Exploitation of Sugarcane Bagasse and Environmentally Sustainable Production, Purification, Characterization, and Application of Lovastatin by Aspergillus terreus AUMC 15760 under Solid-State Conditions.

Using the internal transcribed spacer (ITS) region for identification, three strains of Aspergillus terreus were identified and designated AUMC 15760, AUMC 15762, and AUMC 15763 for the Assiut University Mycological Centre culture collection. The ability of the three strains to manufacture lovastatin in solid-state fermentation (SSF) using wheat bran was assessed using gas chromatography-mass spectroscopy (GC-MS). The most potent strain was strain AUMC 15760, which was chosen to ferment nine types of lignocellulosic waste (barley bran, bean hay, date palm leaves, flax seeds, orange peels, rice straw, soy bean, sugarcane bagasse, and wheat bran), with sugarcane bagasse turning out to be the best substrate. After 10 days at pH 6.0 at 25 °C using sodium nitrate as the nitrogen source and a moisture content of 70%, the lovastatin output reached its maximum quantity (18.2 mg/g substrate). The medication was produced in lactone form as a white powder in its purest form using column chromatography. In-depth spectroscopy examination, including 1H, 13C-NMR, HR-ESI-MS, optical density, and LC-MS/MS analysis, as well as a comparison of the physical and spectroscopic data with published data, were used to identify the medication. At an IC50 of 69.536 ± 5.73 µM, the purified lovastatin displayed DPPH activity. Staphylococcus aureus and Staphylococcus epidermidis had MICs of 1.25 mg/mL, whereas Candida albicans and Candida glabrata had MICs of 2.5 mg/mL and 5.0 mg/mL, respectively, against pure lovastatin. As a component of sustainable development, this study offers a green (environmentally friendly) method for using sugarcane bagasse waste to produce valuable chemicals and value-added commodities.

Lovastatin production by an oleaginous fungus, Aspergillus terreus KPR12 using sago processing wastewater (SWW).

BACKGROUND: Lovastatin is one of the first statins to be extensively used for its cholesterol-lowering ability. It is commercially produced by fermentation. Species belonging to the genus Aspergillus are well-studied fungi that have been widely used for lovastatin production. In the present study, we produced lovastatin from sago processing wastewater (SWW) under submerged fermentation using oleaginous fungal strains, A. terreus KPR12 and A. caespitosus ASEF14. RESULTS: The intra- and extracellular concentrations of lovastatin produced by A. terreus KPR12 and A. caespitosus ASEF14 were lactonized. Because A. caespitosus ASEF14 produced a negligible amount of lovastatin, further kinetics of lovastatin production in SWW was studied using the KPR12 strain for 9 days. Lovastatin concentrations in the intra- and extracellular fractions of the A. terreus KPR12 cultured in a synthetic medium (SM) were 117.93 and 883.28 mg L-1, respectively. However, these concentrations in SWW were 142.23 and 429.98 mg L-1, respectively. The yeast growth inhibition bioassay confirmed the antifungal property of fungal extracts. A. terreus KPR12 showed a higher inhibition zone of 14 mm than the ASEF14 strain. The two-way analysis of variance (ANOVA; p < 0.01) showed significant differences in the localization pattern, fungal strains, growth medium, and their respective interactions. The lovastatin yield coefficient values were 0.153 g g-1 on biomass (YLOV/X) and 0.043 g g-1 on the substrate, starch (YLOV/S). The pollutant level of treated SWW exhibited a reduction in total solids (TS, 59%), total dissolved solids (TDS, 68%), biological oxygen demand (BOD, 79.5%), chemical oxygen demand (COD, 57.1%), phosphate (88%), cyanide (65.4%), and void of nutrients such as nitrate (100%), and ammonia (100%). CONCLUSION: The starch-rich wastewater serves as a suitable medium for A. terreus KPR12 for the production of lovastatin. It simultaneously decontaminates the sago processing wastewater, enabling its reuse for irrigation/recreation.

Analysis of Monacolins and Berberine in Food Supplements for Lipid Control: An Overview of Products Sold on the Italian Market.

