Using High-Throughput Screening to Evaluate Perturbations Potentially Linked to Neurobehavioral Outcomes: A Case Study Using Publicly Available Tools on FDA Batch-Certified Synthetic Food Dyes.

There is growing evidence from human and animal studies indicating an association between exposure to synthetic food dyes and adverse neurobehavioral outcomes in children. However, data gaps persist for potential mechanisms by which the synthetic food dyes could elicit neurobehavioral impacts. We developed an approach to evaluate seven US FDA-batch-certified food dyes using publicly available high-throughput screening (HTS) data from the US EPA's Toxicity Forecaster to assess potential underlying molecular mechanisms that may be linked to neurological pathway perturbations. The dyes were screened through 270 assays identified based on whether they had a neurological-related gene target and/or were mapped to neurodevelopmental processes or neurobehavioral outcomes, and were conducted in brain tissue, targeted specific hormone receptors, or targeted oxidative stress and inflammation. Some results provided support for neurological impacts found in human and animal studies, while other results showed a lack of correlation with in vivo findings. The azo dyes had a range of activity in assays mapped to G-protein-coupled receptors and were active in assays targeting dopaminergic, serotonergic, and opioid receptors. Assays mapped to nuclear receptors (androgen, estrogen, and thyroid hormone) also exhibited activity with the food dyes. Other molecular targets included the aryl hydrocarbon receptor, acetylcholinesterase, and monoamine oxidase. The Toxicological Prioritization Index tool was used to visualize the results of the Novascreen assays. Our results highlight certain limitations of HTS assays but provide insight into potential underlying mechanisms of neurobehavioral effects observed in in vivo animal toxicology studies and human clinical studies.

Prenatal exposure to artificial food colorings alters NMDA receptor subunit concentrations in rat hippocampus.

Exposure to artificial food color additives (AFCAs) has been implicated in the etiology of certain childhood hyperactivity and learning disabilities. N-methyl-D-aspartate receptors and alpha-7 nicotinic acetylcholine receptor (α7 nAChR) are involved in learning and memory. We administered a mixture of AFCAs (erythrosine, ponceau 4R, allura red AC, sunset yellow FCF, tartrazine, amaranth, brilliant blue, azorubine, and indigotine) to female rats during gestation to investigate the effects of prenatal exposure to AFCAs on neurobehavior, spatial learning, and memory in their offspring. We also investigated whether AFCAs modulate NR2A, NR2B, and α7 nAChR protein levels in their offsprings' hippocampi. Although spatial learning and memory were not altered, the offspring of rats exposed to AFCAs exhibited decreased motivation and increased despair-related behavior. NR2A and NR2B protein levels were significantly reduced in female offspring in the experimental group (p < 0.05), whereas α7 nAChR level was not significantly altered. Our results suggest that prenatal exposure to AFCAs may lead to sex-dependent alterations in glutamatergic signaling which may continue into adolescence.

Sunset yellow degradation product, as an efficient water-soluble inducer, accelerates 1N4R Tau amyloid oligomerization: In vitro preliminary evidence against the food colorant safety in terms of "Triggered Amyloid Aggregation".

Today, Alzheimer's disease (AD) as the most prevalent type of dementia turns into one of the most severe health problems. Neurofibrillary tangle (NFT), mostly comprised of fibrils formed by Tau, is a hallmark of a class of neurodegenerative diseases. Tau protein promotes assembly and makes stable microtubules that play a role in the appropriate function of neurons. Polyanionic cofactors such as heparin, and azo dyes, can induce aggregation of tau protein in vitro. Sunset Yellow is a food colorant used widely in food industries. In the current work, we introduced degradation product (DP) of Sunset Yellow as an effective inducer of Tau aggregation. Two Tau aggregation inducers were produced, and then the aggregation kinetics and the structure of 1N4R Tau amyloid fibrils were characterized using ThT fluorescence spectroscopy, X-Ray Diffraction (XRD), circular dichroism (CD) and atomic force microscopy (AFM). Also, the toxic effects of the induced aggregates on RBCs and SH-SY5Y cells were demonstrated by hemolysis and LDH assays, respectively. Both inducers efficiently accelerated the formation of the amyloid fibril. Along with the confirmation of the ß-sheets structure in Tau aggregates by Far-UV CD spectra, X-ray diffractions revealed the typical cross-ß diffraction pattern. The oligomer formation in the presence of DPs was also confirmed by AFM. The possible in vivo effect of artificial azo dyes on Tau aggregation should be considered seriously as a newly opened dimension in food safety and human health.

