Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.     

Production of citrinin by various species ofMonascus

Summary The production of citrinin by variousMonascus species was determinated using various culture mediums and conditions. The maximal production was obtained in fermentor usingM. ruber with concentrations of 380 mg/l. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives fromMonascus sp. avoid the occurrence of citrinin; so, we argue that some nitrogen sources are unfavorable to the production of citrinin.     

Kinetics of product inhibition in alcohol fermentation-temperature effect in the “sake” brewing

One of the kinteic equations derived previously from a series of sophisticated batch and continuous alcohol fermentations by using a respiration-deficient mutant of baker's yeast is as follows: where dp/dt = ethanol production rate, v0 = specific rate of ethanol production at p = 0, k₂ = empirical constant, K′_s = saturation constant, S = glucose concentration, and X = cell mass concentration. The above equation was confirmed in the previous paper to fit, the brewing of “sake.” The temperature of the specific brewing is not always constant (10 to 18°C). The effect of temperature on v0 was assessed from the Arrhenius plot, assuming that k₂ was independent of temperature. Values of dp/dt taken from the “sake” brewing data were rearranged, taking the temperature change into account. These datu, corrected for the temperature, were found to follow quite favorably the kinetic equation mentioned above. So far, a prediction of the ethanol production rate in practice was rectified to the extent of p = 19%.     

Growth kinetics of biopigment production by Thai isolated <Emphasis Type="Italic">Monascus purpureus</Emphasis> in a stirred tank bioreactor

Monascus purpureus is a biopigment-producing fungi whose pigments can be used in many biotechnological and food industries. The growth kinetics of biopigment production were investigated in a liquid fermentation medium in a 5-l stirred tank bioreactor at 30°C, pH 7, for 8 days with 100 rpm agitation and 1.38 × 10⁵ N/m² aeration. Thai Monascus purpureus strains TISTR 3002, 3180, 3090 and 3385 were studied for color production, growth kinetics and productivity. Citrinin as a toxic metabolite was measured from the Monascus fermentation broth. The biopigment productions were detected from fermentation broth by scanning spectra of each strain produced. Results showed a mixture of yellow, orange and red pigments with absorption peaks of pigments occurring at different wavelengths for the four strains. It was found that for each pigment color, the color production from the strains increased in the order TISTR 3002, 3180, 3090, 3385 with 3385 production being approximately 10 times that of 3002. Similar results were found for growth kinetics and productivity. HPLC results showed that citrinin was not produced under the culture conditions of this study. The L*, a* and b* values of the CIELAB color system were also obtained for the yellow, orange and red pigments produced from the TISTR 3002, 3180, 3090 and 3385 strains. The colors of the pigments ranged from burnt umber to deep red.     

Accumulation of anthocyanin in callus cultures of red-pod okra [<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> (L.) Hongjiao] in response to light and nitrogen levels

Nitrogen and light are critical determinants of biomass accumulation and secondary metabolite production under in vitro culture conditions. In this study, we analyzed the effects of varied concentrations of total nitrogen in Murashige and Skoog (MS) medium and light intensity on the production of biomass, anthocyanin pigments, and bioactive antioxidants in callus cultures of Abelmoschus esculentus cv. ‘Hongjiao’. Maximum callus biomass accumulation (3 g FW) was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 μmol m^??2 s^??1 light intensity. In contrast, maximum values of total anthocyanin accumulation (TA; 7.3 CV/g FW), total phenolic content (TP; 12.07 mg/100 g FW), total flavonoid content (TF; 2.47?±?0.15 mg/100 g FW), and total antioxidant activity (TAA; 56.10 μmol Trolox/g FW) were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 μmol m^??2 s^??1 light intensity. In addition, callus grown under same culture condition exhibited high flavonoid content along with increased phenolic content and antioxidant activity. High performance liquid chromatography (HPLC) was performed for qualitative and quantity analysis of callus cultures. Most of the pigments from the callus extracts were identical with pod anthocyanins, and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues. These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.     

Production of patulin and citrinin by <Emphasis Type="Italic">Penicillium expansum</Emphasis> from British Columbia (Canada) apples

Twenty-four isolates of Penicillium expansum Link from British Columbia (Canada) apples were cultured in yeast-extract sucrose (YES) at 25°C for 28 days to investigate production of patulin and citrinin. These isolates proved to be potent producers of citrinin, patulin, or in most cases, both mycotoxins. In every isolate, citrinin, patulin, or both compounds were produced at levels as high as 565 μg/mL (mean 269 μg/mL) and 100 μg/mL (mean 31 μg/mL), respectively. Of the 24 isolates, 4 produced citrinin only, and 2 produced patulin only. Overall, 83% of the isolates formed patulin and 91% formed citrinin. YES broth proved to be an effective medium for patulin and citrinin production. Other workers have noted that production of these mycotoxins in culture often presages production in fruits, so these results might help Canadian fruit processors evaluate and minimize mycotoxin levels in their products.     

Improvement of monacolin K, γ-aminobutyric acid and citrinin production ratio as a function of environmental conditions of<Emphasis Type="Italic"> Monascus purpureus</Emphasis> NTU 601

Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement. Its metabolic products monacolin K and -aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public. In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied. When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg. In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design. When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30°C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.     

Citrinin-producing capacity of Monascus purpureus in response to low −frequency magnetic fields

Citrinin is a type of mycotoxin that negatively affects Monascus products. Application of a low-frequency magnetic field (LF-MF) decreased citrinin production by M. purpureus in liquid-state fermentation during shake flask culture. Under 30 °C incubation, six different magnetic field induction intensity (MF-II), four different exposure times and five exposure time periods were tested to discover optimal treatment conditions. The cultures were exposure to a MF-II of 1.6 mT from 0 to 2 d of incubation time. With LF-MF treatment, peak citrinin production decreased by 46.7% while yellow, red, orange pigments and monacolin K production increased by 31.3%, 40.3%, 41.7% and 29.3%, respectively, compared with control groups at 12 d of incubation. Moreover, the relative expression levels of the citrinin biosynthesis genes pksCT and ctnA were 0.46 and 0.43 times lower, respectively, than the control values relative to the GAPDH reference gene. This study provides evidence that LF-MF is a preferable way to alter M. purpureus metabolism to reduce citrinin production and to increase pigments and monacolin K production without affecting cell growth. Therefore, LF-MF may be used as a tool to process Monascus products to obtain important functional food additive while reducing the adverse effects of citrinin.     

Effect of microelements on the biosynthesis of secondary metabolites by the fungusPenicillium citrinum thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondaryO-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions slightly affected fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

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.     

Monacolin K production by citrinin‐free Monascus pilosus MS‐1 and fermentation process monitoring

Monacolin K (MK) is a naturally occurring hypocholesterolemic agent that specifically inhibits HMG‐CoA reductase. As a natural source of MK, Monascus‐fermented products are of special interest; however, some Monascus strains could produce citrinin, which is a nephrotoxin, as a contaminant in Monascus‐derived products. A Monascus pilosus strain (MS‐1) that produces high amounts of MK, but no citrinin, was screened in previous investigations. Herein, liquid‐state fermentation parameters of the MS‐1 strain were optimized using statistical methods to maximize the MK yield with potato juice as a basic medium. The maximum MK yield (326.74 μg/mL) was predicted with 50 mL of medium in a 250‐mL conical flask containing 30 g/L sucrose, 38.75 g/L soybean flour, 0.00105 mol/L Mg²⁺ at pH 5.48, and 8% v/v seed inoculum precultured for 42 h at 30°C, incubated at 30°C for 3 days, followed by further incubation for 11 days at 24.7°C. The verified MK yield was 390.68 μg/mL and the MK yield increased to 565.64 μg/mL after 21 days of fermentation. No citrinin was detected in MS‐1‐fermented products. The results suggest that citrinin‐free MK can be obtained from natural medium through liquid‐state fermentation in an economical way. This method will be of practical value to the industrial production of MK.     

Lipids of Algae

The carotenoid pigments prepared from acetone extracts of chlorella were separated into epiphasic and hypophasic fractions by partition between petroleum ether and 90% methanol. Each fraction was subjected to column chromatography, using aluminium oxide, magnesium oxide, calcium hydroxide or calcium carbonate as adsorbent. The absorption maxima of the separated pigments in hexane and in carbon disulfide were compared with those of the known pigments. Some of the separated pigments were identified as those previously known which follow: α-carotene, β-carotene, rhodoxanthin, sarcinaxanthin, lutein and neoxanthin. Two unknown pigments with absorption maxima not yet reported were separated. The first showed absorption maxima at 478 m_μ in hexane and 518 m_μ in carbon disulfide, and the second at 383, 402 and 425 m_μ in hexane and 428 and 450 m_μ in carbon disulfide.     

Improvement of red pigment/citrinin production ratio as a function of environmental conditions by monascus ruber

The growth and metabolic behaviour of the filamentous fungus Monascus ruber were studied in submerged cultures under various aeration and agitation conditions. Improving the oxygen supply, by increasing either the air input or the agitation speed, resulted in modified metabolism: the biomass yield, the consumption of the nitrogen source (monosodium glutamate), and the production of secondary metabolites (red pigment and citrinin) all increased. However, the citrinin production increased more than that of the red pigment. In consequence, a low oxygen transfer coefficient was required to improve the red pigment/citrinin production ratio. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 497–501, 1999.     

Pigments and citrinin biosynthesis by fungi belonging to genus Monascus

Citrinin is a mycotoxin, which is produced by fungi belonging to the genus Monascus, known in biotechnology as producers of azaphilone pigments. The relation between biosynthesis of these secondary metabolites was investigated in different species of the genus Monascus in batch-culture at the following cultivation conditions: T = 28 degrees C, agitation 220 rpm, and a medium, which induce citrinin production, containing ethanol as a carbon source. The screening was carried out with 16 fungal strains and the biosynthesis of citrinin and pigments was monitored quantitatively at the standard conditions mentioned above. Some kinetic parameters of the process have been determined. The values of the growth yield coefficient Y(X/C) were between 0.32 and 0.57. The amount of the extracellular red and orange pigments at the end of cultivation varied for the different strains between 0.09 and 1.33 OU/ mg dry weight, and 0.15 and 0.96 OU/mg dry weight, respectively. The amount of the total pigments measured was between 0.16 and 3.6 OU/mg dry weight, and between 0.21 and 3.39 OU/mg dry weight. The determined ratio 500 nm/400 nm, characterizing the pigment production, ranged between 0.60 and 1.06. Twelve of the investigated strains produced citrinin and pigments, two of them produced only pigments. Two strains were not able to produce neither pigments nor citrinin. Thus, the biosynthesis of citrinin appeared to be strain-specific and does not correlate with the pigments' biosynthesis by the fungal strains belonging to the genus Monascus.     

Elimination of the mycotoxin citrinin production in the industrial important strain Monascus purpureus SM001

The application of the high-producing pigments industrial strain Monascus purpureus SM001 has been greatly limited by the synchronous production of mycotoxin citrinin. Here we have tried both traditional mutagenesis and metabolic engineering methods to eliminate the production of citrinin. Traditional chemical and physical mutagens were applied to induce mutagenesis, and a bio-screening method based on the antibacterial activity of citrinin against Bacillus subtilis was designed to select mutants. Among the resulting four citrinin-free mutants, only mutant MU2411 can maintain the similar pigments yield. A binary vector system was constructed and successfully disrupted the polyketide synthase gene pksCT in M. purpureus SM001 through the Agrobacterium tumefaciens-mediated transformation. The resulting citrinin-free ΔpksCT mutants maintained the same level of pigments yield. The established Monascus genetic system was comprehensively evaluated and showed high efficiency and specificity, which provides us a potential approach to manipulate and improve industrial Monascus strains.     

Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 μmol m⁻² s⁻¹ by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 μmol m⁻² s⁻¹ than at 65 μmol m⁻² s⁻¹, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 μmol m⁻² s⁻¹ stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata.

Graphical abstract

     

The behaviour of yeast flocs in hindered settling and fluidized bed regimes

Summary Sedimentation and fluidization of yeast flocs were found to be non-synonymous processes. The analysis of Richardson and Zaki (1954) was found not to hold when applied to yeast flocs in both regimes. Partial support and channelling were implicated in the deviations from idela behaviour. Other factors responsible for the behaviour of yeast flocs in these regimes are discussed.Symbols D bed height (cm) - g gravitational constant (981 cm·s^-1) - n constant (-) - R retardation factor (s) - S constant (-) - v liquid/particle velocity (cm·s^-1) - V _o particle terminal velocity (cm·s^-1) - bed voidage (-)     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

Batch xylitol production by<Emphasis Type="Italic"> Candida guilliermondii</Emphasis> FTI 20037 from sugarcane bagasse hemicellulosic hydrolyzate at controlled pH values

Batch fermentation of sugarcane bagasse hemicellulosic hydrolyzate by the yeast Candida guilliermondii FTI 20037 was performed using controlled pH values (3.5, 5.5, 7.5). The maximum values of xylitol volumetric productivity (Q _p=0.76 g/l h) and xylose volumetric consumption (Q _s=1.19 g/l h) were attained at pH 5.5. At pH 3.5 and 7.5 the Q _p value decreased by 66 and 72%, respectively. Independently of the pH value, Y _x/s decreased with the increase in Y _p/s suggesting that the xylitol bioconversion improves when the cellular growth is limited. At the highest pH value (7.5), the maximum specific xylitol production value was the lowest (q _pmax=0.085 g/l h.), indicating that the xylose metabolism of the yeast was diverted from xylitol formation to cell growth.List of symbols P _max xylitol concentration (g/l) - Q _x volumetric cell production rate (g/l h) - Q _s volumetric xylose uptake rate (g/l h) - Q _p volumetric xylitol production rate (g/l h) - q _pmax specific xylitol production (g/g h) - q _smax specific xylose uptake rate (g/g h) - _max specific cell growth rate (h^–1) - Y _p/s xylitol yield coefficient, g xylitol per g xylose consumed (g/g) - Y _p/x xylitol yield coefficient, g xylitol per g dry cell mass produced (g/g) - Y _x/s cell yield coefficient, g dry cell mass per g xylose consumed (g/g) - _cell percentage of the cell yield from the theoretical value (%) - _xylitol percentage of xylitol yield from the theoretical value (%)     

Inhibitory effect of dihydroxyacetone onGluconobacter oxydans: Kinetic aspects and expression by mathematical equations

Summary Microbial conversion of glycerol into dihydroxyacetone (DHA) byGluconobacter oxydans was subjected to inhibition by excess substrate. Comparison of cultures containing increasing initial DHA contents (0 to 100 g l^–1) demonstrated that DHA also inhibited this fermentation process. The first effect was on bacterial growth (cellular development stopped when DHA concentration reached 67 gl^–1), and then on oxidation of glycerol (DHA synthesis only occurred when the DHA concentration in the culture medium was lower than 85 g l^–1). Productivity, specific rates and, to a lesser extent, conversion yields decreased as initial concentrations of DHA increased. The changes in the specific parameters according to increasing initial DHA contents were described by general equations. These formulae satisfactorily express the concave aspect of the curves and the reduction in biological activity when the cells were in contact with DHA concentrations of up to 96 g l^–1.Abbreviations X, S, P biomass, substrate, product concentrations - r _x,r _s,r _p rates of growth, consumption and production - ,q _s,q _p specific rates of growth, glycerol consumption and DHA production - Y _x/s, Y_p/s conversion yields of substrate into biomass and product - K _s constant of affinity of cells to the substrate - K _ip product inhibition constant - P _m threshold concentration of DHA in substrate