that the astaxanthin-containing microparticles had stronger DPPH free radical scavenging power and FRAP reducing ability than unembedded astaxanthin.(4)The protective effect of astaxanthin-loading microparticles on Caco-2 cells under oxidative stress were studied.The results showed that when the concentration of astaxanthin was 2 ?g/mL,AST,AP and APLN and two empty carriers(BP and BPLN)cultured with Caco-2 cells for 24 h,there was no toxic damage to Caco-2 cell survival rate and LDH leakage rate,indicating they can be used to explore the role of antioxidant protection in Caco-2 cells under oxidative stress.The strong pro-oxidant(400 ?M t-BOOH)was used to establish an oxidative stressed Caco-2 cells model with 2 h.The results showed that the two astaxanthin-loading microparticles could increase the survival rate,reduce LDH leakage rate,inhibit excessive ROS production and excessive accumulation of protein carbonylation products,and reduce the activity of four key intracellular antioxidant enzymes(SOD,CAT,GPx,and GR)of oxidative stressed Caco-2 cells.The primary mechanism of astaxanthin-loading microparticles to protect oxidative stressed Caco-2 cells was through scavenging free radicals in cells. |
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