The biotechnology (genetic transformation and molecular biology) of Bixa orellana L. (achiote).

MAIN CONCLUSION: Genetic transformation allows for greater bixin or norbixin production in achiote. Knowledge of genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants. Annatto is a natural dye or coloring agent derived from the seeds, or their arils, of achiote (Bixa orellana L.), and is commercially known as E160b. The main active component of annatto dye is water-insoluble bixin, although water-soluble norbixin also has commercial applications. Relative to other antioxidants, bixin is light- and temperature stable and is thus safe for human consumption. Bixin is, therefore, widely applied as a dye and as an antioxidant in the medico-pharmaceutical, food, cosmetic, and dye industries. Even though bixin has also been isolated from leaves and bark, yield is lower than from seeds. More biotechnology-based research of this industrial and medicinal plant is needed. Building on provisional genetic transformation studies, it would be advantageous to transform genes that could result in greater bixin or norbixin production. Reliable protocols for the extraction of bixin and norbixin, as well as deeper knowledge of the genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants.

Beneficial Effects of Monascus sp. KCCM 10093 Pigments and Derivatives: A Mini Review.

The production of Monascus pigments and related byproducts, via microbial fermentation, has been broadly utilized as coloring by traditional food industries and as a natural textile dye. In addition to these traditional purposes, Monascus pigments have been recently favored for a variety of commercial and academic purposes. Pigments and derivatives formed during Monascus fermentation have pharmaceutical and clinical properties that can counteract common diseases, including obesity, type-2 diabetes, and cancer. Various research attempts have investigated the optimum conditions for this derived compound synthesis, as well as the still-unknown bio-functional effects. Recently, several studies were conducted using Monascus sp. KCCM 10093 and its derivatives. These experimental outcomes potentially reflect the bio-functional features of Monascus sp. KCCM 10093. However, no publication to date provides an overview of Monascus sp. KCCM 10093's unique metabolite products, functionalities, or biological pathways. In order to develop profitable commercial applications of Monascus sp. KCCM 10093, it is necessary not only to conduct continuous research, but also to systematically organize previous Monascus studies. The goals of this review are to investigate the current derivatives of Monascus sp. KCCM 10093 pigments-some of which have demonstrated newly-identified functionality-and the relevant uses of these molecules for pharmaceutical or nutraceutical purposes.

Kinetic of orange pigment production from Monascus ruber on submerged fermentation.

Pigments produced by species of Monascus have been used to coloring rice, meat, sauces, wines and beers in East Asian countries. Monascus can produce orange (precursor), yellow and red pigments. Orange pigments have low solubility in culture media and when react with amino groups they become red and largely soluble. The orange pigments are an alternative to industrial pigment production because the low solubility facilitates the downstream operations. The aim of this work was to study the kinetic on the production of orange pigments by Monascus ruber CCT 3802. The shaking frequency of 300 rpm was favorable to production, whereas higher shaking frequencies showed negative effect. Pigment production was partially associated with cell growth, the critical dissolved oxygen concentration was between 0.894 and 1.388 mgO2 L-1 at 30 °C, and limiting conditions of dissolved oxygen decreased the production of orange pigments. The maintenance coefficient (mo) and the conversion factor of oxygen in biomass (Yo) were 18.603 mgO2 g x-1  h-1 and 3.133 gx gO 2-1 and the consideration of these parameters in the oxygen balance to estimate the biomass concentration provided good fits to the experimental data.

Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin.

Anthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

Novel natural food colourant G8000 benefits LDL- and HDL-cholesterol in humans.

