Leucine interference in the production of water-soluble red Monascus pigments

The formation of soluble Monascus red pigments is strongly positively and negatively regulated by different amino acids. Leucine, valine, lysine, and methionine had strong negative effects on pigment formation. Leucine supported poor pigment formation when used as sole nitrogen source in fermentations, yet it neither repressed pigment synthase(s) nor inhibited its action. The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced. Therefore, the low level of red pigments produced when leucine was the nitrogen source was not due to feed-back regulation by cell-bound leucine pigments. The negative effect of leucine was caused by enhanced decay of pigment synthase(s). The enhanced decay was not due simply to de novo synthesis of a leucine-induced protease.Abbreviations mSG Monosodium glutamate - MOPS 3-(N-morpholine)propane sulfonic acid - DCW dry cell weight     

Effect of nutrition of Monascus sp. on formation of red pigments

Summary A higher producer of ascospores and pigments, Monascus strain TTWMB 6042, was used to study regulation of pigment production by nutrients. An initial medium containing 4% glucose, 0.3% NH₄NO₃ (75 mm nitrogen) and inorganic salts was used. We found that the formation of red pigments in this strain, measured by optical density at 500 nm (OD₅₀₀) was strongly stimulated by monosodium glutamate (MSG) as the sole nitrogen source. The choice of carbon source and an initial pH of pH 5.5 were also important. High concentrations of phosphate and MgSO₄ were inhibitory to pigment production. A new chemically defined medium was devised containing 5% maltose, 75 mm MSG, phosphate and MgSO₄ at lower concentrations plus other mineral salts, which yielded a tenfold increase in OD₅₀₀ and a reversal of the pigment location from predominantly cell-bound, including both intracellular and surface-bound pigments, to mainly extracellular. Offsprint requests to: A. L. Demain     

Submerged fermentation in wheat substrates for production of Monascus pigments

Cereal grains are normally used as solid substrates for the production of Monascus metabolites. However, solid fermentation in these substrates requires complex control systems, whereas in liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods. A flour composed of a mixed variety of Canadian hard wheat was used as sole nutrient source to produce the pigments of Monascus purpureus Went (IMI 210765). Supplementation with NH₄Cl promoted biomass and orange dye formation, whereas the use of zinc sulphate favoured red dyes production. In submerged fermentations significant differences in final pigment yields were observed in the use of wheat-based broth at different concentrations in the presence of bran particles and/or gluten protein. It has been found that the viscosity of the broth had a significant effect on the growth morphology and production of pigments. Gluten-free wheat flour at concentrations of 3–5% was found to be the most suitable for liquid Monascus culture. The subsequent use of passive immobilization of Monascus served to enhance red pigment yields and to facilitate the downstream processing of the dyes.     

Optimization of submerged fermentation medium for citrinin-free monascin production by Monascus

Microbial fermentation of citrinin-free Monascus pigments is in favor in the development of food industry. This study investigated the influences of carbon source, nitrogen source, and mineral salts on the cell growth, monascin (MS), and citrinin (CT) production in Monascus M9. A culture medium composition was established for maximizing the production of citrinin-free MS in submerged culture, as follows: 50?g/L Japonica rice powder, 20?g/L NH₄NO₃, 3?g/L NaNO₃, 1.5?g/L KH₂PO₄, 1?g/L MgSO₄?·?7H₂O, 0.2?g/L MnSO₄. Under these conditions, no CT was detectable by high performance liquid chromatography. The yield of MS reached 14.11?mg/g, improving approximately 30% compared with before optimization.     

Analyses of Monascus pigment secretion and cellular morphology in non‐ionic surfactant micelle aqueous solution

