New strategies to study in depth the metabolic mechanism of astaxanthin biosynthesis in Phaffia rhodozyma.

Astaxanthin, a ketone carotenoid known for its high antioxidant activity, holds significant potential for application in nutraceuticals, aquaculture, and cosmetics. The increasing market demand necessitates a higher production of astaxanthin using Phaffia rhodozyma. Despite extensive research efforts focused on optimizing fermentation conditions, employing mutagenesis treatments, and utilizing genetic engineering technologies to enhance astaxanthin yield in P. rhodozyma, progress in this area remains limited. This review provides a comprehensive summary of the current understanding of rough metabolic pathways, regulatory mechanisms, and preliminary strategies for enhancing astaxanthin yield. However, further investigation is required to fully comprehend the intricate and essential metabolic regulation mechanism underlying astaxanthin synthesis. Specifically, the specific functions of key genes, such as crtYB, crtS, and crtI, need to be explored in detail. Additionally, a thorough understanding of the action mechanism of bifunctional enzymes and alternative splicing products is imperative. Lastly, the regulation of metabolic flux must be thoroughly investigated to reveal the complete pathway of astaxanthin synthesis. To obtain an in-depth mechanism and improve the yield of astaxanthin, this review proposes some frontier methods, including: omics, genome editing, protein structure-activity analysis, and synthetic biology. Moreover, it further elucidates the feasibility of new strategies using these advanced methods in various effectively combined ways to resolve these problems mentioned above. This review provides theory and method for studying the metabolic pathway of astaxanthin in P. rhodozyma and the industrial improvement of astaxanthin, and provides new insights into the flexible combined use of multiple modern advanced biotechnologies.

Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (Tm) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.

Fermentation of waste water from agar processing with Bacillus subtilis by metabolomic analysis.

Fungal infection has become a major threat to crop loss and affects food safety. The waste water from agar processing industries extraction has a number of active substances, which could be further transformed by microorganisms to synthesize antifungal active substances. In this study, Bacillus subtilis was used to ferment the waste water from agar processing industries extraction to analyze the antifungal activity of the fermentation broth on Alternaria alternata and Alternaria spp. Results showed that 25% of the fermentation broth was the most effective in inhibited A. alternata and Alternaria spp., with fungal inhibition rates of 99.9% and 96.1%, respectively, and a minimum inhibitory concentration (MIC) was 0.156 µg/mL. Metabolomic analysis showed that flavonoid polyphenols such as coniferyl aldehyde, glycycoumarin, glycitin, and procyanidin A1 may enhance the inhibitory activity against the two pathogenic fungal strains. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that polyphenols involved in the biosynthesis pathways of isoflavonoid and phenylpropanoid were upregulated after fermentation. The laser confocal microscopy analyses and cell conductivity showed that the cytoplasm of fungi treated with fermentation broth was destroyed. This study provides a research basis for the development of new natural antifungal agents and rational use of seaweed agar waste. KEY POINTS: • Bacillus subtilis fermented waste water has antifungal activity • Bacillus subtilis could transform active substances in waste water • Waste water is a potential raw material for producing antifungal agents.

Transcriptomics analysis and fed-batch regulation of high astaxanthin-producing Phaffia rhodozyma/Xanthophyllomyces dendrorhous obtained through adaptive laboratory evolution.

Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma. ONE-SENTENCE SUMMARY: A high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.

Metabolomics of astaxanthin biosynthesis and corresponding regulation strategies in Phaffia rhodozyma.

Astaxanthin is a valuable carotenoid and is used as antioxidant and health care. Phaffia rhodozyma is a potential strain for the biosynthesis of astaxanthin. The unclear metabolic characteristics of P. rhodozyma at different metabolic stages hinder astaxanthin's promotion. This study is conducted to investigate metabolite changes based on quadrupole time-of-flight mass spectrometry metabolomics method. The results showed that the downregulation of purine, pyrimidine, amino acid synthesis, and glycolytic pathways contributed to astaxanthin biosynthesis. Meanwhile, the upregulation of lipid metabolites contributed to astaxanthin accumulation. Therefore, the regulation strategies were proposed based on this. The addition of sodium orthovanadate inhibited the amino acid pathway to increase astaxanthin concentration by 19.2%. And the addition of melatonin promoted lipid metabolism to increase the astaxanthin concentration by 30.3%. It further confirmed that inhibition of amino acid metabolism and promotion of lipid metabolism were beneficial for astaxanthin biosynthesis of P. rhodozyma. It is helpful in understanding metabolic pathways affecting astaxanthin of P. rhodozyma and provides regulatory strategies for metabolism.