The aim of this study was to investigate the phenolic composition of a natural food colourant (G8000™) as well as its effects on plasma markers after 28-day consumption by healthy individuals at a dietary dose (70 g). Parameters of total cholesterol and its fractions, triglycerides and plasma enzymes biomarkers of muscle injury were measured. Major compounds identified in G8000™ by ESI-MS showed the presence of anthocyanins, organic acids, phenolic acids as well as monosaccharides. HDL levels significantly increased from 43 ± 10.2 mg/dL to 95 ± 16.9 mg/dL. LDL levels significantly decreased from 110 ± 40.9 mg/dL to 69 ± 39 mg/dL (p < 0.001). No significant statistical differences (p > 0.05) were observed for total cholesterol, triglycerides and VLDL. After the intake, plasma enzyme CK-MB decreased from 20 ± 12.1 U/L to 10 ± 1.9 U/L while LDH levels increased from 275 ± 124.4 U/L to 317 ± 114.7 U/L (p < 0.005). No significant differences were observed for CK levels. Taken together, dietary intake of natural colourant G8000™ was able to exert beneficial effects on atherosclerosis biomarkers.

Inclusion of Palmaria palmata (red seaweed) in Atlantic salmon diets: effects on the quality, shelf-life parameters and sensory properties of fresh and cooked salmon fillets.

BACKGROUND: The use of Palmaria palmata (PP) as a natural ingredient in farmed Atlantic salmon diets was investigated. The effect of salmon diet supplementation with P. palmata (0, 5, 10 and 15%) or synthetic astaxanthin (positive control, PC) for 16 weeks pre-slaughter on quality indices of fresh salmon fillets was examined. The susceptibility of salmon fillets/homogenates to oxidative stress conditions was also measured. RESULTS: In salmon fillets stored in modified atmosphere packs (60% N2 /40% CO2 ) for up to 15 days at 4 °C, P. palmata increased surface -a* (greenness) and b* (yellowness) values in a dose-dependent manner, resulting in a final yellow/orange flesh colour. In general, the dietary addition of P. palmata had no effect on pH, lipid oxidation (fresh, cooked and fillet homogenates) and microbiological status. 'Eating quality' sensory descriptors (texture, odour and oxidation flavour) in cooked salmon fillets were not influenced by dietary P. palmata. Salmon fed 5% PP showed increased overall acceptability compared with those fed PC and 0% PP. CONCLUSION: Dietary P. palmata was ineffective at providing red coloration in salmon fillets, but pigment deposition enhanced fillets with a yellow/orange colour. Carotenoids from P. palmata may prove to be a natural pigment alternative to canthaxanthin in salmon feeds.

Binding properties of food colorant allura red with human serum albumin in vitro.

Allura red (AR) is a widely used colorant in food industry, but may have a potential security risk. In this study, the properties of interaction between AR and human serum albumin (HSA) in vitro were determined by fluorescence, UV-Vis absorption and circular dichroism (CD) spectroscopy combining with multivariate curve resolution-alternating least squares (MCR-ALS) chemometrics and molecular modeling approaches. An expanded UV-Vis data matrix was resolved by MCR-ALS method, and the concentration profiles and pure spectra for the three reaction components (AR, HSA, and AR-HSA complex) of the system were then successfully obtained to evaluate the progress interaction of AR with HSA. The calculated thermodynamic parameters indicated that hydrogen binding and hydrophobic interactions played major roles in the binding process, and the interaction induced a decrease in the protein surface hydrophobicity. The competitive experiments revealed that AR mainly located in Sudlow's site I of HSA, and this result was further supported by molecular modeling studies. Analysis of CD spectra found that the addition of AR induced the conformational changes of HSA. This study have provided new insight into the mechanism of interaction between AR and HSA.

Recent advances in engineering microorganisms for the production of natural food colorants.

Food colorants are frequently added to processed foods since color is an important tool in the marketing of food products, influencing consumer perceptions, preferences, and purchasing behavior. While synthetic dyes currently dominate the food colorant market, consumer concern regarding their safety and sustainability is driving a demand for their replacement with naturally derived alternatives. However, natural colorants are costly compared to their synthetic counterparts as the pigment content in the native sources is usually very low and extraction can be challenging. Recent advances in the engineering of microbial metabolism have sparked interest in the development of cell factories capable of producing natural colorants from renewable resources. This review summarizes major developments within metabolic engineering for the production of nature-identical food colorants by fermentation. Additionally, it highlights common applications, formulations, and physicochemical characteristics of prevalent pigment classes. Lastly, it outlines a workflow for accelerating the optimization of cell factories for the production or derivatization of nature-identical food colorants.

