Promotion of astrocyte-neuron glutamate-glutamine shuttle by SCFA contributes to the alleviation of Alzheimer's disease.

The brain is particularly susceptible to oxidative damage which is a key feature of several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease. The shuttling of glutathione (GSH) precursors from astrocytes to neurons has been shown to be instrumental for the neuroprotective activity. Here, we revealed that short chain fatty acids (SCFA), which have been related to AD and PD, could promote glutamate-glutamine shuttle to potentially resist oxidative damage in neurons at cellular level. Furthermore, we performed nine-month-long dietary SCFA supplementations in APPswe/PS1dE9 (APP/PS1) mice, and showed that it reshaped the homeostasis of microbiota and alleviated the cognitive impairment by reducing Aß deposition and tau hyperphosphorylation. Single-cell RNA sequencing analysis of the hippocampus revealed SCFA can enhance astrocyte-neuron communication including glutamate-glutamine shuttle, mainly by acting on astrocyte in vivo. Collectively, our findings indicate that long-term dietary SCFA supplementations at early aging stage can regulate the neuroenergetics to alleviate AD, providing a promising direction for the development of new AD drug.

Construction and heterologous expression of the di-AFN A1 biosynthetic gene cluster in Streptomyces model strains.

Natural cyclohexapeptide AFN A1 fromStreptomyces alboflavus 313 has moderate antibacterial and antitumor activities. An artificial designed AFN A1 homodimer, di-AFN A1, is an antibiotic exhibiting 10 to 150 fold higher biological activities, compared with the monomer. Unfortunately, the yield of di-AFN A1 is very low (0.09 ± 0.03 mg·L-1) in the engineered strain Streptomyces alboflavus 313_hmtS (S. albo/313_hmtS), which is not friendly to be genetically engineered for titer improvement of di-AFN A1 production. In this study, we constructed a biosynthetic gene cluster for di-AFN A1 and increased its production through heterologous expression. During the collection of di-AFN A1 biosynthetic genes, the afn genes were located at three sites of S. alboflavus 313 genome. The di-AFN A1 biosynthetic gene cluster (BGC) was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24, which produced di-AFN A1 at a titer of 0.43 ± 0.01 mg·L-1. To further increase the yield of di-AFN A1, the di-AFN A1 BGC was multiplied and split to mimic the natural afn biosynthetic genes, and the production of di-AFN A1 increased to 0.62 ± 0.11 mg·L-1 in S. lividans TK24 by the later strategy. Finally, different Streptomyces hosts were tested and the titer of di-AFN A1 increased to 0.81 ± 0.17 mg·L-1, about 8.0-fold higher than that in S. albo/313_hmtS. Successful heterologous expression of di-AFN A1 with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.

Isolation and Functional Characterization of Multiple NADPH-Cytochrome P450 Reductase Genes from Camellia sinensis in View of Catechin Biosynthesis.

Catechins are critical constituents for the sensory quality and health-promoting benefits of tea. Cytochrome P450 monooxygenases are required for catechin biosynthesis and are dependent on NADPH-cytochrome P450 reductases (CPRs) to provide reducing equivalents for their activities. However, CPRs have not been identified in tea, and their relationship to catechin accumulation also remains unknown. Thus, three CsCPR genes were identified in this study, all of which had five CPR-related conserved domains and were targeted to the endoplasmic reticulum. These three recombinant CsCPR proteins could reduce cytochrome c using NADPH as an electron donor. Heterologous co-expression in yeast demonstrated that all the three CsCPRs could support the enzyme activities of CsC4H and CsF3'H. Correlation analysis indicated that the expression level of CsCPR1 (or CsCPR2 or CsCPR3) was positively correlated with 3',4',5'-catechin (or total catechins) content. Our results indicate that the CsCPRs are involved in the biosynthesis of catechins in tea leaves.

CsUGT78A15 catalyzes the anthocyanidin 3-O-galactoside biosynthesis in tea plants.