The use of dietary supplements for the prevention and management of diseases associated with excess of lipids is spreading in Western countries. Supplements containing red yeast rice (RYR) and extracts from Berberis species, characterized, respectively, by the active compounds monacolin K (MK) and berberine (BBR), are sold in pharmacies as over the counter medicines (OTC) and in regular markets without the need of medical prescription and medical surveillance. However, MK is chemically identical to lovastatin, a drug commonly used to treat hypercholesterolemia, and is characterized by the same mechanism of action, pharmacokinetic profile and toxicity. On the other hand, although BBR-containing supplements are considered to be well-tolerated and safe, they frequently show poor standardization of active ingredients, and this could lead to lack of effects. In this work, with the aim to give an overview on the potency of RYR- and BBR-containing supplements available on the Italian market, we analyzed a pool of supplements bought from both local pharmacies and markets. Results confirm the data already published by other authors, showing scarce standardization of bioactives and discrepancy between the doses of bioactives reported by the manufacturers and the amounts resulting from analysis of the same products. Overall, our data represent a further proof that a strict legislation regulating the production and marketing of dietary supplements and a close monitoring of these products by food and drug regulatory organs is mandatory.

On the Identification and Quantification of Ergothioneine and Lovastatin in Various Mushroom Species: Assets and Challenges of Different Analytical Approaches.

In recent years, mushrooms have drawn the attention of agro-industries and food-industries as they were considered to be valuable natural sources of health promoting compounds such as ß-glucans, ergothioneine, and lovastatin. The detection and quantification of such compounds by implementing reliable analytical approaches is of the utmost importance in order to adjust mushrooms' cultivation conditions and maximize the production in different species. Toward this direction, the current study focuses on the comparison of ultraviolet-visible (UV-Vis) spectrometry and liquid chromatography-mass spectrometry (LC-MS) methods (a) by evaluating the content of ergothioneine and lovastatin in mushrooms and (b) by highlighting any possible substrate-based interferences that hinder the accurate determination of these two compounds in order to propose the technique-of-choice for a standardized bioactive compounds monitoring. For this purpose, mushrooms produced by three species (i.e., Agaricus bisporus, Pleurotus ostreatus, and P. citrinopileatus) on various cultivation substrates, namely wheat straw (WS), winery (grape marc (GM)), and olive oil (OL) by-products, were examined. Among the two applied techniques, the developed and validated LC-MS methods, exhibiting relatively short analysis time and higher resolution, emerge as the methods-of-choice for detecting ergothioneine and lovastatin in mushrooms. On the contrary, UV-Vis methods were hindered due to co-absorbance of different constituents, resulting in invalid results. Among the studied mushrooms, P. citrinopileatus contained the highest amount of ergothioneine (822.1 ± 20.6 mg kg-1 dry sample), whereas A. bisporus contained the highest amounts of lovastatin (1.39 ± 0.014 mg kg-1 dry sample). Regarding the effect of different cultivation substrates, mushrooms produced on OL and WS contained the highest amount of ergothioneine, while mushrooms deriving from GM-based substrates contained the highest amount of lovastatin.

Submerged fermentation of Ginkgo biloba seed powder using Eurotium cristatum for the development of ginkgo seeds fermented products.

BACKGROUND: Ginkgo biloba seeds are well known for the significant curative effects on relieving cough and asthma. However, the development of products from ginkgo seeds still falls behind at present, resulting in a great waste of ginkgo seeds' resource. In this work, submerged fermentation of ginkgo seed powder using Eurotium cristatum was studied to investigate its feasibility as a new processing method. RESULTS: To promote the growth of E. cristatum, the optimum fermentation medium was 80.0 g L-1 of ginkgo seed powder with addition of 5.0 g L-1 calcium chloride (CaCl2 ), 4.0 g L-1 magnesium sulfate (MgSO4 ), 1.25 g L-1 zinc sulfate (ZnSO4 ) and 0.65 g L-1 iron(II) sulfate (FeSO4 ). The optimum fermentation conditions were pH 5.8 ± 0.1, inoculum size 5.1 × 106 CFU mL-1 , liquid medium volume 100 mL in 250-mL Erlenmeyer flask and fermentation 4 days. Through fermentation, the production of lovastatin in fermentation broth could reach up to 32.97 ± 0.17 µg mL-1 and the total antioxidant capacity was improved by more than two-fold. In addition, 40.15% of the ginkgotoxin in ginkgo seed powder was degraded while the entire degradation of ginkgolic acids was obtained. Moreover, fermented ginkgo seed powder suspension presented pleasant fragrances, and the activities of amylase and protease were enhanced to 11.30 ± 0.10 U mL-1 and 23.01 ± 0.20 U mL-1 , respectively. CONCLUSIONS: Submerged fermentation using E. cristatum could significantly enhance the functional value and safety of ginkgo seed powder, and had great potential to become a novel processing method for the development of ginkgo seeds fermented products. © 2020 Society of Chemical Industry.