Characterisation of marine bacterium Microbulbifer sp. ALW1 with Laminaria japonica degradation capability.

Marine bacterium Microbulbifer sp. ALW1 was revealed to be able to effectively degrade Laminaria japonica thallus fragments into fine particles. Polysaccharide substrate specificity analysis indicated that ALW1 could produce extracellular alginate lyase, laminarinase, fucoidanase and cellulase. Based on alignment of the 16 S rRNA sequence with other reference relatives, ALW1 showed the closest relationship with Microbulbifer aggregans CCB-MM1T. The cell morphology and some basic physiological and biochemical parameters of ALW1 cells were characterised. ALW1 is a Gram-negative, rod- or oval-shaped, non-spore-forming and non-motile bacterium. The DNA-DNA relatedness values of ALW1 with type strains of M. gwangyangensis (JCM 17,800), M. aggregans (JCM 31,875), M. maritimus (JCM 12,187), M. okinawensis (JCM 16,147) and M. rhizosphaerae (DSM 28,920) were 28.9%, 43.3%, 41.2%, 35.4% and 45.6%, respectively. The major cell wall sugars of ALW1 were determined to be ribose and galactose, which differed from other closely related species. These characteristics indicated that ALW1 could be assigned to a separate species of the genus Microbulbifer. The complete genome of ALW1 contained one circular chromosome with 4,682,287 bp and a GC content of 56.86%. The putative encoded proteins were categorised based on their functional annotations. Phenotypic, physiological, biochemical and genomic characterisation will provide insights into the many potential industrial applications of Microbulbifer sp. ALW1.Key points.

Characterization of the inhibition of aldose reductase with p-coumaric acid ethyl ester.

The inhibition of aldose reductase is an effective strategy to alleviate symptoms of diabetic complications. The p-coumaric acid ethyl ester (p-CAEE) was taken as an example to investigate the inhibition of aldose reductase from p-coumaric acid derivations. The results showed p-CAEE strongly inhibited aldose reductase with the half inhibitory concentration of 1.92 µM, following the noncompetitive manner with a Ki value of 0.94 µM. After binding with p-CAEE, the enzyme showed increased ß-sheet content, and the α-helix content, random coil content, and intrinsic fluorescence strength decreased. p-CAEE bonded with aldose reductase at the anionic, hydrophobic, and selective pockets of the enzyme, via hydrogen bond and hydrophobic interactions with Thr113, Cys80, Trp111, and Leu300, etc. The strong inhibition was related to the high oil-water partition coefficient and special esterify group. This study provides new information to develop aldose reductase inhibitors from p-coumaric acid derivations. PRACTICAL APPLICATIONS: Inhibition of aldose reductase is an effective strategy to alleviate and control the symptoms of diabetic complications. In this study, it has been shown that p-coumaric acid ethyl ester could strongly inhibit the aldose reductase. In addition, the inhibition of aldose reductase was been correlated with structures and oil-water partition coefficients of p-coumaric acid derivatives. It provides a theoretical basis for the development of effective aldose reductase inhibitors.

Characterization of a Thermostable and Surfactant-Tolerant Chondroitinase B from a Marine Bacterium Microbulbifer sp. ALW1.

