Proteomic response to intracellular proteins of Monascus pilosus grown under phosphate-limited complex medium with different growth rates and pigment production

Monascus pigments are important colorings in food applications. Rice containing potassium phosphate and sodium nitrate was reported as a good pigment-producing medium for Monascus in previous studies. We found that the lack of potassium phosphate in this medium depressed red pigment production in cultivated Monascus pilosus. However, the influence of phosphate limitation on the biochemical metabolisms concerning culture growth and pigment production in Monascus remains unknown. Here, we used proteomic analysis by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), tandem mass spectrometry (MS/MS), and database interrogation to separate and identify the proteins of M. pilosus grown between the lack of potassium phosphate and the control media. Phosphate limitation to this complex medium induced an up-regulation of aldehyde dehydrogenase and several glycolytic enzymes in Monascus relative to the control. In contrast, the metabolic enzymes such as glucosamine:fructose-6-phosphate aminotransferase and ADP-ribosylation factor 1 were up-regulated in the control.     

Proteome changes in Caco-2 cells treated with Monascus-fermented red mold rice extract

Monascus-fermented red mold rice has been extensively used as a folk medicine for thousands of years. Monascus secondary metabolites, including monacolin K, monascorubrin, and ankaflavin, have been reported to have an antiproliferative effect on cancer cells. However, the cell machinery responsible for the antiproliferation of Monascus-fermented red mold rice treatment in cancer cells remains unclear. A proteomic approach using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry, and tandem mass spectrometry was used to identify proteins with modified expression in Caco-2 cells treated with Monascus-fermented red mold rice extract. A total of 20 proteins were identified with significantly altered expression (P < 0.05) in response to Monascus-fermented red mold rice extract treatment. The deregulated proteins that were identified included heat shock protein 70, protein kinase C epsilon type, clusterin-associated protein 1, and two tumor suppressors (N-chimaerin and calponin-2). Our results suggested the involvement of heat shock protein 70-mediated cytotoxicity in the Caco-2 cells treated with Monascus-fermented red mold rice extract.     

Proteomic analysis of Caco-2 cells treated with monacolin K

Monascus species is an important traditional fermentation fungus used on food. Monacolin K (a secondary metabolite of Monascus, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase) in inhibition of mevalonate synthesis may result in reductions of isoprenoid prenylation in cells. Impairment of protein isoprenoid prenylation has been related to anticancer effect in cancer cells. As a functional food for Monascus, however, the molecular mechanisms responsible for the anti-proliferate effect of monacolin K in cancer cells are not clear. We used proteomic analysis by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), tandem mass spectrometry (MS/MS), and database interrogation to separate and identify the proteins of Caco-2 cells treated with monacolin K. The results showed that monacolin K inhibited the proliferation of Caco-2 cells in a dose-dependent manner. The identified proteins in proteomic analysis included anti-oxidation enzymes related to reactive oxygen species stress, cytoskeleton proteins, glycolytic enzymes, and enzymes involved in mediating protein interactions. Furthermore, glutathione S-transferase P 1 and cytoskeleton-8, -18, and -19 revealed a down-regulation in a dose-dependent manner in exposure of Caco-2 cells to monacolin K.     

Two new Monascus metabolites with strong blue fluorescence isolated from red yeast rice

Red yeast rice obtained as cultures of Monascus AS3.4444 on rice was extracted and analyzed by high-performance liquid chromatography (HPLC). Two new Monascus metabolites with similar fluorescence spectra (lambda ex = 396 nm, lambda em = 460 nm) and UV absorption spectra (lambda max = 386 nm) were detected. They were isolated by rechromatography on a silica gel column and semipreparative HPLC, and two strong blue fluorescent compounds were obtained. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), intensive ESI-MS, and nuclear magnetic resonance spectroscopy ( (1)H NMR, (13)C NMR, COSY, and HMBC) studies. High-resolution mass spectrometry indicated the molecular formulas C 21H 24O 5 and C 23H 28O 5. The two new compounds, named monasfluore A and monasfluore B, respectively, contain a alkyl side chain, gamma-lactone, and propenyl group, whereas the more lipophilic compound, monasfluore B, is a higher homologue of monasfluore A, with the more lipophilic octanoyl instead of the hexanoyl side chain.     

Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA- mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.     

New monascus metabolite isolated from red yeast rice (angkak,red koji)

Red yeast rice (angkak, red koji) obtained as cultures of Monascus purpureus on rice was extracted and analyzed by HPLC. In addition to the known red, orange, and yellow pigments and the mycotoxin citrinin, a new Monascus metabolite was detected. It is present in the original red yeast rice and formed in higher amounts when red yeast rice is heated. High-resolution mass spectrometry indicated the molecular formula C(15)H(12)O(4). The chemical structure was elucidated by analysis of NMR data. The new compound, named monascodilone, is characterized by a propenyl group on a pyrone ring, an aromatic ring, and a gamma-lactone group.     

Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose

Heat treatment of dairy products leads to structural changes of proteins, which can severely decrease the nutritional value [Mauron, J. J. Nutr. Sci. Vitaminol. (Tokyo) 1990, 36 (Suppl. 1), S57-69]. In this study, model solutions of the two main whey proteins, alpha-lactalbumin and beta-lactoglobulin, respectively, were incubated with lactose, and modifications were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Lactulosyl residues were the most abundant modifications of alpha-lactalbumin and beta-lactoglobulin. Up to four of these adducts were identified on the proteins. Enzymatical digest with endoproteinase AspN prior to mass spectrometric analysis allowed the detection of further modifications and their localization in the amino acid sequence. Most prominent modifications were lactulosyllysine, Nepsilon-carboxymethyllysine, oxidation of lysine to aminoadipic semialdehyde, oxidation of methionine to methionine sulfoxide, cyclization of N-terminal glutamic acid to a pyrrolidone, and oxidation of cysteine or tryptophan. The presence of methionine oxidation was deduced from a control protein that had been oxidized by hydrogen peroxide. These studies establish MALDI-TOF-MS as a reliable tool to monitor chemical modifications of nutritional proteins during food processing.     

Comparison of MALDI-TOF mass spectrometric to enzyme colorimetric quantification of glucose from enzyme-hydrolyzed starch

Successful quantification of the glucose produced by enzyme hydrolysis of starch was achieved by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol, using sorbitol as an internal standard. The starch contents measured by MALDI-TOF MS of corn starch, fiber-enriched oat flour derivatives, oat and barley flours, and barley flour/corn starch composites were evaluated in comparison to a widely accepted and validated method of starch determination, which relies on enzyme colorimetry (EC). The average starch content measured in a series of corn starch samples of different masses was 93 and 101% for EC and MALDI-TOF MS, respectively, values that represent the estimated purity of the sample. There was an agreement of 99% between the starch contents determined by the two analytical methods for complex flour-derived samples. Starch values estimated by MALDI-TOF MS consistently showed a greater degree of variability than those determined by EC, but this limitation was readily compensated by rapid acquisition of multiple mass spectra. This study is the first to report the quantification of glucose by MALDI-TOF MS, and it offers new perspectives into the potential utility of MALDI-TOF MS as a definitive tool for monosaccharide analysis and rapid starch determination in complex samples.     

利用蛋白质组学技术研究不同储藏条件稻谷陈化机制

为了研究储藏过程中不同温度和气调条件对稻谷品质劣化的影响,利用蛋白质组学技术探讨稻谷储藏陈化的分子机理,研究温度37℃、25℃和25℃+CO2气调下稻谷储藏90 d品质和蛋白质组的变化.结果表明,较37、25℃贮藏,25℃+CO2气调下稻谷储藏脂肪酸值升高最少,发芽率下降最少(P＜0.05).稻谷储藏产生125个差异蛋白点,其中37个蛋白得到鉴定,根据蛋白质的功能可分为5类,包括代谢(45.9％),细胞结构(29.7％),抗胁迫(2.7％),功能性蛋白(5.4％)和其他蛋白(16.3％).并鉴定出4个目标蛋白,分别为蛋白酶体亚基β-1(B26、D09和F16),葡糖-1-磷酸腺苷酰基转移酶(C01和E07),ADP-葡萄糖焦磷酸化酶大亚基(B04和F04)和乙酰辅酶A(A06和C05).采用蛋白质组学技术分析稻谷储藏过程中蛋白质组变化,结果表明高温储藏促进稻谷差异蛋白表达,CO2气调储藏可降低差异蛋白表达.对差异表达蛋白功能分析表明,稻谷陈化可能与糖代谢紊乱、蛋白质分解能力降低,抗氧化酶活性降低,脂肪水解和氧化增强有关.研究结果为稻谷的合理、安全储藏提供参考.     

Physiological response and protein expression under acid stress of Escherichia coli O157:H7 TWC01 isolated from Taiwan