Lovastatin in lactone and hydroxy acid forms and citrinin in red yeast rice powders analyzed by HPTLC-UV/FLD.

For the analysis of pigment-rich red yeast rice products, a fast quantitative high-performance thin-layer chromatography (HPTLC) method was newly developed and validated. The active ingredient lovastatin, present in lactone (LL) and hydroxy acid forms (LH), as well as the mycotoxin citrinin were analyzed in 19 red yeast rice products, including powders, dietary supplements, and Chinese proprietary medicines (Xuezhikang and Zhibituo). The HPTLC method including sample preparation allows a high throughput of matrix-rich samples (10 min per analysis) and is highly cost-efficient (running costs of 0.5 Euro per analysis). For a fast protocol, application volumes up to 10 µL were selected although higher application volumes will lower still the LODs, which were 30 mg/kg for LL and LH as well as 4 mg/kg for citrinin. Thanks to the minimalistic sample preparation, the overall mean recovery rate was good (109.9% ± 5.9%; repeated measurements of the three analytes per fresh sample preparation at three spike levels). Repeated calibrations (five per analyte) in the red yeast rice matrix showed highly satisfying determination coefficients (≥ 0.9991; mean 0.9996). For three analytes at three concentration levels, the obtained mean intermediate precisions in red yeast rice matrix analyzed over the whole procedure including sample preparation were highly satisfying (≤ 2.6%). Citrinin was not detectable in the samples down to the given LOD of 4.0 mg/kg for the 10-µL sample volume applied. The mean content of lovastatin in 15 RYR powders was 8.7 g/kg, with a rang of 1.5-26.2 g/kg. The content of lovastatin in Zhibituo tablets and Xuezhikang capsules was determined to be 2.7 and 11.1 g/kg, respectively. The two commercially available RYR dietary supplement samples showed the highest lovastatin contents of 40.7 and 41.4 g/kg. By these figures of merit, the HPTLC method was proven to be suited for the control of such matrix-rich, fermented food. Graphical abstract.

Fully automated method based on on-line molecularly imprinted polymer solid-phase extraction for determination of lovastatin in dietary supplements containing red yeast rice.

A fully automated method for the determination of lovastatin in dietary supplements containing red yeast rice has been developed. It uses a sequential injection analysis system combined with solid-phase extraction applying highly selective molecularly imprinted polymer sorbent. A miniaturized column for on-line extraction was prepared by packing 4.5 mg of the sorbent in a 5.0 × 2.5-mm-i.d. cartridge, which was used in the flow manifold. Sequential injection analysis manifold enabled all steps of lovastatin extraction and continuous spectrophotometric detection at 240 nm. A limit of detection of 60 µg g-1, a limit of quantitation of 200 µg g-1, and a linear calibration range of 200-2000 µg g-1 were achieved. Intra-day and inter-day precision values (RSD) were ≤ 6.7% and ≤ 4.9%, respectively, and method recovery values of spiked red yeast rice extracts at 200, 1000, and 2000 µg g-1 concentration levels were 82.9, 95.2, and 87.7%. Our method was used for determination of lovastatin lactone in four dietary supplements containing red yeast rice as a natural source of lovastatin, also known as monacolin K. The extracted samples were subsequently analyzed by the reference UHPLC-MS/MS method. Statistical comparison of results (F test, t test, α = 0.05) obtained by both methods did not reveal significant difference. A substantial advantage of the new automated approach is high sample throughput thanks to the analysis time of 7.5 min, miniaturization via down-scaling the extraction column, and smaller sample and solvent consumption, as well as reduced generation of waste. Graphical abstract ᅟ.

Quality and Authenticity Control of Functional Red Yeast Rice-A Review.

Red yeast rice (RYR) is made by fermenting the rice with Monascus. It is commonly used in food colorants, dyeing, and wine making in China and its neighboring countries. Nowadays RYR has two forms on the market: common RYR is used for food products, the other form is functional RYR for medicine. However, some researchers reported that commercial lovastatin (structure is consistent with monacolin K) is illegally added to common RYR to meet drug quality standards, so as to imitate functional RYR and sell the imitation at a higher price. Based on current detection methods, it is impossible to accurately distinguish whether functional RYR is adulterated. Therefore, it is especially important to find a way to authenticate functional RYR. In the current review, the advances in history, applications, components (especially monacolins, monacolins detection methods), quality standards, authentication methods and perspectives for the future study of RYR are systematically reviewed.