Anthocyanins are a group of natural water-soluble pigments in plants that contribute to the pink-purple color of a range of tissues. Because anthocyanins have various biological activities in human health, there is great research interest in the development of anthocyanin-rich foods and beverages, including purple shoot tea. Anthocyanidin 3-O-galactosides have been identified as one of the main anthocyanin components in purple shoot tea, but the enzyme responsible for their biosynthesis remains unclear. UDP-galactose anthocyanidin 3-O-galactosyltransferase (UA3GalT) is presumed to catalyze the galactosylation of anthocyanidin. Therefore, we assayed the UA3GalT activity in five tea samples with varying degrees of purple color and found that its activity was strongly positively correlated (r = 0.929, p < 0.05) with anthocyanin content. Phylogenetic analysis and sequence alignment suggested that CsUGT78A15 encoded a UA3GalT enzyme. Enzymatic assays indicated that rCsUGT78A15 could catalyze the synthesis of cyanidin 3-O-galactoside and delphinidin 3-O-galactoside using UDP-galactose as a sugar donor, and it showed higher catalytic efficiency towards delphinidin than cyanidin. These results indicate that CsUGT78A15 acts as a UA3GalT in vitro. Subcellular localization showed that CsUGT78A15 was located in the endoplasmic reticulum (ER) and nucleus, consistent with the location of anthocyanin synthesis. Transient overexpression of CsUGT78A15 in the fruit of mature 'Granny Smith' apples showed that the upregulation of CsUGT78A15 promoted cyanidin 3-O-galactoside accumulation in apple skins. These results suggested that CsUGT78A15 could catalyze galactosylation of anthocyanidins in planta. Our findings provide insight into the biosynthesis of anthocyanins in tea plants.

Peniterpenoids A-C, new sesquiterpenoid metabolites from a wheat cyst nematode Penicillium janthinellum.

Three new sesquiterpenoids, peniterpenoids A - C (1-3), together with six known metabolites (4-9) were isolated from an entomogenous fungus Penicillium janthinellum (LB1.20090001) collected from a wheat cyst nematode. The structures of the new compounds were elucidated based on NMR and HRESIMS spectroscopic analyses. The absolute configuration of the C-8 secondary alcohol of peniterpenoid B (2) was determined by [Rh2(OCOCF3)4]-induced ECD experiment. Subsequently, the antimicrobial and DPPH scavenging activities were determined. Compounds 6-8 exhibited moderate antibacterial activities against Staphylococcus aureus (CGMCC1.2465) with MIC values of 25.0, 50.0 and 12.5 µg/mL, respectively.

Identification and α-Glucosidase Inhibitory Activity of Meroterpenoids from Hericium erinaceus.

Hericium erinaceus is a very popular edible and medicinal mushroom used for the treatment of enervation and gastrointestinal diseases in Eastern Asia. Chemical investigation on the fruiting body of Hericium erinaceus led to the isolation of 4 new (1:  - 4: ) and 10 known meroterpenoids (5:  - 14: ). The structures of new compounds were determined via analysis of NMR and MS data in combination with chemical derivatization. The inhibitory activities of 1:  - 14: against α-glucosidase were evaluated using p-nitrophenyl-α-D-glucopyranoside, sucrose, or maltose as substrate. Compounds 6, 9, 11:  - 13: were demonstrated to show the α-glucosidase inhibitory activities. This work confirms the potential of H. erinaceus in the treatment of diabetes.

Cordycepamides A-E and cordyglycoside A, new alkaloidal and glycoside metabolites from the entomopathogenic fungus Cordyceps sp.

Five new alkaloidal metabolites cordycepamides A-E (1-5), and one glycoside metabolite cordyglycoside A (6), together with six known compounds (7-12) were isolated from the entomopathogenic fungus Cordyceps sp. (LB1.18060004) from unidentified insect collected in Baoshan City, Yunnan Province, People's Republic of China. The structures were characterized by NMR and HRESIMS spectroscopic analyses. Cordycepamides A and B (1 and 2) were mixtures of two isomers in 5:4 ratio by integration of 1H NMR spectra. In additional, the structure of cordycepamide A (1) was further confirmed by X-ray crystallography as a pair of enantiomers. Absolute configurations of sugar moiety of cordyglycoside A (6) was confirmed by the acid hydrolysis and subsequent HPLC analysis. The isolated metabolites were evaluated for antimicrobial, cytotoxicity, and the DPPH scavenging assay, only 4 showed modest antioxidant effects in the DPPH scavenging assay (IC50 = 51.42 ± 3.08 µM).

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

The tea plant reference genome and improved gene annotation using long-read and paired-end sequencing data.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future.

Parabacteroides distasonis Alleviates Obesity and Metabolic Dysfunctions via Production of Succinate and Secondary Bile Acids.