Escherichia coli O157:H7 has an unusually high resistance to acidic environments. Some research has revealed that acid-adapted cells, by exposure to moderately acidic conditions, are more resistant to a subsequent strong acidic challenge or other stress. This study was conducted to understand the protein expression regulation of acid tolerance response (ATR) of a local isolated E. coli O157:H7 TWC01 (TWC01) induced by an acidic environment. TWC01 cells were acid adapted by using hydrochloric acid (HCl) or lactic acid as acidifier to induce ATR. The total proteins of adapted cells were extracted for proteomic analysis and protein identification by matrix-assisted laser desorption ionization quadrupole time-of-flight tandem mass spectrometry (MALDI-Q-TOF MS/MS). Furthermore, the effects of acid adaptation on shiga-like toxin (stx) secretion were examined. Results revealed that acid adaptation depressed stx production of E. coli O157:H7 TWC01 during adaptation and did not improve post-stress toxin production. Image analysis of the gel indicated that numerous proteins were up-regulated and that lactic acid had a greater effect than HCl did (percentages of up-regulated proteins were 57.64 and 35.47%, respectively). Analysis of proteins by mass spectrometry revealed that most of the up-regulated proteins were metabolism-related, including phosphoglycerate kinase (PGK), glutamate decarboxylases alpha and beta (GadA, GadB), adenine phosphoribosyltransferase (APRT), and dihydrodipicolinate synthase (DHDPS). Others were related to translation (e.g., elongation factor Tu, elongation factor G), protein folding (e.g., alkyl hydroperoxide reductase), and membrane proteins (e.g., ompA precursor and ompR). The variation of protein expression showed that acid resistance was induced in TWC01 and was primarily manifested via expression of up-regulated proteins that contribute to increased energy conservation and polypeptide synthesis.     

Characterization of reaction products formed in a model reaction between pentanal and lysine-containing oligopeptides

Aldehydes formed as a result of lipid oxidation form fluorophores after binding to proteins. The structure of the fluorophores formed by reaction between saturated aldehydes and lysine has not yet been identified. The reaction products formed in the reaction between pentanal and oligopeptides were studied by fluorescence spectroscopy and mass spectrometry. The emission spectra showed an increase in fluorescence intensity with incubation time, and the rates were linear with the concentration of pentanal. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the reaction products suggested a molar relation for peptide:pentanal of 1:4. Further tandem mass spectrometry analysis of one of the modified peptides (Pro-Thr-His-Ile-Lys-Trp-Gly-Asp) strongly suggested binding of one pentanal molecule to the amino terminal proline and three pentanal molecules bound to the lysine residue. The latter species is suggested to be the actual fluorophore, through the formation of conjugated double bonds, and a possible reaction pathway through a combination of aldol condensation of pentanal and Schiff base formation with the lysine is suggested.     

Changes in the charged metabolite and sugar profiles of pasteurized and unpasteurized Japanese sake with storage

Japanese sake (rice wine) is commonly heat treated (pasteurized) to maintain its quality. In this study, temporal changes in the metabolite profiles of pasteurized and unpasteurized sake were investigated during storage. Metabolomic analyses were conducted for eight sets of pasteurized and unpasteurized sake obtained from single process batches stored at 8 or 20 °C for 0, 1, 2, or 4 months. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry were used to obtain charged metabolite and sugar profiles, respectively. The total amino acid concentration decreased with storage, and the decrease was faster in pasteurized sake than in unpasteurized. The organic acid concentrations were relatively constant in both types of sake. Peptide and glucose concentrations increased and polysaccharide concentrations decreased in unpasteurized sake, while they were relatively constant in pasteurized sake. Rather than stabilizing the sake metabolite profile during storage, pasteurization results in characteristic changes compared to unpasteurized sake.     

Changes in free amino acid and sugar levels of dried figs during aflatoxin B1 production by Aspergillus flavus and Aspergillus parasiticus

An aqueous slurry of gamma-irradiated sterilized dried figs was inoculated with toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. During incubation at 28 degrees C, pH, fructose, glucose, and free amino acids were determined by high-performance liquid chromatography and liquid chromatography (LC)/mass spectrometry, respectively, over 13 time points (1-20 days). At the same 13 time points using a LC/time-of-flight mass spectrometry screening method, aflatoxin B 1 and other secondary metabolites were simultaneously monitored. During the course of incubation, the pH significantly decreased and aflatoxin B 1 formation correlated with a reduction in proline content for both fungi. Of the 22 free amino acids that were monitored, only proline and cystine were found to be critical in supporting aflatoxin production. Levels of fructose and glucose steadily declined during incubation, until glucose was almost exhausted after 21 days. These time-course experiments confirmed the need for carbon and nitrogen sources for aflatoxin production in dried figs, and the favorable composition of figs as a fungal growth medium.     

Commercially Produced Rice and Maize Starches Contain Nonhost Proteins,as Shown by Mass Spectrometry

To evaluate the presence of contaminating nonhost proteins in commercially prepared rice and maize starch samples, we initiated a direct sequencing mass spectrometric proteomics survey. We discovered nonhost proteins from a variety of species, including Phytophthora cinnamomi, Homarus americanus, and Ovis aries. Our documentation of H. americanus proteins in these starch samples may have food safety implications with regard to shellfish allergies. We hypothesize that these proteins were introduced to the starch samples via process wash water used in the milling and deproteination steps in the commercial preparation of the starches analyzed. The introduction of nonhost proteins during commercial processing of starch samples that are used routinely in analytical studies indicates that these studies are using impure materials. Therefore, further study and documentation of the starch samples is required to ensure that all components of the samples are properly catalogued.     