Determination of lovastatin, mevastatin, rosuvastatin and simvastatin with HPLC by means of gradient elution.

A novel HPLC method with UV detection was developed and validated in skin penetration (in vitro) studies to identify and quantify lovastatin, mevastatin, rosuvastatin and simvastatin. A Venusil XBP C18 (2), 150 x 4.6 mm, 5 µm column (Agela Technologies, Newark, DE) was used with gradient elution (start at 45 % acetonitrile and increase linearly to 90 % after 1 min; hold at 90 % until 6 min and then re-equilibrate at start conditions) and the mobile phase consisted of (A) Milli-Q ® water and 0.1% orthophosphoric acid, and (B) HPLC grade acetonitrile. The flow rate was set at 1 ml/min, 240 nm UV detection and an injection volume of 10 µl. Linearity was obtained over a range of 0.50-200.00 µg/ml and correlation coefficients ranging from 0.998-1.000 were obtained. Average recovery ranged from 95.9-100.6 %. The LOD and LOQ values obtained from the slope of a calibration curve and the standard deviation of the response ranged from 0.0138-0.0860 µg/ml and 0.0419-0.2615 µg/ml, respectively, where lovastatin and simvastatin could be detected at a concentration similar to the other statins, but could only be quantified at a higher concentration than the remaining statins. The specificity of the method was proved as accurate and quantification of statins was found, even within the incorporation of other compounds.

Optimization of process variables for increased production of lovastatin in Aspergillus terreus PU-PCSIR1 and its characterization.

During intrinsic cholesterol formation 3-hydroxy-3-methylgutaryl coenzyme A reductase (HMGCR) converts HMGCoA to mevalonate, in biosynthetic cascade of cholesterol. Statins, competitive inhibitors of HMGCR, now-a-days commonly used to lower the blood-cholesterol level in the hyper-cholesterolemic patients. Lovastatin, one of the most potent natural statins, was produced from wild-type indigenous isolate Aspergillus terreus PU-PCSIR-1, through solid state fermentation (SSF). This study was carried out to investigate different parameters influencing lovastatin production such as pH, carbon source, nitrogen source and media components etc. Each parameter was investigated separately to optimize lovastatin production. Maximum yield of 2860mg/Kg of total lovastatin, comprising 1700 and 1160mg/Kg of hydroxy and lactone forms respectively, was achieved after incubating for 14 days, pH 5.5 and at 28°C. The integrity of biotechnologically-produced lovastatin was analyzed using high performance liquid chromatography (HPLC). Lovastatin was purified by preparative HPLC, and was characterized by FT-IR and LC-MS analyses. The study revealed that A. terreus PU-PCSIR-1 has been proved to be a potent strain for the production of lovastatin that has great pharmaceutical and commercial applications.

Rapid determination of lovastatin in the fermentation broth of Aspergillus terreus using dual-wavelength UV spectrophotometry.

CONTEXT: Lovastatin, a hypocholesterolemic drug, is produced by submerged fermentation of Aspergillus terreus Thom (Trichocomaceae). High performance liquid chromatography is usually used to determine lovastatin in samples of the fermentation broth. However, this method is inconvenient and costly, especially in the context of high-throughput sample analysis. OBJECTIVE: A direct and simple dual-wavelength ultraviolet spectrophotometric method for quantifying lovastatin in the fermentation broth of A. terreus was developed. MATERIALS AND METHODS: A. terreus Z15-7 was used for all experiments. The liquid fermentation was conducted at 30 °C in a rotary shaker at 150 rpm for 15 d. Silica gel and neutral alumina column chromatography were used for the separation and purification of lovastatin from the fermentation broth. RESULTS: The limits of detection of lovastatin were 0.320 µg/ml in the lovastatin standard solution and 0.490 µg/ml in the fermentation broth sample and the limits of quantification of lovastatin were 1.265 µg/ml in the lovastatin standard solution and 3.955 µg/ml in the fermentation broth sample. The amounts of lovastatin in the fermentation broth ranged from 876.614 to 911.967 µg/ml, with relative standard deviations from 1.203 to 1.709%. The mean recoveries of lovastatin using silica gel and neutral alumina column chromatography were 84.2 ± 0.82 and 87.2 ± 0.21%, respectively. DISCUSSION AND CONCLUSION: Dual-wavelength UV spectrophotometry is a rapid, sensitive, accurate, and convenient method for quantifying lovastatin in fermentation broth. Neutral alumina column chromatography is more efficient than silica gel column chromatography for the purification and determination lovastatin using the developed dual-wavelength UV spectrophotometry method.