Synchronous Bioproduction of Betanin and Mycoprotein in the Engineered Edible Fungus Fusarium venenatum.

Sustainable production of edible microbial proteins and red food colorants is an important demand for future food. Therefore, creation of a chassis strain that can efficiently synthesize both products is extremely necessary and meaningful. To realize this envision, a CRISPR/Cas9-based visual multicopy integration system was successfully developed in Fusarium venenatum. Subsequently, the de novo synthesis of the red food colorant betanin was achieved in the engineered F. venenatum using the above system. After fermentation optimization, the final yields of betanin and mycoprotein reached 1.91 and 9.53 g/L, respectively, when the constant pH naturally decreased from 6 to 4 without the addition of acid after 48 h of fermentation. These results determine a highly suitable chassis strain for the microbial biomanufacturing of betanin, and the obtained engineered strain here is expected to expand the application prospect and improve economic returns of F. venenatum in the field of future food.

A food color-based colorimetric assay for Cryptococcus neoformans laccase activity.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

PURPOSE: This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. METHODS: Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. RESULTS: Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻6 and (3.2 ± 0.3) × 10⁻6 cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 µL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 µM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. CONCLUSIONS: Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Deletion of pigR gene in Monascus ruber leads to loss of pigment production.

Pigments produced by Monascus are traditional food colorants and are widely used as dietary supplements. Since genes involving in pigment biosynthesis have not been reported, we describe the identification of a putative pigment-regulatory gene (pigR) obtained by molecular analysis of an albino strain of Monascus ruber M7. In the pigR-deleted strain (ΔpigR), neither the pigments nor pigR expression were detected by HPLC or reverse-transcription PCR, respectively, whereas the introduction of the pigR, together with a constitutive trpC promoter into ΔpigR, caused it to produce 5.4 U of red pigments/g dry mycelia, about 12-fold higher than Monascus ruber M7 (0.46 U/g dry mycelia). Thus pigR up-regulates pigment production in Monascus ruber M7.

Monascus secondary metabolites: production and biological activity.

The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of ß-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.

Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria.

BACKGROUND: The phycobiliprotein C-phycocyanin (C-PC) is used in cosmetics, diagnostics and foods and also as a nutraceutical or biopharmaceutical. It is produced in the cyanobacterium Arthrospira platensis grown phototrophically in open cultures. C-PC may alternatively be produced heterotrophically in the unicellular rhodophyte Galdieria sulphuraria at higher productivities and under improved hygienic standards if it can be purified as efficiently as C-PC from A. platensis. RESULTS: Ammonium sulfate fractionation, aqueous two-phase extraction, tangential flow ultrafiltration and anion exchange chromatography were evaluated with respect to the purification of C-PC from G. sulphuraria extracts. Galdieria sulphuraria C-PC showed similar properties to those described for cyanobacterial C-PC with respect to separation by all methodologies. The presence of micelles in G. sulphuraria extracts influenced the different procedures. Only chromatography was able to separate C-PC from a second phycobiliprotein, allophycocyanin. CONCLUSION: C-PC from heterotrophic G. sulphuraria shows similar properties to cyanobacterial C-PC and can be purified to the same standards, despite initial C-PC concentrations being low and impurity concentrations high in G. sulphuraria extracts.

Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica.

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l-1 betanin and 55 ± 7 mg l-1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr-1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering.

Monascus pigments have various food industry applications and are pharmacologically active. Genome sequencing-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been implemented to increase pigment production in Monascus. To increase pigment production in M. ruber KACC46666, the CRISPR/Cas9 system was used to introduce mutations in two negative regulator genes (MpigI and MpigI'), among other genes involved in the Monascus pigment biosynthesis pathway. Dual single-guide RNAs were constructed to inactivate MpigI and MpigI'. After CRISPR/Cas9 inactivation, yellow, orange, and red pigment expression in the resulting △MpigI16-7 strain (among several Cas9-mediated mutants studied) was 2.5-, 12.4-, and 18.5-fold, respectively, higher than that in the wild-type strain. This study provides valuable information regarding CRISPR-guided metabolic engineering for natural colorant production.