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural characterization. The recombinant ChSase B6 showed optimum activity at 40 °C and pH 8.0, with enzyme kinetic parameters of Km and Vmax against chondroitin sulfate B (CSB) to be 7.85 µg/mL and 1.21 U/mg, respectively. ChSase B6 demonstrated thermostability under 60 °C for 2 h with about 50% residual activity and good pH stability under 4.0-10.0 for 1 h with above 60% residual activity. In addition, ChSase B6 displayed excellent stability against the surfactants including Tween-20, Tween-80, Trion X-100, and CTAB. The degradation products of ChSase B6-treated CSB exhibited improved antioxidant ability as a hydroxyl radical scavenger. Structural analysis and site-directed mutagenesis suggested that the conserved residues Lys248 and Arg269 were important for the activity of ChSase B6. Characterization, structure, and molecular dynamics simulation of ChSase B6 provided a guide for further tailoring for its industrial application for chondroitin sulfate bioresource development.

Simultaneous Inhibitory Effects of All-Trans Astaxanthin on Acetylcholinesterase and Oxidative Stress.

Alzheimer´s disease is a global neurodegenerative health concern. To prevent the disease, the simultaneous inhibition of acetylcholinesterase and oxidative stress is an efficient approach. In this study, the inhibition effect of all-trans astaxanthin mainly from marine organisms on acetylcholinesterase and oxidative stress was evaluated by a chemical-based method in vitro and cell assay model. The results show that all-trans astaxanthin was a reversible competitive inhibitor and exhibited a strong inhibition effect with half inhibitory concentration (IC50 value) of 8.64 µmol/L. Furthermore, all-trans astaxanthin inhibited oxidative stress through reducing malondialdehyde content and increasing the activity of superoxide dismutase as well as catalase. All-trans astaxanthin could induce the changes of the secondary structure to reduce acetylcholinesterase activity. Molecular-docking analysis reveals that all-trans astaxanthin prevented substrate from binding to acetylcholinesterase by occupying the space of the active pocket to cause the inhibition. Our finding suggests that all-trans astaxanthin might be a nutraceutical supplement for Alzheimer´s disease prevention.

Toward Understanding the Alginate Catabolism in Microbulbifer sp. ALW1 by Proteomics Profiling.

The bacterial strain of Microbulbifer sp. ALW1 has demonstrated visible ability of degrading the cell wall of Laminaria japonica, and biochemical characterization has been performed on some individual enzymes to elucidate its genetic basis. However, it still remains elusive how strain ALW1 successfully breaks down the major cell wall component alginate polysaccharide and colonizes on its marine host. In this study, a mass spectrometry-based quantitative analysis of the extracellular and intracellular proteomes was introduced to elucidate the alginate degradation pathway in ALW1 strain. Mass spectrometry and biochemical assays indicated that strain ALW1 could effectively degrade alginate polysaccharide into disaccharides and trisaccharides within 12 h. Proteome analysis identified 156 and 1,047 proteins exclusively localized in extracellular and intracellular compartments, respectively, with 1,086 protein identities of dual localization. Functional annotation of the identified proteins suggested the involvement of diverse catalytic enzymes and non-catalytic molecules for the cleavage and metabolism of alginate polysaccharide. A simplified pathway was constructed to demonstrate the extracellular digestion, active transport, and intracellular conversion of alginate polysaccharide and its fragmented oligosaccharides, casting a picture of genetic loci controlling alginate catabolism by ALW1 strain. This study aims to provide a guide for utilization and genetic manipulation of the bacterial strain ALW1 for efficient alginate oligosaccharides production by fermentation.

The identification of biotransformation pathways for removing fishy malodor from Bangia fusco-purpurea using fermentation with Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is effective in reducing the fishy malodor of sea products. However, the biotransformation pathways are still unclear. The seaweed B. fusco-purpurea was taken as an example to investigate the chemical transformation pathways for the deodorization process with S. cerevisiae fermentation. Sensory evaluation, GC-MS, GC-MS-O and odor activity value (OAV) analyses showed the fishy odorants were 1-octen-3-ol, (E)-2-nonenal, 2,4-decadienal, 2-pentylfuran, 2-octen-1-ol and nonanal. The removal of fishy malodor was related to the reactions of reduction, dehydrogenation, deformylation-oxygenation and ester syntheses via catalysis of aldehyde dehydrogenase, alcohol dehydrogenases, epoxide hydrolase, aldehyde deformylating-oxygenase, enone reductase, oxidases, dehydrogenases, aldo-keto reductases, ester synthase and acyltransferase. Interestingly, for the first time, it was found that 3,5-octadien-2-one transformed to 6-octen-2-one; and 2-pentylfuran transformed to o-cymene and hexyl acetate. Our findings enrich the knowledge for the removal of fishy malodor from sea products such as seaweeds.