Generation of statin drug metabolites through electrochemical and enzymatic oxidations.

The generation of key drug metabolites for the purpose of their complete structural characterization, toxicity testing, as well as to serve as standards for quantitative studies, is a critical step in the pharmaceutical discovery and development cycle. Here, we utilized electrochemistry/mass spectrometry for the detection and subsequent generation of six phase I metabolites of simvastatin and lovastatin. Both simvastatin and lovastatin are widely used for the treatment of hypercholesterolemia. There are known drug-drug interaction issues of statin therapy, and it has been suggested that the oxidative metabolites may contribute to the cholesterol-lowering effect of both statins. Of the known phase I metabolites of simvastatin and lovastatin, none are commercially available, and chemical means for the synthesis of a very few of them have been previously reported. Here, we report that electrochemical oxidation of less than 1 mg each of simvastatin and lovastatin led to the generation of three oxidative metabolites of each parent to allow complete nuclear magnetic resonance characterization of all six metabolites. The yields obtained by the electrochemical approach were also compared with incubation of parent drug with commercially available bacterial mutant CYP102A1 enzymes, and it was found that the electrochemical approach gave higher yields than the enzymatic oxidations for the generation of most of the observed oxidative metabolites in this study.

Simplification of pharmacopoeial liquid chromatography methods for related substances of statins by hyphenated ultraviolet and charged aerosol detection.

For a more comprehensive characterization of a drug substance and its impurities, multidetector approaches are a helpful tool in liquid chromatography. In particular, the relatively inexpensive hyphenation of the ultraviolet (UV) with the charged aerosol detector (CAD) extends the scope from UV-active to non- or weak chromophore analytes, respectively. In this study, the chromatographic methods of the test for related substances of simvastatin and lovastatin in the European Pharmacopoeia were adapted to UV-CAD and thus allowing a more sophisticated detection of the weak chromophore dihydro impurity besides the other UV-active impurities. The compendial gradient program for simvastatin had to be modified (lowered initial acetonitrile percentage and increased gradient slope) because an additional critical peak pair emerged with the Hypersil C18 BDS column used here. Therefore, a Plackett-Burman design with 11 factors (including 4 dummy factors) was chosen to evaluate robustness of the adapted method. The flow rate, initial acetonitrile percentage, and column temperature were identified as three critical parameters that had to be carefully observed. Finally, the validity of the method for simultaneous detection of dihydrosimvastatin with CAD and of lovastatin and simvastatin as examples of UV detection was verified according to ICH Q2 (R1). In the case of lovastatin, the direct comparison with the pharmacopoeial method reveal that a determination with CAD is the more sensitive method.

Micellar liquid chromatography as a sustainable tool to quantify three statins in oral solid dosage forms.

A method based on micellar liquid chromatography has been developed to determine rosuvastatin, lovastatin and simvastatin in oral solid dosage forms. Samples were solved in mobile phase up to the target concentration, filtered and directly injected. The three statins were resolved in 30 min, using an aqueous solution of 0.10 M sodium dodecyl sulfate - 7.0% 1-butanol, buffered at pH 3 with 0.01 M phosphate salt as mobile phase, running under isocratic mode at 1 mL/min through a C18 column. Detection was at 240 nm. The effect of sodium dodecyl sulfate on elution strength was more important than that of the organic solvent. The procedure was successfully validated by the guidelines of the International Council for Harmonization in terms of: specificity, linearity (r2 > 0.990), calibration range (1.5 - 15 mg/L for rosuvastatin, 0.5-10 mg/L for lovastatin and simvastatin), limit of detection (0.4, 0.2 and 0.15 mg/L for rosuvastatin, lovastatin and simvastatin, respectively), trueness (98.8-101.7%), precision (<2.7%), carry-over effect, robustness, and stability. Values were inside the acceptance criteria of the Methods, Method Verification and Validation, Food and Drug Administration-Office of Regulatory Affairs, thus ensuring the reliability of the results. The main feature was the low proportion of organic solvent used, thus making the procedure sustainable and green. Besides, it was easy-to-conduct and with high sample-throughput, and then useful for routine analysis in pharmaceutical quality control. Finally, it was applied to commercial pharmaceutical preparations.