The Effect of Light on the Accumulation of Lipids in Rice Seeds

To investigate the effects of light on the accumulation of assimilate in rice seeds, ears were allowed to grow in darkness 10 d after their formation. The lipid content of rice bran on the 40th day increased by cultivation of ears in darkness whereas the dry weight and starch and sucrose contents of the rice seeds decreased. In the light, the triacylglyceride (TG) content of rice seeds was lowest on the 20th day but then increased continuously for the next 20 d, while the diacylglyceride (DG) content reached a maximum value on the 20th day. In darkness, in contrast, rice seeds showed a higher TG content, with a lower DG content and lower levels of other lipids on the 20th day compared with seeds exposed to light. These results suggest that the biosynthesis of starch and lipids in rice seeds is regulated by light.     

CO2浓度增高对水稻籽粒淀粉代谢相关酶活性的影响

以高产优质粳稻松粳9号和稻花香2号为材料,利用开放式空气CO₂浓度富集系统（FACE）实验平台,研究CO₂浓度增高对水稻籽粒淀粉代谢相关酶活性的影响。试验设正常大气CO₂浓度（400±40μmol·mol-1）和高CO₂浓度（600±60μmol·mol-1）,测定开花后两个水稻品种籽粒中ADPG焦磷酸化酶、淀粉合成酶和淀粉分支酶活性的变化。结果表明,CO₂浓度增高对不同灌浆进程中酶活性的影响程度有显著差异,对乳熟期之后ADPG焦磷酸化酶、可溶性和颗粒型淀粉合成酶活性的表达均有较明显的促进作用,仅阻碍了乳熟期籽粒中淀粉分支酶活性的表达;淀粉代谢相关酶活性对CO₂浓度增高的响应因品种而异,松粳9号籽粒中ADPG焦磷酸化酶活性受CO₂浓度增高的影响较大,而稻花香2号淀粉合成酶活性受其影响更大。说明随着灌浆进程的推进,CO₂浓度增高对淀粉生物合成途径中关键酶活性表达的影响程度存在明显的时段特征,且不同品种的响应程度有显著差异,总体来看,CO2浓度增高可在一定程度上促进淀粉代谢相关酶活性的表达。     

Effect of Alkaline‐Soluble Proteins on Pasting Properties of Nonwaxy Rice Flour

The pasting properties of rice flours and reconstituted rice flours from mixing a common starch with proteins extracted from different rice cultivars at different total protein content levels were studied. Results showed that not only the total protein content but also the protein composition had an effect on the pasting properties of the rice flours. Among the different strands of rice proteins, globulin had the strongest influence on the pasting properties, followed by glutelin, whereas prolamin had the least influence. At the subunit level of the proteins, proteins with a molecular weight of 17,000, most likely from globulin, had the strongest effect on the peak viscosity of the rice flour, followed by those of 33,000. In comparison with that of the rice starch, the influence of proteins in rice was limited. The effect of interactions between the rice proteins and the starch, such as the role of starch‐granule‐associated proteins, was not isolated in this study, and further investigation is required to quantify this effect.     

Improving the ratio of monacolin K to citrinin production of Monascus purpureus NTU 568 under dioscorea medium through the mediation of pH value and ethanol addition

Using dioscorea root as substrate of Monascus species was found to stimulate monacolin K (cholesterol-lowering agent) formation in our previous study, but the mycotoxin-citrinin has never been studied. This study used dioscorea root as the liquid medium to culture Monascus purpureus NTU 568 using a 6.6 L jar fermentor. Culture pH value, dioscorea concentration, and ethanol concentration were used as the factors of response surface methodology (RSM) to investigate the optimal culture condition for high monacolin K production and low citrinin formation. Monacolin K and citrinin formation of M. purpureus NTU 568 under submerged dioscorea medium were respectively found to be significantly formed by 148% and 147%, as compared to that under submerged rice medium. The reason is due to the pH value (3.5) of dioscorea medium involved in the formation of Monascus cell amount and secondary metabolite. RSM results further indicated that lowering the pH value to 2.5 would result in high monacolin K and citrinin concentrations as well as high biomass in fixed dioscorea amount, implying that pH value may stimulate the formation of monacolin K and citrinin through increasing Monascus cell amount. Lowering dioscorea and ethanol concentration was able to increase the ratio of monacolin K level to citrinin level. The optimal culture condition (pH 5.7, 1% dioscorea concentration, and 0.5% ethanol concentration) would increase monacolin K levels to 27.9 mg/g (by 47%) and decrease citrinin level to 2.15 microg/g (by 54%), as compared to control conditions (pH 3.5, 5% dioscorea, and ethanol free).