We demonstrated the metabolic benefits of Parabacteroides distasonis (PD) on decreasing weight gain, hyperglycemia, and hepatic steatosis in ob/ob and high-fat diet (HFD)-fed mice. Treatment with live P. distasonis (LPD) dramatically altered the bile acid profile with elevated lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) and increased the level of succinate in the gut. In vitro cultivation of PD demonstrated its capacity to transform bile acids and production of succinate. Succinate supplementation in the diet decreased hyperglycemia in ob/ob mice via the activation of intestinal gluconeogenesis (IGN). Gavage with a mixture of LCA and UDCA reduced hyperlipidemia by activating the FXR pathway and repairing gut barrier integrity. Co-treatment with succinate and LCA/UDCA mirrored the benefits of LPD. The binding target of succinate was identified as fructose-1,6-bisphosphatase, the rate-limiting enzyme in IGN. The succinate and secondary bile acids produced by P. distasonis played key roles in the modulation of host metabolism.

Characterization of a heat responsive UDP: Flavonoid glucosyltransferase gene in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) accumulates abundant flavonoid glycosides that are the major bioactive ingredients in tea. Biosynthesis of flavonoid glycosides are catalyzed by UDP-glucosyltransferases (UGTs) that are widely present in plants. Among one hundred and seventy-eight UGTs genes that we have previously identified in tea plant, few of them have been functionally characterized. In the present study, we further identified UGT73A17 gene that is responsible for the biosynthesis of a broad range of flavonoid glycosides. Sequence analysis revealed that the deduced UGT73A17 protein showed high identity with 7-O-glycosyltransferases at amino acid level and it was clustered into the clade containing several 7-O-glycosyltransferases from other plant species. Enzymatic assays revealed that the recombinant UGT73A17 protein (rUGT73A17) exhibited activity toward flavonols (kaempferol, quercetin, and myricetin), flavones (apigenin, luteolin, and tricetin), flavanone (naringenin), isoflavones (genistein) and epicatechin gallate, yielding 7-O-glucosides as the major in vitro products. In particular, rUGT73A17 displayed higher activity at high temperatures (eg. 50°C) than at low temperatures, which was consistent with its relatively high expression level at high temperatures. Two amino acid substitutions at I296L and V466A improved the enzymatic activity of rUGT73A17. Our study demonstrated that UGT73A17 is responsible for the biosynthesis of a broad range of flavonoid glucosides, which is also involved in heat response and quality of tea plant.

Effects of vitro sucrose on quality components of tea plants (Camellia sinensis) based on transcriptomic and metabolic analysis.

BACKGROUND: Tea plants [Camellia sinensis (L.) O. Kuntze] can produce one of the three most widely popular non-alcoholic beverages throughout the world. Polyphenols and volatiles are the main functional ingredients determining tea's quality and flavor; however, the biotic or abiotic factors affecting tea polyphenol biosynthesis are unclear. This paper focuses on the molecular mechanisms of sucrose on polyphenol biosynthesis and volatile composition variation in tea plants. RESULTS: Metabolic analysis showed that the total content of anthocyanins, catechins, and proanthocyanidins(PAs) increased with sucrose, and they accumulated most significantly after 14 days of treatment. Transcriptomic analysis revealed 8384 and 5571 differentially expressed genes in 2-day and 14-day sucrose-treated tea plants compared with control-treated plants. Most of the structural genes and transcription factors (TFs) involved in polyphenol biosynthesis were significantly up-regulated after 2d. Among these transcripts, the predicted genes encoding glutathione S-transferase (GST), ATP-binding cassette transporters (ABC transporters), and multidrug and toxic compound extrusion transporters (MATE transporters) appeared up regulated. Correspondingly, ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) analysis revealed that the content of non-galloylated catechins and oligomeric PAs decreased in the upper-stem and increased in the lower-stem significantly, especially catechin (C), epicatechin (EC), and their oligomeric PAs. This result suggests that the related flavonoids were transported downward in the stem by transporters. GC/MS data implied that four types of volatile compounds, namely terpene derivatives, aromatic derivatives, lipid derivatives, and others, were accumulated differently after in vitro sucrose treatment. CONCLUSIONS: Our data demonstrated that sucrose regulates polyphenol biosynthesis in Camellia sinensis by altering the expression of transcription factor genes and pathway genes. Additionally, sucrose promotes the transport of polyphenols and changes the aroma composition in tea plant.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality.

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Structural Modification of Natural Product Ganomycin I Leading to Discovery of a α-Glucosidase and HMG-CoA Reductase Dual Inhibitor Improving Obesity and Metabolic Dysfunction in Vivo.