Lovastatin production by Aspergillus terreus using agro-biomass as substrate in solid state fermentation.

Ability of two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) for production of lovastatin in solid state fermentation (SSF) using rice straw (RS) and oil palm frond (OPF) was investigated. Results showed that RS is a better substrate for production of lovastatin in SSF. Maximum production of lovastatin has been obtained using A. terreus ATCC 74135 and RS as substrate without additional nitrogen source (157.07 mg/kg dry matter (DM)). Although additional nitrogen source has no benefit effect on enhancing the lovastatin production using RS substrate, it improved the lovastatin production using OPF with maximum production of 70.17 and 63.76 mg/kg DM for A. terreus ATCC 20542 and A. terreus ATCC 74135, respectively (soybean meal as nitrogen source). Incubation temperature, moisture content, and particle size had shown significant effect on lovastatin production (P < 0.01) and inoculums size and pH had no significant effect on lovastatin production (P > 0.05). Results also have shown that pH 6, 25°C incubation temperature, 1.4 to 2 mm particle size, 50% initial moisture content, and 8 days fermentation time are the best conditions for lovastatin production in SSF. Maximum production of lovastatin using optimized condition was 175.85 and 260.85 mg/kg DM for A. terreus ATCC 20542 and ATCC 74135, respectively, using RS as substrate.

Analysis of the differences in the chemical composition of monascus rice and highland barley monascus.

Monascus rice (MR) and highland barley monascus (HBM), the monascus fermented products, are applied in food and medicine to reduce cholesterol and promote digestion. Due to the fermentation substrates, their compositions are different. However, the exact differences have not been reported to date. By UPLC-Q-Orbitrap HRMS analysis, multiple components of twenty batches of MR and HBM samples were identified. In total, 100 components were confirmed (e.g., monacolins, pigments, decalin derivatives, amino acids). Then, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to filter the components of MR and HBM. In the PCA model, 88.1% of the total variance was uncovered. The OPLS-DA model showed better discrimination between MR and HBM, and the values of R2X, R2Y, and Q2 were 0.837, 0.996, and 0.956, respectively. Based on the value of the variable importance in projection (VIP) and the result of the t-test, 424 components (VIP > 1, p < 0.05) were acquired. Finally, 11 differential components were selected as the characteristic compounds to discriminate between MR and HBM: the content of 9 monacolins (3-hydroxy-3,5-dihydrodehydromonacolin K, monacolin K, dehydromonacolin K, dehydromonacolin J hydroxy acid, monacophenyl, dihydromonacolin J hydroxy acid, monacolin L, dehydromonacolin J, and monacolin R) in HBM was higher than the content in MR, but the content of 2 pigments (ankaflavin and monascin) was lower in HBM and higher in MR. The findings revealed the similarities and differences in the chemical composition between MR and HBM, which is expected to provide a basis for quality control of HBM.

Synchronous high-performance liquid chromatography with a photodiode array detector and mass spectrometry for the determination of citrinin, monascin, ankaflavin, and the lactone and acid forms of monacolin K in red mold rice.

The Monascus fermentation product red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK) in both its lactone (MKL) and acid (MKA) forms and the mycotoxin citrinin (CT). The yellow pigments in RMR, namely, monascin (MS) and ankaflavin (AK), have been reported to exhibit antimetastatic and antiangiogenic activities. Currently, MK and these yellow pigments are usually detected in RMR by different analytical methods that are inconvenient, expensive, and time-consuming. The goal of this study was to establish a rapid, synchronous analytical method for determination of the MKA, MKL, MS, AK, and CT levels in RMR. MKA, MKL, MS, AK, and CT were extracted by the same extraction method, then separated by RP-HPLC with a C18 column. The effluent from the column was passed through a photodiode array detector and then introduced directly into a fluorescence detector. The results showed that high recovery rates of MKA, MKL, MS, AK, and CT are possible if RMR powder is extracted with 75% ethanol (10 mL) at 80 degrees C for 30 min. With regard to the optimal conditions of the HPLC, the peaks of MKA, MKL, MS, AK, and CT can be clearly separated from any noise peaks by isocratic elution with a mobile phase comprising 0.05% trifluoroacetic acid in acetonitrile-water (62.5 + 37.5, v/v).