Removal of the fishy malodor from Bangia fusco-purpurea via fermentation of Saccharomyces cerevisiae, Acetobacter pasteurianus, and Lactobacillus plantarum.

The present study aims to evaluate the deodorization of Bangia fusco-purpurea using microorganism fermentation with commercial starter cultures of Saccharomyces cerevisiae, Acetobacter pasteurianus, and Lactobacillus plantarum. The results showed the fermentation with S. cerevisiae, A. pasteurianus, and L. plantarum resulted in significantly decreases (p < .05) of the fishy malodor in B. fusco-purpurea. Among the three strains, S. cerevisiae was the best for reducing the fishy malodor. The optimal inoculum size and fermentation time were 0.2% and 4 hr, respectively. After the fermentation with the S. cerevisiae, the content of 1-octen-3-ol, (E)-2-octen-1-ol, hexanal, non-(2E)-enal, (E,E)-2,4-decadienal, 3,5-octadien-2-one, and 2-pentyl-furan were hard to be detected in the seaweed, whereas increases were observed in the concentrations of 2-butyl-1-octanol, cedrol, diisobutyl phthalate, and 2,4-di-t-butylphenol. The odor active value analysis indicated the removal of fishy odor was related to the reduction, dehydrogenation, and deformylating oxygenation of hexanal, nonanal, non-(2E)-enal, and (E,E)-2,4-decadienal and esterification of 1-octen-3-ol and (E)-2-octen-1-ol. Our findings provide a technical and scientific basis for the removal of fishy odor from B. fusco-purpurea. PRACTICAL APPLICATIONS: Bangia fusco-purpurea is a seaweed that can reduce the risks of cardiovascular and chronic metabolic diseases in human body. However, the seaweed has a strong fishy malodor, which largely declines consumer's acceptance. In this study, the commercial starters of Saccharomyces cerevisiae, Acetobacter pasteurianus, and Lactobacillus plantarum were shown to reduce the fishy malodor in B. fusco-purpurea via fermentation. After the fermentation with the microorganisms especially with the S. cerevisiae, the fishy malodor significantly reduced, and the overall aroma acceptance of B. fusco-purpurea products greatly improved. Therefore, this study provides a technical basis for the removal of fishy odor from B. fusco-purpurea and processing value-added products from it and facilitating its health benefits for human.

Tyrosinase inhibition by p-coumaric acid ethyl ester identified from camellia pollen.

A tyrosinase inhibitor was separated from camellia pollen with the aid of solvent fraction, macroporous adsorptive resin chromatography, and high-speed countercurrent chromatography. The inhibitor was identified to be p-coumaric acid ethyl ester (p-CAEE) by nuclear magnetic resonance and mass spectrum. Its inhibitory activity (IC50 = 4.89 µg/ml) was about 10-fold stronger than arbutin (IC50 = 51.54 µg/ml). The p-CAEE inhibited tyrosinase in a noncompetitive model with the K I and K m of 1.83 µg/ml and 0.52 mM, respectively. Fluorescence spectroscopy analysis showed the p-CAEE quenched an intrinsic fluorescence tyrosinase. UV-Vis spectroscopy analysis showed the p-CAEE did not interact with copper ions of the enzyme. Docking simulation implied the p-CAEE induced a conformational change in the catalytic region and thus changed binding forces of L-tyrosine. Our findings suggest that p-CAEE plays an important role in inhibiting tyrosinase and provides a reference for developing pharmaceutical, cosmetic, and fruit preservation products using pollen.

Effectively Improve the Astaxanthin Production by Combined Additives Regulating Different Metabolic Nodes in Phaffia rhodozyma.