It is a great challenge to develop drugs for treatment of metabolic syndrome. With ganomycin I as a leading compound, 14 meroterpene derivatives were synthesized and screened for their α-glucosidase and HMG-CoA reductase inhibitory activities. As a result, a α-glucosidase and HMG-CoA reductase dual inhibitor (( R, E)-5-(4-( tert-butyl)phenyl)-3-(4,8-dimethylnona-3,7-dien-1-yl)furan-2(5 H)-one, 7d) with improved chemical stability and long-term safety was obtained. Compound 7d showed multiple and strong in vivo efficacies in reducing weight gain, lowering HbAlc level, and improving insulin resistance and lipid dysfunction in both ob/ob and diet-induced obesity (DIO) mice models. Compound 7d was also found to reduce hepatic steatosis in ob/ob model. 16S rRNA gene sequencing, SCFA, and intestinal mucosal barrier function analysis indicated that gut microbiota plays a central and causative role in mediating the multiple efficacies of 7d. Our results demonstrate that 7d is a promising drug candidate for metabolic syndrome.

Insight into Catechins Metabolic Pathways of Camellia sinensis Based on Genome and Transcriptome Analysis.

Tea is an important economic crop with a 3.02 Gb genome. It accumulates various bioactive compounds, especially catechins, which are closely associated with tea flavor and quality. Catechins are biosynthesized through the phenylpropanoid and flavonoid pathways, with 12 structural genes being involved in their synthesis. However, we found that in Camellia sinensis the understanding of the basic profile of catechins biosynthesis is still unclear. The gene structure, locus, transcript number, transcriptional variation, and function of multigene families have not yet been clarified. Our previous studies demonstrated that the accumulation of flavonoids in tea is species, tissue, and induction specific, which indicates that gene coexpression patterns may be involved in tea catechins and flavonoids biosynthesis. In this paper, we screened candidate genes of multigene families involved in the phenylpropanoid and flavonoid pathways based on an analysis of genome and transcriptome sequence data. The authenticity of candidate genes was verified by PCR cloning, and their function was validated by reverse genetic methods. In the present study, 36 genes from 12 gene families were identified and were accessed in the NCBI database. During this process, some intron retention events of the CsCHI and CsDFR genes were found. Furthermore, the transcriptome sequencing of various tea tissues and subcellular location assays revealed coexpression and colocalization patterns. The correlation analysis showed that CsCHIc, CsF3'H, and CsANRb expression levels are associated significantly with the concentration of soluble PA as well as the expression levels of CsPALc and CsPALf with the concentration of insoluble PA. This work provides insights into catechins metabolism in tea and provides a foundation for future studies.

Six phenylalanine ammonia-lyases from Camellia sinensis: Evolution, expression, and kinetics.

Phenylalanine ammonia-lyase (PAL), the branch point enzyme controlling the flow of primary metabolism into second metabolism, converts the L-phenylalanine (L-Phe) to yield cinnamic acid. Based on the sequencing data available from eight transcriptome projects, six PAL genes have been screened out, cloned, and designated as CsPALa-CsPALf. The phylogenetic tree showed that CsPALs were divided into three subgroups, PALa and PALb, PALc and PALd, and PALe and PALf. All six CsPALs exhibited indiscriminate cytosolic locations in epidermis cells and mesophyll cells. Then, the expression profiles of six PAL genes were qualitatively investigated and they displayed tissue-/induced-expression specificity in several tissues or under different exogenous treatments. Furthermore, in vitro enzymatic assays showed that all six recombinant proteins were characterized by the strict substrate specificity toward L-Phe, but no activity toward L-Tyr, and they displayed subtle differences in kinetics and enzymatic properties. These results indicate that CsPALs play both distinct and overlapping roles in plant growth and responses to environmental cues.

Sesquiterpenoids with PTP1B Inhibitory Activity and Cytotoxicity from the Edible Mushroom Pleurotus citrinopileatus.

One new perhydrobenzannulated 5,5-spiroketal sesquiterpene, pleurospiroketal F (1), as well as six new modified bisabolene sesquiterpenes pleurotins A-F (2-7) were isolated from solid-state fermentation of Pleurotus citrinopileatus. The structures of compounds 1-7 were determined by NMR and MS spectroscopic analysis. The absolute configuration of 1 was determined by X-ray diffraction analysis, while the absolute configurations of 3-7 were assigned using the in situ dimolybdenum circular dichroism method and circular dichroism data comparison. Protein tyrosine phosphatase 1B plays a crucial role as a negative regulator of the insulin-dependent signal cascades. Therefore, the protein tyrosine phosphatase 1B inhibitor can be used for treating type 2 diabetes mellitus and obesity. Compounds 2 and 6 showed moderate inhibitory effects on protein tyrosine phosphatase 1B with IC50 s of 32.1 µM and 30.5 µM, respectively. The kinetic study confirmed compound 2 to be a noncompetitive inhibitor. Compounds 1-7 did not show cytotoxic activity against cancer cell lines (IC50 > 50 µM).

[Effects of plant fermentation extract (PFE) on biofilms of Pseudomonas aeruginosa].

Objective: We studied the effects of a plant fermentation extract on destroying biofilms of P. aeruginosa, to provide basic information for treatment of P. aeruginosa related infection diseases. Methods: Strains of P. aeruginosa in clinical specimens were isolated by streaking plate method and identified by PCR and sequencing. Virulence factors were examined using reporter strains, and biofilms were measured by test tube method and a confocal laser scanning microscopy. Results: A total of 16 strains of P. aeruginosa were isolated from clinical specimens from a local hospital, among them PA007 strain showed a maximum response when treated with plant fermentation extract. It shows that 1% plant fermentation extract significantly reduced the production of biofilm, pyocyanine and 3-oxo-C12-HSL (P<0.05). Besides, 1% plant fermentation extract also deceased the bioactivity of LasA protease and survival rate of persisters (P<0.05). The qRT-PCR result indicated that the expressions of lasI and pqsA genes were also markedly inhibited at the presence of 1% plant fermentation extract (P<0.05). Conclusion: The studied plant fermentation extract has anti-infection effect against some P. aeruginosa strains, suggesting a great potential to work as natural antibiotics.

Lanostane Triterpenes from the Tibetan Medicinal Mushroom Ganoderma leucocontextum and Their Inhibitory Effects on HMG-CoA Reductase and α-Glucosidase.

Sixteen new lanostane triterpenes, ganoleucoins A-P (1-16), together with 10 known tripterpenes (17-26), were isolated from the cultivated fruiting bodies of Ganoderma leucocontextum, a new member of the Ganoderma lucidum complex. The structures of the new compounds were elucidated by extensive spectroscopic analysis and chemical transformation. The inhibitory effects of 1-26 on HMG-CoA reductase and α-glucosidase were tested in vitro. Compounds 1, 3, 6, 10-14, 17, 18, 23, 25, and 26 showed much stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin. Compounds 13, 14, and 16 presented potent inhibitory activity against α-glucosidase from yeast with IC50 values of 13.6, 2.5, and 5.9 µM, respectively. In addition, the cytotoxicity of 1-26 was evaluated against the K562 and PC-3 cell lines by the MTT assay. Compounds 1, 2, 6, 7, 10, 12, 16, 18, and 25 exhibited cytotoxicity against K562 cells with IC50 values in the range 10-20 µM. Paclitaxel was used as the positive control with an IC50 value of 0.9 µM. This is the first report of secondary metabolites from this medicinal mushroom.

Process optimization for extraction of carotenoids from medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Natural carotenoids have attracted great attention for their important beneficial effects on human health and food coloring function. Cordyceps militaris, a well-known edible and medicinal fungus, is a potential source of natural carotenoids. The present study aimed to optimize the process parameters for carotenoid extraction from this mushroom. The effects of different methods of breaking the fungal cell wall and organic solvents were studied by the one-factor-at-a-time method. Subsequently, the process parameters including the duration of the extraction time, the number of extractions, and the solvent to solid ratio were optimized by using the Box-Behnken design. The optimal extraction conditions included using an acid-heating method to break the cell wall and later extracting three times, each for a 1 h duration, with a 4:1 mixture of acetone: petroleum ether and a solvent: solid ratio of 24:1. The carotenoid content varied from 2122.50 to 3847.50 µg/g dry weights in different commercially obtained fruit bodies of C. militaris. The results demonstrated that the C. militaris contained more carotenoid content in its fruit bodies than other known mushrooms. Stability monitoring by HPLC demonstrated that the carotenoids could be stored at 4°C for 40 d. It is suggested that the carotenoid content should be considered as the quality standard of commercial products of this valued mushroom. These findings will facilitate the exploration of carotenoids from C. militaris.