Monascus pigments produced by Monascus spp. are widely used as natural food colourants. Extractive fermentation technology can facilitate the secretion of intracellular Monascus pigments into extracellular non‐ionic surfactant micelle aqueous solution, so as to avoid the feedback inhibition and decomposition. In this study, behaviour of the trans‐membrane secretion of Monascus pigments was investigated using morphological and spectroscopic analyses. Laser scanning confocal microscopy (LSCM) traced that pigment secretion occurred through rapid trans‐membrane permeation in 4 min, with a simultaneous conversion in pigment characteristics. Approximately 50% of intracellular pigments (AU₄₇₀) extracted to extracellular broth with 40 g l^?1 Triton X‐100, indicating the capacity for pigment extraction was limited by the saturation concentrations of surfactant. Scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging showed some damage in the cell wall but an intact cell membrane with a slightly increased mycelial diameter. However, the physiological properties of the cell membrane, including integrity, fluorescence intensity and permeability, were altered. A diagram was provided to demonstrate the behaviour of Monascus pigment secretion induced by Triton X‐100. This study lays a foundation for the further investigation of Monascus pigment metabolism and secretion in extractive fermentation.     

Optimization for producing cell-bound lipase from <Emphasis Type="Italic">Geotrichum</Emphasis> sp. and synthesis of methyl oleate in microaqueous solvent

An integrated optimization strategy involving a combination of different designs was employed to optimize producing conditions of cell-bound lipase (CBL) from Geotrichum sp. Firstly, it was obtained by a single factorial design that the most suitable carbon source was a mixture of olive oil and citric acid and the most suitable nitrogen source was a mixture of corn steep liquor and NH₄NO₃. Then, the Plackett–Burman design was used to evaluate the effects of 13 variables related to CBL production, and three statistically significant variables namely, temperature, olive oil concentration, and NH₄NO₃ concentration, were selected. Subsequently, the levels of the three variables for maximum CBL production were determined by response surface analysis as follows: 1.64% (v/v) olive oil, 1.49% (w/v) NH₄NO₃, and temperature 33.00°C. Such optimization resulted in a high yield of CBL at 23.15 U/ml, an enhanced 4.45-fold increase relative to the initial result (5.2 U/ml) in shake flasks. The dried CBL was used to synthesize methyl oleate in microaqueous hexane, resulting in 94% conversion after 24 h, and showed reusability with 70% residual activity and 69% conversion after eight cycles of batch operation, which indicating that CBL, as a novel and natural form of immobilized enzyme, can be effectively applied in repeated synthesis of methyl oleate in a microaqueous solvent.     

Influence of trace elements and nitrogen sources on versicolorin production by a mutant strain of Aspergillus parasiticus

A mutant strain of Aspergillus parasiticus blocked in aflatoxin biosynthesis accumulates versicolorin A and versicolorin C. The effect of trace elements on the growth and versicolorin production by this strain was studied in a defined medium. The omission of manganese was slightly stimulatory to versicolorin production; when zinc was omitted from the medium, no detectable versicolorins were produced. Experiments on nitrogen sources in a highsucrose medium indicated that fourfold to fivefold increases in versicolorin yields could be obtained by substituting 3 ml/l corn steep liquor or 0.1 M NH₄NO₃ for the 0.023 M (NH₄)₂SO₃ used previously as the nitrogen source in studies on versicolorin production by this strain. These improved yields will facilitate attempts to accumulate enough versicolorin A and versicolorin C for toxicity and carcinogenicity testing. Chromatographic profiles of mycelial extracts of cultures grown in a defined medium with 0.1 M NH₄NO₃ as the nitrogen source revealed 2 previously unrecognized compounds. The accumulation of these new metabolites in a mutant blocked in aflatoxin production may indicate that they are biosynthetically related to aflatoxin.     

MACROALGAL RESPONSES TO NITROGEN SOURCE AND AVAILABILITY: AMINO ACID METABOLIC PROFILING AS A BIOINDICATOR USING GRACILARIA EDULIS (RHODOPHYTA)1

The use of macroalgae as biological indicators of dissolved nutrient source and availability in the water column was investigated. Total tissue nitrogen (N) content, pigments, and amino acids of the red alga Gracilaria edulis (Gmelin) Silva were compared to N source and availability in laboratory and field incubations to identify responses that would serve as bioindicators of N. Field-collected algae were preincubated (6–8 wk) in low-nutrient seawater to deplete their luxury reserves ofN. Incubations were then conducted for periods of 3 d in laboratory aquaria (N-spiked seawater) and in the field using macroalgal incubation chambers. After incubation in different N sources (NH₄⁺, NO₃^?, and urea) in laboratory aquaria, photosynthetic pigments (phycoerythrin and chlorophyll a) and total tissue N increased, in response to increasing [NH₄⁺] but not to [NO₃^?] or [urea]. Incubation in two ranges of [NH₄⁺], one from 0 to 80 μM and the other from 0 to 800 μM, in laboratory aquaria increased the total amino acid pool. Citrulline concentrations were the most responsive to [NH₄⁺] (r²= 0. 84). NH₄⁺ source treatments produced increases in citrulline, phenylalanine, serine, and free NH₄⁺ and decreases in alanine; NO₃^? treatments produced increases in glutamic acid, citrulline, and alanine; and urea treatments produced increases in free NH₄⁺ and decreases in phenylalanine and serine. The observed variations in amino acid content facilitated the development of an index for each N source based on relative concentrations of various amino acids (i. e. metabolic profiling). Gracilaria edulis was incubated along a field N gradient in the Brisbane River (three sites) and Moreton Bay (four sites), Queensland, Australia. Both phycoerythrin and tissue N appeared to respond equally to NH₄⁺ and NO₃^? availability in the field. N source indices, based on amino acid concentration, were effective predictors of both [NH₄⁺] and [NO₃^?] over a wide range of concentrations along the field gradient. Macroalgal physiological responses, particularly amino acid content, to changes in source and availability of N appear to be useful as sensitive bioindicators of N.     

INORGANIC NITROGEN NUTRITION OF THE SEEDLINGS OF THE ORCHID,CATTLEYA

When grown in vitro in a medium containing NH₄NO₃ as the sole source of nitrogen, seeds ro the orchid, Cattleya (C. labiata ‘Wonder’ X C. labiata ‘Treasure'), germinated readily and proceeded to form small plantlets. Development of the embryos was accompanied by an increase in their total nitrogen and a decline in the percent dry weight. Growth responses of the seedlings in other ammonium salts like (NH₄)₂SO₄, (NH₄)₂HPO₄, NH₄Cl, ammonium acetate and ammonium oxalate were similar to that in NH₄NO₃. However, when grown in a medium containing NaNO₃, development of the seedlings was drastically inhibited; KNO₃, Ca(NO₃)₂, KNO₂ and NaNO₂ also were poor nitrogen sources. Attempts to grow the seedlings in NaNO₃ by changing the pH or by addition of kinetin, molybdenum or ascorbic acid as supplements were completely unsuccessful. When seedlings growing in NH₄NO₃ for varying periods were transferred to NaNO₃, it was found that those plants allowed to grow for 60 or more days in NH₄NO₃ could resume normal growth thereafter in NaNO₃. Determination of the nitrate reductase activity in seedlings of different ages grown in NaNO₃, after NH₄NO₃, showed that the ability of the seedlings to assimilate inorganic nitrogen was paralleled by the appearance of the enzyme.     

The Effect of Several Protein Amino Acids and Some Inorganic Nitrogen Sources on the Growth of Sphagnum nemoreum

The nitrogen metabolism of a bog moss, Sphagnum nemoreum Scop., has been studied in aseptic cultures. The effect of several protein amino acids, especially those found in peat, has been investigated. NH₄NO₃ (1.25 mM) was the best nitrogen source but NH₄⁺ ions were more effectively utilized than NO₃⁻ ions when given as the only nitrogen source. Some of the amino acids (2.5 mM) allowed fairly satisfactory growth (arginine and alanine) when given as the only nitrogen source, but some of them were not utilized at all (leucine, lysine, isoleucine and methionine). Given at low concentrations (0.001 and 0.25 mM) together with NH₄NO₃ (2.5 mM), most of the protein amino acids failed to reveal any growth-promoting or -inhibiting effect. Only lysine (0.25 mM) clearly inhibited growth under these conditions. The nitrogen metabolism of Sphagnum nemoreum seems to be rather flexible and this species is more tolerant of organic nitrogen, especially hydroxyproline, than the higher plants.     

Production of water-soluble yellow pigments via high glucose stress fermentation of Monascus ruber CGMCC 10910

Monascus pigments are secondary metabolites of Monascus species and are mainly composed of yellow pigments, orange pigments and red pigments. In this study, a larger proportion of Monascus yellow pigments could be obtained through the selection of the carbon source. Hydrophilic yellow pigments can be largely produced extracellularly by Monascus ruber CGMCC 10910 under conditions of high glucose fermentation with low oxidoreduction potential (ORP). However, keeping high glucose levels later in the culture causes translation or a reduction of yellow pigment. We presume that the mechanism behind this phenomenon may be attributed to the redox level of the culture broth and the high glucose stress reaction of M. ruber CGMCC 10910 during high glucose fermentation. These yellow pigments were produced via high glucose bio-fermentation without citrinin. Therefore, these pigments can act as natural pigments for applications as food additives.

     

Production of red pigments byMonascus purpureus in solid-state culture

To maximize and sustain the productivity ofMonascus pigments, various environmental and nutritional parameters, such as the initial moisture content, pH, inoculum size, sample size, and nutrient supplement, that influence pigment production were evaluated in solid-state cultures as follows: initial moisture content, 50%; pH, 6.0; inoculum size 1 x 10⁴ spore cells (grams of dry solid substrate)⁻¹; sample size, 300 g. All supplementary nutrients (carbon, nitrogen, and mineral sources) added has inhibitory effects on the cell growth and red pigment production. In open tray culture the maximum biomass yield and specific productivity of red pigments were 223 mg DCW (grams of initial dry substrate)⁻¹ and, 47.6 OD₅₀₀ (DCW grams)⁻¹ h⁻¹, respectively.     

Role of inorganic nitrogen in the synthesis and degradation of chlorophyll and carotenoids in maize leaves

Supply of KNO₃, NH₄Cl or NH₄NO₃ to the maize seedlings increased total chlorophyll and carotenoids (over zero nitrogen control) amounts per fresh matter unit of the primary leaves. The increase was most apparent when the pigment level in control plants started declining,i.e. between days 14 and 16. Supply of inorganic nitrogen increased pigment synthesis in excised leaf segments from dark grown seedlings, although the increase during a 24 h incubation was not as high as with either glycine or glutamine. Salicylic acid at 0.01 to 1 mM concentration increased and at 10 mM lowered the pigment biosynthesis. The increase at lower concentrations of salicylic acid was unaffected by KNO₃ but it was suppressed by NH₄Cl. When 12-day seedlings were transferred to the dark, total chlorophyll and carotenoids declined appreciably, but the supply of inorganic nitrogen protected them from decline to same extent. Thus inorganic nitrogen, especially the nitrate, is more important in protecting from degradation of chlorophyll and carotenoids than in increasing their biosyntheses.     

Resting cell studies on formation of water-soluble red pigments byMonascus sp.

Summary A resting cell system was developed for the biosynthesis of soluble red pigments byMonascus. The medium contains glucose, glycine, ZnSO₄ and MnSO₄ in pH 7.0 MOPS buffer containing cycloheximide to prevent protein synthesis. The linear production observed over a period of at least four h was due to de novo polyketide synthesis and biological methylation, as shown by inhibition with cerulenin, iodoacetamide and ethionine. Production was inhibited by carbonyl reagents and stimulated by pyridoxamine suggesting that the conversion of endogenous intracellular orange pigments to extracellular red pigments involves Schiff base intermediates and vitamin B₆ a cofactor. The resting cell system was used to study the mode of action of nutritional effectors previously pinpointed by experiments with growing cells. The negative effects of high concentrations of phosphate and Mg⁺⁺ are due to inhibition of pigment synthase action, not to repression or inactivation of these enzymes. The positive effects of trace metals, especially Zn⁺⁺, are due to stimulation of growth and enzyme action, not to induction or stabilization of the synthases.     

NITROGEN ALLOCATION AND STORAGE PATTERNS IN GRACILARIA TIKVAHIAE (RHODOPHYTA)1

Internal nitrogen pools in thalli of Gracilaria tikvahiae McLachlan were examined in three experiments as a function of total nitrogen content of the thallus, nitrogen deprivation, and nitrogen resupply. Amino acids and proteins appeared to form the major nitrogen storage pools in G. tikvahiae, while DNA appeared to be relatively unimportant in this regard. Inorganic nitrogen in the forms of NH₄⁺ and NO₃^? was found in the thalli; however, its contribution to the total nitrogen, pools was small. Within the protein pool, the phycoerythrin pigments appear important as a source of nitrogen when thalli are initially becoming nitrogen limited. In general, there was an inverse relationship between the levels of nitrogen and the carbohydrate content of the algal thalli.     

The Influence of Ammonium Nitrate,pH and Indole Butyric Acid on Root Induction and Survival in Soil of Micropropagated <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

Rooting of Eucalyptus globulus shoots was influenced by the concentration of the indole butyric acid (IBA) and NH₄ ⁺ in the root-induction medium. Optimum plantlet vigor and survival were achieved using low concentrations (1 – 2.5 μM) of IBA and when NH₄NO₃ was removed. Removal of NH₄ ⁺ also had a significant effect on medium pH, its presence caused a decrease in pH as the culture period proceeded. When different nitrate compounds (excluding NH₄NO₃) were used as the nitrogen source, the medium pH was more stable and this was associated with higher root production. The higher root production, in association with appropriate IBA concentrations, produced plantlets with higher survival and better growth on transfer to soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nitrate and ammonium as nitrogen sources for lupins prior to nodulation

Two cultivars of Lupinus angustifolius L. were grown in a glasshouse in solutions containing NO₃ ^-, NH₄ ⁺ or NH₄NO₃ with a total nitrogen concentration of 2.8 M m^-3 in each treatment. One cultivar chosen (75A-258) was relatively tolerant to alkaline soils whereas the other (Yandee) was intolerant to alkalinity. Controlled experiments were used to assess the impact of cationic vs. anionic forms of nitrogen on the relative performance of these cultivars. Relative growth rates (dry weight basis) were not significantly different between the two cultivars when grown in the presence of NO₃ ^-, NH₄ ⁺ or NH₄NO₃. However, when NO₃ ^- was supplied, there was a modest decline in relative growth rates in both cultivars over time. When plants grown on the three sources of nitrogen for 9 days were subsequently supplied with ¹⁵NH₄NO₃ or NH₄ ¹⁵NO₃ for 30 h, NH₄ ⁺ uptake was generally twice as fast as NO₃ ^- uptake, even for plants grown in the presence of NO₃ ^-. Low rates of NO₃ ^- uptake accounted for the decrease in growth rates over time when plants were grown in the presence of NO₃ ^-. It is concluded that the more rapid growth of 75A-258 than Yandee in alkaline conditions was not due to preferential uptake of NH₄ ⁺ and acidification of the external medium. In support of this view, acidification of the root medium was not significantly different between cultivars when NH₄ ⁺ was the sole nitrogen source.     

Perstraction of intracellular pigments by submerged cultivation of <Emphasis Type="Italic">Monascus</Emphasis> in nonionic surfactant micelle aqueous solution

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.     

Effects of carbohydrates and nitrogen on the development of anthocyanins of a red leaf peach (Prunus persica (L.) Batsch) in vitro

Heterozygous red leaf peach (Prunus persica (L.) Batsch) shoots were implanted on media with varying nitrogen and carbohydrate regimes to identify a combination which elicited maximum anthocyanin production in explants. A medium with relatively low nitrogen (5 mM NH₄₊ and 10 mM NO_3-) and high sucrose (234 mM) was most effective in stimulating anthocyanin production. Sucrose was more effective as a carbon source than glucose, fructose, or starch under given nitrogen levels. The major anthocyanin in red leaf peach was tentatively identified as cyanidin 3-glucoside based on PC and HPLC analysis.     

Nitrogen metabolism of Spirodela oligorrhiza

Summary Spirodela oligorrhiza grown in axenic culture was able to use either ammonium, nitrate, or nitrite as sole source of nitrogen, although the morphology of the plants was affected. Plants utilizing ammonium contained higher levels of NH₄, arginine, asparagine and glutamine than did those utilizing NO₃, whereas concentrations of other amino acids were similar.The utilization of NH₄ inhibited that of NO₃ by inhibiting, at least partially, NO₃ uptake, and by inhibiting almost completely the reduction of NO₃ to NO₂. NO₂ also inhibited the utilization of NO₃. NH₄ and NO₂ were taken up and assimilated simultaneously when they were supplied together in the medium.