Astaxanthin is an important natural resource that is widely found in marine environments. Metabolic regulation is an effective method for improving astaxanthin production in Phaffia rhodozyma. Most studies have focused on single regulators, which have limited effects. In this study, 16 metabolic regulators were screened to improve astaxanthin production in high-yield and wild-type strains. Fluconazol and glutamic acid increased astaxanthin volumetric yield in MVP14 by 25.8 and 30.9%, respectively, while ethanol increased astaxanthin volumetric yield in DSM626, 29.3%. Furthermore, six additives that inhibit the competing pathways and promote the main pathway for astaxanthin synthesis were selected for combination treatment. We found that the optimal combination was penicillin, ethanol, triclosan, and fluconazol, which increased astaxanthin cell yield by 51%. Therefore, we suggest that simultaneously promoting the master pathways (mevalonate) and inhibiting competing pathways (fatty acid synthesis and ergosterol) is the best strategy to improve astaxanthin cell yield. Moreover, regulators of the biomass pathway should be avoided to improve cell yield. This study provides a technical basis for the utilisation of astaxanthin in P. rhodozyma.

Effects of Lactobacillus fermentum CQPC04 on Lipid Reduction in C57BL/6J Mice.

Probiotics are functional foods that can effectively regulate lipid reduction and maintain body health. In this study, a strain of Lactobacillus fermentum CQPC04 (LF-CQPC04) isolated from traditional naturally fermented vegetables (Sichuan pickles) was studied, and its effects on lipid reduction in mice, as well as its mechanism of action, were observed. The results of this experiment show that LF-CQPC04 can reduce the abnormal weight gain and abnormal visceral index of mice caused by a high-fat diet. LF-CQPC04 can decrease TG (triglycerides), TC (total cholesterol), LDL-c (low-density lipoprotein cholesterol), AST (aspartate transaminase), ALT (alanine aminotransferase), and AKP (alkaline phosphatase) levels and increase HDL-c (high-density lipoprotein cholesterol) levels in the serum of high-fat mice. LF-CQPC04 can also decrease the levels of inflammatory cytokines, such as IL-6 (interleukin-6), IL-1ß (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IFN-γ (interferon gamma), and increase IL-4 and IL-10 levels in the serum of high-fat mice. The results of RT-qPCR (real-time quantitative polymerase chain reaction) and western blot experiments show that LF-CQPC04 can also down-regulate the expression of PPAR-γ (peroxisome proliferator-activated receptor gamma), C/EBP-α (CCAAT/enhances binding protein alpha) mRNA, and protein in the liver tissue of high-fat mice, while up-regulating the expression of Cu/Zn-SOD (copper/zinc superoxide dismutase), Mn-SOD (manganese superoxide dismutase), CAT (catalase), CYP7A1 (cholesterol 7 alpha hydroxylase), PPAR-α (peroxisome proliferator-activated receptor alpha), CPT1 (carnitine palmitoyl transferase 1), LPL (lipoprotein lipase), and ABCA1 (ATP-binding cassette transporter A1). Moreover, LF-CQPC04 shows stronger effects in regulating lipid reduction in mice than L-carnitine and commercial LB (Lactobacillus delbrueckii subsp. Bulgaricus) bacteria. LF-CQPC04 is beneficial for lipid reduction in animals and has good probiotic potential.

Two-dimensional liquid chromatography analysis of all-trans-, 9-cis-, and 13-cis-astaxanthin in raw extracts from Phaffia rhodozyma.

An effective two-dimensional liquid chromatography method has been established for the analysis of all-trans-astaxanthin and its geometric isomers from Phaffia rhodozyma employing a C18 column at the first dimension and a C30 column in the second dimension, connected by a 10-port valve using the photo-diode array detector. The regression equation of astaxanthin calibration curve was established, and the precision and accuracy values were found to be in the range of 0.32-1.14% and 98.21-106.13%, respectively. By using two-dimensional liquid chromatography, it was found that day light, ultrasonic treatment, and heat treatment have significant influence on the content of all-trans-astaxanthin in the extract from P. rhodozyma due to the transformation of all-trans-astaxanthin to cis-astaxanthin. The day light and ultrasonic treatments more likely transform all-trans-astaxanthin to 9-cis-astaxanthin, and the thermal treatment transforms all-trans-astaxanthin to 13-cis-astaxanthin. These results indicate that the two-dimensional liquid chromatography method can facilitate monitoring astaxanthin isomerization in the raw extract from P. rhodozyma. In addition, the study will provide a general reference for monitoring other medicals and bioactive chemicals with geometric isomers.