Screening of gamma-aminobutyric acid-producing lactic acid bacteria and its application in Monascus-fermented rice production.

BACKGROUND: Gamma-aminobutyric acid (GABA), with an antidepressant effect, and Monacolin K, with a cholesterol-lowering effect, are the main bioactive ingredients in Monascus-fermented rice (MFR). The simultaneous enrichment of both ingredients can effectively enhance the health benefits of MFR. However, the capacity of Monascus spp. to produce GABA is limited. METHODS: Seventeen lactic acid bacteria (LAB) strains were preliminarily screened for GABA-producing by whole-cells bioconversion of L-glutamate, followed by rescreening through fermentation with the addition of the precursor L-glutamic acid. Subsequently, the bioconversion conditions (temperature, metal ions, and pH) for the conversion of L-monosodium glutamate (MSG) were investigated. Additionally, the GABA-producing LAB was co-inoculated with a monacolin K producing strain Monascus anka 20-2, and the ratio of M. anka 20-2 to LAB in microbial consortia was optimized for MFR production. RESULTS: The strain Lactobacillus plantarum 8014 was screened out for its ability to produce GABA. At an optimal temperature of 33°C and pH 7.5, with the addition of 0.05 g/L ZnSO4, the strain showed an L-glutamate conversion rate of 100%. The ratio optimization of M. anka 20-2 to L. plantarum 8014 in microbial consortia showed that when the dry cell ratio was 2:1, the content of monacolin K and GABA in the MFR simultaneously reached 2.22 mg/g and 29.9 mg/g, respectively. CONCLUSIONS: A two-stage fermentation using microbial consortia containing M. anka 20-2 and L. plantarum 8014 was developed for the production of bioactive MFR, in which the active ingredients monacolin K and GABA were simultaneously enriched, with good consumer acceptability due to the aromatic scent produced by lactic acid bacteria.

Protective effect and mechanism of Monascus-fermented red yeast rice against colitis caused by Salmonella enterica serotype Typhimurium ATCC 14028.

Red yeast rice (RYR), a traditional Chinese fermented food, has the effect of lowering blood lipid and cholesterol, but little information is available about whether RYR can inhibit pathogenic bacterial infection in vivo. The present study explored the effect of RYR on Salmonella enterica-induced intestinal inflammation and gut microbiota dysbiosis in mice as well as the underlying anti-inflammatory mechanism. Results showed that RYR can alleviate S. enterica infection in vivo and Monascus pigments are the main functional components. The analysis of microbiota, gene expression profile and serological immunology revealed that RYR can regulate the intestinal flora and increase the relative abundance of beneficial bacteria such as Lactobacillus and Akkermansia. Meanwhile, RYR is also found to regulate the expression of pro-inflammatory factors and tight junction-related genes to inhibit the NO and NF-κB-mediated inflammatory response and maintain the integrity of the intestinal barrier. This study provides a new dietary intervention strategy for the prevention of pathogenic bacterial infection.

Ultrasound assisted ionic liquid dispersive liquid phase extraction of lovastatin and simvastatin: a new pretreatment procedure.

A fast and novel sample preparation procedure: ultrasound assisted ionic liquid (IL) dispersive liquid extraction for the concentration of lovastatin and simvastatin in aqueous samples was developed. An IL ([C(6)MIM][PF(6)]) was used as the extraction solvent, and the factors affecting the extraction efficiency such as initial temperature, the volume of IL, pH of water samples, cooling time, and salt concentration were optimized. In combination with HPLC-UV, both lovastatin and simvastatin exhibited a good linear range of 1-100 ng/mL. The limits of detection (LODs) of lovastatin and simvastatin were 0.17 and 0.29 ng/mL, respectively. Precisions of the proposed method (RSDs, n = 9) were 4.12 and 4.52%, respectively. This method has been successfully applied for the analysis of target compounds in three real water samples and good spiking recoveries were obtained in the range of 90.0-102.2% for lovastatin and 80.5-112.0% for simvastatin. These results indicated that ultrasound assisted IL dispersive liquid phase extraction would have good application prospect in the pretreatment of environmental samples.