Enhanced Pigment Production Research Articles

Enhanced anthocyanin production from grape callus in an Air-Lift type bioreactor using a viscous additive-supplemented medium

An N-medium containing carboxymethyl cellulose (CMC) was applied to an air-lift type bioreactor culture of grape ( Vitis vinifera cv. Bailey alicant A.) callus, and anthocyanin production was investigated. Grape callus grew well at an air flow rate of 80 ml/min and anthocyanin production was significantly increased in the N-medium, reaching 17 mg/ l after 7 d of culture. The anthocyanin content of the N-medium was about two times higher than that of the conventional medium without CMC. The effect of air flow rate was also investigated within the range from 40 to 160 ml/min. A twofold increase in anthocyanin content was obtained at all the air flow rates tested in the N-medium. The distribution of grape callus size obtained after 7 d of the bioreactor culture was investigated. The average callus size was 490 μm which was 1.6 times larger than that obtained in the conventional medium. It was found that large calli with a relatively high anthocyanin pigment content were formed in the bioreactor culture using the N-medium. The fluid dynamics in the bioreactor was also investigated at three points (top, middle and bottom) in the bioreactor by laser doppler velocimetry. The average axial velocity of the circulated medium was 0.4 times lower than that of the conventional medium while their average radial velocities were almost the same (zero). The standard deviation of radial velocity fluctuation in the N-medium was also 0.4 times less than that in the conventional medium. These results suggest that turbulent flow occurred in the bioreactor culture using the conventional medium and the degree of turbulent flow decreased significantly when 0.8% CMC was added to the medium to prepare the N-medium. A change of the flow pattern is considered to be the cause of the decrease in hydrodynamic stress, resulting in enhanced pigment production due to the enlargement of the callus.

The fermentation of rice for anka pigment production

Optimal physical parameters of the solid state fermentation of rice to produce anka pigments and their influences on pigment production were studied. Anka pigment production, especially that of two orange anka pigments (rubropunctatin and monascorubrin), was highly sensitive to the moisture content of the rice substrate. Optimal initial moisture content of rice substrate was 24%. Pigment formation was retarded when extra water was added to the inoculated substrate during cultivation. High filling amount of rice substrate in a flask was unfavorable for pigment production. Loosening of the inoculated substrate once a day enhanced pigment production. With a high carbon dioxide level in the incubator, no orange pigments were detected. Freeze drying the fermented material produced a superior yield of anka pigments, while oven drying at 50°C for 24 h was a reasonable alternative. Journal of Industrial Microbiology & Biotechnology (2000) 25, 141–146.

Medium-chain fatty acids affect citrinin production in the filamentous fungus Monascus ruber.

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the (13)C-pigment molecules from mycelia cultivated with [1-(13)C]-, [2-(13)C]-, or [1, 2-(13)C]acetate by (13)C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.

The effect of increased phenazine antibiotic production on the inhibition of economically important soil‐borne plant pathogens by pseudomonas a ureofaciens 30–84

Pseudomonas aureofaciens 30–84 produces three phenazine antibiotics which are responsible for the ability of the strain to compete with the indigenous microflora in the rhizosphere. An additional consequence of phenazine production is the inhibition of several fungal plant pathogens in vitro. Phenazine production is regulated by a positive activator (PhzR) that acts in conjuction with a diffusable signal produced by Phzl. Introduction of additional copies of PhzR into strain 30–84 resulted in ca. a five‐fold increase in the level of phenazine production and increased the ability of strain 30–84 to inhibit the growth of nine economically important fungal pathogens in vitro. Six of the nine fungal strains were inhibited significantly more in the presence of the over‐producing strain. Gaeumannomyces graminis, was inhibited greater than 50%, regardless of the level of phenazines produced. The addition of a specific carbon source in Kanner medium (medium B) enhanced pigment production and inhibition of the fungal strains as compared to 1/5 potato dextrose agar medium (medium A), consistent with the proposed role of nutrients in the regulation of phenazine biosynthesis. The sclerotia formation of Sclerotinia sclerotiorum was 0% in the presence of strain 30–84 and strain 30–85 on medium B. However, 30–84 could not significantly reduce sclerotia formation on medium A, where less phenazine pigments were produced. In greenhouse cone assays, 30–84 significantly reduced disease incidence and disease severity. The phenazine deficient mutant strain 30–84 Z, was significantly less effective in cone assay experiments.

Identification of a peptide from mammal albumins responsible for enhanced pigment production by group B streptococci.

The peptide from peptones responsible for enhanced pigment production by Streptococcus agalactiae in culture media has been isolated from a peptic digest of human albumin and has been identified as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe. The related heptapeptide lacking the N-terminal Ile also had pigment-enhancing activity. A sequence similarity search showed that these sequences are present only in mammal albumins.

Morphological change and enhanced pigment production of monascus when cocultured with saccharomyces cerevisiae or aspergillus oryzae

When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture. Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus. However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus. Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp. oryzae. Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes. Culture filtrates of S. cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S. cerevisiae. The hydrolytic enzymes produced by S. cerevisiae, such as amylase, and chitinase, are thought to be the effectors. The commercial enzymes alpha-amylase and protease from Asp. oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production. However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective. The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls. An approximate 10-fold increase in pigment production was observed in liquid cocultures with S. cerevisiae. Copyright 1998 John Wiley & Sons, Inc.

Identification of serum and urine proteins responsible for enhanced pigment production by group B streptococci as amylases.

The serum and urine proteins responsible for enhanced pigment production in Streptococcus agalactiae in culture media were purified by chromatography and were identified as amylases by comparison of their amino acid composition with that calculated for proteins with known sequences. Similar pigment-enhancing activity was displayed by other amylases of nonanimal origin and by maltooligosaccharides.

Photo-Regulated Astaxanthin Production by Phaffia rhodozyma Mutants

Astaxanthin production by Phaffia rhodozyma mutants was photo-inducible. The intracellular astaxanthin concentration during cultivation in the dark was almost constant in the different mutants and did not reflect the concentrations reached in cells grown in the light, which differed considerably among these mutants. The final astaxanthin concentration obtained was determined by the total time of illumination, the growth phase subjected to illumination and the light intensity. Higher astaxanthin levels were obtained when cells were illuminated during the exponential growth phase. Subjecting cells to a 15 min light pulse greatly enhanced pigment production, but the stimulation was of a transient nature and a reduction in total pigment content was observed when the cultures were returned to the dark. The maximum astaxanthin production was obtained when cultures were continuously illuminated at moderate light intensities (5 to 100 µE.m-2·-1). Blue light was primarily responsible for photo-induced astaxanthin production in P. rhodozyma.

Derivation of highly mefloquine-resistant lines from Plasmodium falciparum in vitro.

Serial passage of a multidrug-resistant clone of Plasmodium falciparum in concentrations of mefloquine hydrochloride ranging from 30 to 2,400 ng/ml resulted in the derivation of increasingly resistant parasite lines in vitro. Parasite lines isolated in mefloquine concentrations greater than 300 ng/ml demonstrated increased vacuolization, enhanced pigment production, and increased growth rates as compared with the progenitor clone, W2-mef. Although microdilution incorporation assays demonstrated that the 50% inhibitory concentration (IC50) of mefloquine were similar for all lines, the IC90, IC95, and IC99 levels were significantly increased. Growth rate assays performed in 5% hematocrit suspensions demonstrated different levels of mefloquine resistance among these lines. Under these conditions the most resistant line, Mef 2.4, grew efficiently in approximately 10-fold higher concentrations of mefloquine than the progenitor clone W2-mef. Analysis of drug susceptibility profiles to mefloquine hydrochloride, chloroquine diphosphate, quinine sulfate, and halofantrine hydrochloride indicated that selection for high levels of mefloquine resistance had resulted in significant increases in resistance to halofantrine and increased sensitivity to chloroquine. The phenotypic changes demonstrated in the most resistant line, Mef 2.4, reflect a multidrug resistant-like phenotype, and appear to mimic changes recently reported in drug susceptibility profiles of recrudescent isolates following mefloquine treatment failures in Thailand.

Reinvestigation of Brevibacterium sp. Strain KY-4313 as a Source of Canthaxanthin

The hydrocarbon-utilizing Brevibacterium sp. strain KY-4313 was reevaluated for its potential to produce canthaxanthin, a carotenoid pigment of strong commercial interest. Three approaches were used to optimize the canthaxanthin yield from this organism, i.e., the preparation of mutants, the addition of supposedly carotenogenic chemicals to the growth medium, and growth promotion. Following treatment of the parent strain with N-nitrosomethylurea, a presumed mutant was isolated which showed a 32% increase in cellular canthaxanthin content. No effective carotenogenic chemicals were found in connection with hydrocarbon fermentations, in which mainly growth promotion through periodic medium renewal proved conducive to enhanced pigment production. Carotenogenesis could be stimulated in brain heart infusion broth by adding alcohols or retinol. Improved growth in this medium was generally not associated with higher canthaxanthin yields. Both superior growth and pigment levels were obtained in a newly designed medium based on fumaric acid-molasses. The maximum yields of canthaxanthin in shake flasks were (in milligrams per liter) 4.2 (brain heart infusion broth plus propanol-zinc sulfate), 3.6 (hydrocarbon medium), and 9.3 (fumaric acid-molasses), which represent a significant improvement over the originally reported optimal result (1 mg/liter). The corresponding yields of echinenone, the direct precursor of canthaxanthin, were 1.2, 1.6, and 2.3 mg/liter, respectively. Two-liter hydrocarbon batch fermentations involving medium renewal maximally produced 7.2 mg of canthaxanthin and 3.7 mg of echinenone per liter.

Effects of various agents on the pigment (violacein) and antibiotic production of Alteromonas luteoviolacea.

Effects of various antibiotics on the growth and pigment (violacein) production in eleven strains of Alteromonas luteoviolacea were examined. Antibiotic production by Alteromonas strains was also studied under various conditions. With the lapse of time, actinomycin D, colistin, streptomycin and leucomycin initially inhibited the growth of all the strains, and also their pigment production. Low concentration of chloramphenicol, oleandomycin and erthromycin rather enhanced pigment production in all the strains. Lincomycin, sulfisoxazole and sulfisomezole enhanced their growth, but suppressed their pigment production. When cultured on the ZoBell medium for 24h, the Alteromonas strains produced a compound which inhibited several strains of Vibrio parahaemolyticus, Vibrio alginolyticus, Lactobacillus delbrueckii, Corynebacterium strain 520 and Proteus vulgaris. V. parahaemolyticus and V. alginolyticus were also inhibited by the Alteromonas strains grown on L-valine medium, while Staphylococcus citreus, Micrococcus lysodeikticus, Bacillus subtilis, Escherichia coli and Alteromonas haloplanktis strain 1055-1 were inhibited by the Alteromonas strains grown in a L-tyrosine medium. The optimum pH for antibiotic production was 7. Solid media were found better than broth media for antibiotic production in the Alteromonas strains.

Pigment Production from Immobilized Monascus sp. Utilizing Polymeric Resin Adsorption

Pigment production by the fungus Monascus sp. was studied to determine why Monascus sp. provides more pigment in solid culture than in submerged culture. Adding a sterilized nonionic polymeric adsorbent resin directly to the growing submerged culture did not enhance the pigment production, thus indicating that pigment extraction is probably not a factor. Monascus cells immobilized in hydrogel were studied and exhibited decreased pigment production as a result of immobilization. This result is thought to be due to diffusional resistance of the pigment through the hydrogel beads. Addition of the adsorbent resin to the immobilized Monascus culture increased both the maximum pigment yield and the production rate above those of the free-cell fermentations. The provision of a support for the mycelium may explain enhanced pigment production by the solid-state culture. These results indicate that product diffusion from immobilized cell systems can be the limiting factor and that in situ extraction is one possible way to circumvent this problem.

Pigmentation and autofluorescence of Candida species after growth on tryptophan media.

Candida albicans and six other medically important Candida species were cultured on glucose–salts–biotin media containing tryptophan as the major nitrogen source. Candida albicans, C. tropicalis, and C. parapsilosis produced a pink pigment; C. krusei, C. pseudotropicalis, and C. gulliermondii formed brown pigments. Pigmentation of C. stellatoidea was variable, some strains produced brown pigments and others a pink pigment. Production of pink pigment was catalyzed by light. Cultures incubated in the dark produced only trace amounts of the pink pigment. Iron enhanced pigment production. Intense autofluorescence was observed for strains producing the brown pigment and weak autofluorescence for strains producing the pink pigment.

Porphyrin production by Arthrobacter globiformis.

The production by Arthrobacter globiformis of an extracellular red pigment in synthetic mineral salts medium was governed by both the conditions of aeration and the trace metals present. Pigment production was the same with either ammonium or nitrate as the nitrogen source. Stand flasks gave approximately the same cell yield as shake cultures but growth was much slower and no pigment was produced. Mn++ was required for maximal pigment production. Zn++ and Co++ enhanced pigment production in the presence of Mn++ but could only partially replace it. The pigment was ether-extractable from culture supernatant fluid acidified with acetic acid.Absorption spectra showed peaks at 620, 565, 527, 495, and 395 mμ. These as well as the extraction properties of the pigment coincided with those reported for coproporphyrin-III.