A Quantitative Analysis Model Established to Determine the Concentration of Each Source in Mixed Astaxanthin from Different Sources.

Astaxanthin from different sources possesses different biological activities and optical isomers. The ingredients of astaxanthin mixtures from different sources on the market have often been mislabeled. Therefore, it is important to determine the sources of astaxanthin and their respective concentrations in a mixture. To solve this problem, a quantitative analysis model was established and further verified. The results showed that the deviation between the calculated concentration and the actual concentration ranged from 0 to 7 µg/mL, and the recovery rate was between 88.90% and 103.56%. This indicates that the quantitative analysis model of astaxanthin was feasible and reliable. This study not only has important applications in the astaxanthin mixture component determination but may also shed light on the quantitative analysis of other sample mixtures with stereoisomers from different sources.

Identification and Characterization of the Tyrosinase Inhibitory Activity of Caffeine from Camellia Pollen.

Tyrosinase inhibitors are important in cosmetic, medical, and food industries due to their regulation of melanin production. A tyrosinase inhibitor was purified from Camellia pollen using high-speed countercurrent chromatography and preparative high-performance liquid chromatography and was identified as caffeine by NMR and mass spectrometry. It showed strong mushroom tyrosinase inhibitory activity with an IC50 of 18.5 ± 2.31 µg/mL in a noncompetitive model. The caffeine did not interact with copper ions in the active center of the enzyme but could quench fluorescence intensity and change the secondary conformation of this tyrosinase. A molecular dynamics simulation showed that caffeine bound this tyrosinase via Lys379, Lys 376, Asp357, Glu356, Thr308, Gln307, Asp312, and Trp358, thus changing the binding sites of l-tyrosine and the loop conformation adjacent to the active center. In vitro cell model analysis revealed that caffeine exhibited significant inhibitory effects on both intracellular tyrosinase activity and melanin production of B16-F10 melanoma cells in a concentration-dependent manner. These comprehensive results suggest that caffeine is a strong tyrosinase inhibitor that has the potential to be developed as skin-whitening agents in the cosmetics and pharmaceutical industries or as antibrowning agents in the food industry.

Antioxidant capacity and prebiotic effects of Gracilaria neoagaro oligosaccharides prepared by agarase hydrolysis.

Gracilaria is a genus of red algae widely cultivated in Asia and is notable for its economic importance as food ingredients. Neoagaro oligosaccharides (NAOSs) are products of Gracilaria that have excellent water solubility, antioxidant activity and prebiotic effect. In this study, Gracilaria crude polysaccharide was treated with agarase and hydrolyzed into NAOSs with different degrees of polymerization (DP). The compositions of the hydrolyzed NAOSs were analyzed by electrospray ionization-time of flight-mass spectrometry and thin layer chromatography. The antioxidant capacity and prebiotic effects of NAOSs with different DPs were investigated and the results showed that DP could affect the antioxidant capacity of NAOSs. The prebiotic effects of NAOSs with different DP were evaluated based on the influence on the growth of four intestinal bacteria. NAOSs promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus and Sterptococcus thermophilus. The protective effect of Gracilaria NAOSs in simulated gastrointestinal juice was also studied. Finally, NAOSs with best prebiotic effects were used in Procambarus feeding experiment and exhibited promotion effect on Procambarus growth. The present study showed that Gracilaria NAOSs can be utilized as antioxidant and prebiotic additive, which had a considerable potential in food and feed industry.

Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography.

An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100mg crude extract of P. rhodozyma was separated to yield 20.6mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS.