Formation mechanism of blue pigment in boiled lotus rhizome discs: Insight into the chelation of polyphenols and iron.

Boiled lotus rhizome discs (BLRDs), as common processed products of lotus rhizome, have gained increasing attention from consumers and food manufacturers. However, the blue pigment formed during boiling affects its appearance and reduces the appetite of BLRDs. In this study, the effects of polyphenols and iron contents on blue pigment formation in BLRDs in different regions and months were investigated. Results revealed that blue variation was more serious in March and April of the second year in Wuhan, and polyphenols and iron contents in these two months were significantly higher than those in other months. Then, UPLC and UV-Vis analysis showed that polyphenols causing the formation of blue pigment in BLRDs were L-dopa, gallocatechin, catechin, epigallocatechin, chlorogenic acid and epicatechin, among which L-dopa (52.450 mg/100 g in fresh lotus rhizome (FLR)) and gallocatechin (36.210 mg/100 g in FLR) possessed the greatest effect. Moreover, the ESI-Q-TOF-MS analysis of L-dopa-iron chelate and gallocatechin-iron chelate suggested that the blue pigment of BLRDs was mainly in the form of bis-complexes under boiling conditions. The study on formation mechanism of blue pigment in BLRDs can provide a reference for lotus rhizome processing.

Enzymatic synthesis of Vaccinium blue using vaccinoside as a bifunctional precursor.

Since Tang dynasty in China, the fresh leaves of Vaccinium bracteatum (VBL) have been applied as natural pigment to produce black rice. However, detailed information on its biosynthetic mechanism still remained unclear. Following rice dyeing capacity assay, vaccinoside, one of iridoid glycosides, was identified as the key active compound. Increased methodical research demonstrated vaccinoside as a distinct bifunctional precursor, which could be catalyzed by polyphenol oxidase or ß-glucosidase independently, followed by reaction with 15 amino acids to give blue pigments (VBPs; λmax 581-590 nm) of different hues. Two synthetic pathways of VBPs were proposed, using multiple techniques such as HPLC, HPSEC, UV-Vis spectrum and colorimeter as analysis tools. Black rice was interpreted to be prepared by cooking, using vaccinoside, intrinsic enzymes from fresh VBL and rice protein in combination. These findings promote the understanding of VBP formation mechanisms and provide an efficient method of producing novel Vaccinium blue pigments.

Influence of Compounding Parameters on Color Space and Properties of Thermoplastics with Ultramarine Blue Pigment.

The incorporation of thermoplastics with pigments imparts diverse aesthetic qualities and properties to colored thermoplastic products. The selection of pigment type and content, along with specific processing conditions, plays a pivotal role in influencing color properties and overall product performance. This study focuses on optimizing these parameters to ensure the desired color quality and product functionality. Two types of polypropylene copolymer (PPCP) with different melt flow rates (MFRs) and acrylonitrile butadiene styrene (ABS) were compounded with ultramarine blue pigment masterbatch (MB) in concentrations ranging from 1 to 5 wt.% using a twin-screw extruder. The compounding process was conducted at a constant screw speed of 200 rpm and a die temperature of 210 °C. The effects of screw speed and die temperature were investigated at a constant MB of 3 wt.%. Colored samples were fabricated by injection molding. Microscopic analysis revealed a well-dispersed pigment within the PPCP matrix when using the MB. Rheological properties, assessed through the power law index, confirmed effective pigment dispersion, facilitated by shear thinning behavior and controlled shear rate via the manipulation of screw speed and die temperature. The effects of masterbatch contents and processing conditions on color spaces were evaluated using CIELAB and CIELCH, with one-way ANOVA employed to identify statistical significance. Higher opacity in high-MFR PPCP and ABS resulted in increased lightness and color strength, surpassing low-MFR PPCP by 15-40% at equivalent MB contents. Masterbatch content emerged as a significant factor influencing the color spaces of all colored thermoplastics. Further analysis, including Fisher pairwise comparisons of one-way ANOVA, revealed that screw speed influenced the redness and hue of low-MFR PPCP, whereas die temperature affected the lightness and hue of high-MFR PPCP and ABS. Interestingly, the blueness and chroma of colored thermoplastics were minimally affected by both screw speed and die temperature. Notably, regardless of processing conditions, the flexural properties of colored thermoplastics remained comparable to the neat polymer when incorporated with ultramarine blue pigment masterbatch.

Total Synthesis of Azulene Derivative, a Blue Pigment Isolated from Lactarius indigo, and Colorant Application of Its Aqueous Dispersion.

Safety concerns in the food industry have increased the demand for natural food colorants. However, the application ranges of natural blue colorants are insufficient because they are scarce in nature, and the currently available natural blue dyes are limited to water-soluble products. In this study, we investigated a fat-soluble azulene derivative isolated from the mushroom Lactarius indigo as a potential candidate for a natural blue colorant. We developed its first total synthesis, where the azulene skeleton was constructed from a pyridine derivative and an ethynyl group was converted into an isopropenyl group using zirconium complexes. Moreover, nanoparticles of the azulene derivative were prepared via reprecipitation method, and their colorant ability was investigated in aqueous solutions. The new candidate food colorant exhibited a deep-blue color in an organic solvent and aqueous dispersion.

Optimization of fermentation, purification, and properties of blue pigment produced from Quambalaria cyanescens QY229.

AIM: Blue pigments have broad applications in foods, cosmetics, and clothing. However, natural blue pigments are rare. At present, the majority of blue pigments for sale are chemically synthetic. Owing to the safety risks of chemical pigments, it is an urgent demand to develop novel natural blue pigments. METHODS AND RESULTS: The fermentation medium and culture conditions of blue pigment produced by Quambalaria cyanescens QY229 were optimized by Plackett-Burman (PB) experimental design and response surface methodology (RSM) for the first time. The stability, bioactivity, and toxicity of the obtained blue pigment were studied after isolation and purification. CONCLUSION: The results showed that the optimal fermentation parameters were 34.61 g·L-1 of peptone concentration, 31.67°C of growing temperature, and 72.33 mL of medium volume in a 250-mL flask, and the yield of blue pigment reached 348.2 ± 7.1 U·mL-1. QY229 blue pigment is stable to light, heat, pH, most metal ions, and additives, and has certain antioxidant and inhibitory activity of α-glucosidase in vitro. QY229 blue pigment at concentrations of 0-1.25 mg·mL-1 was nontoxic to Caenorhabditis elegans in an acute toxicity trial.

Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System.

Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein's catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. The reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.

The Polysaccharidic Nature of the Skeleton of Marennine as Determined by NMR Spectroscopy.

The water-soluble blue-green pigment marennine, produced and partly excreted by the diatom Haslea ostrearia, and known for a long time for its role in the greening of oysters, was isolated from the culture medium, purified, and analyzed by Nuclear Magnetic Resonance (NMR) in order to gain insight into its chemical structure. The spectra show mainly carbohydrates of a complex composition, apparently highly branched, and with a mass in the order of 10 kDa. There are, in addition, some signals of aliphatic and, much weaker, aromatic groups that present aglycons. The latter might be responsible for the color. These carbohydrates are always associated with the blue-green color and cannot be separated from it by most treatments; they are interpreted as constituting the frame of the pigment. NMR after hydrolysis identifies the most abundant monosaccharides in marennine as galactose, xylose, mannose, rhamnose, and fucose.

Compositional analysis and immunomodulatory activity of blue pigment fraction (BPF) from Laba garlic.

Laba garlic is a kind of garlic (Allium sativum L.) product and blue pigment fraction (BPF) is the characteristic fraction of Laba garlic. The objective of the study was to isolate BPF from Laba garlic and explore its stability, composition, antioxidant activity, and immunomodulatory activity. The results suggested BPF was unstable under alkaline conditions. Twenty-four constituents including 9 peptides and 10 saponins were detected in BPF by Q Exactive HF LC/MS anlaysis. BPF showed antioxidant activity in a dose-dependent manner. It also showed effective immunomodulatory activity at a concentration of 5 µg/mL at the cellular level and the morphology of RAW 264.7 cells changed to a polygonal and dendritic-like structure. BPF could significantly increase NO production (P < 0.05), and up-regulate the mRNA levels of TNF-α, IL-6, iNOS and NF-κB in the RT-QPCR analysis. The present study systematically analyzed the compositions of BPF for the first time, and the results suggested that BPF might be a potential immunomodulator candidate, which is beneficial for the development and application of garlic products and natural pigments.

Isolation of the Thermostable ß-Glucosidase-Secreting Strain Bacillus altitudinis JYY-02 and Its Application in the Production of Gardenia Blue.

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by ß-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable ß-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of ß-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding ß-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant ß-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant ß-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-ß-d-glucopyranoside (pNPG) was used as the substrate. The recombinant ß-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE ß-Glucosidases are enzymes capable of hydrolyzing ß-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial ß-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable ß-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant ß-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified ß-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.

Relation of shear stress and KLa on bikaverin production by Fusarium oxysporum CCT7620 in a bioreactor.

This study involved evaluating the effects of rotational impeller speed agitation (N) and specific air flow rate (Фair) on bikaverin production and on the growth of Fusarium oxysporum employing 11 bench-scale bioreactor assays. The results showed that the maximum bikaverin production (close to 300 mg L-1) was achieved after 48 h of fermentation in rice medium (20 g L-1 milled rice in water) at 28 °C with a volumetric oxygen transfer coefficient (KLa) and shear stress values of approximately 20 h-1 and 17 N m-2, respectively. We reached this combination of parameters using an N of 340 rpm and Фair of 0.935 vvm. These KLa and shear stress values can be used as references when upscaling this process. Thus, this study was important to demonstrate how the main parameters in bioreactors affect bikaverin production and it presented important indications for upscaling this bioprocess.

Evaluation of the physicochemical properties and in vitro digestibility of the complex formed between rice starch and a novel pigment from Vaccinium bracteatum Thunb. leaf.

This study investigated the effect of Vaccinium bracteatum Thunb. leaf (VBTL) dark blue pigment on the physicochemical properties and in vitro digestibility of different rice starches. The results showed that glutinous rice starch (GRS) had a higher pigment loading capacity than indica rice starch (IRS) and japonica rice starch (JRS). Fourier transform infrared spectroscopy showed that these binary complexes between pigment and starch molecules bind through strong hydrogen bonds. All starch-pigment complexes displayed a lower gelatinization enthalpy than the controls. The addition of this pigment reduced the maximum starch in vitro digestion amount. Furthermore, the addition of pigment significantly increased the resistant starch and decreased the rapidly digestible starch in 'Wu mi', rice grains dyed by VBTL. This study provided important evidence of the digestion resistibility of VBTL dark blue pigment on 'Wu mi' to promote glycaemic regulation as a healthy cereal product.

(S)-3-aminopiperidine-2,6-dione is a biosynthetic intermediate of microbial blue pigment indigoidine.

The biosynthetic investigations of microbial natural products continuously provide powerful biocatalysts for the preparation of valuable chemicals. Practical methods for preparing (S)-3-aminopiperidine-2,6-dione (2), the pharmacophore of thalidomide (1) and its analog drugs, are highly desired. To develop a biocatalyst for producing (S)-2, we dissected the domain functions of IdgS, which is responsible for the biosynthesis of indigoidine (3), a microbial blue pigment that consists of two 2-like moieties. Our data supported that the L-glutamine tethered to the indigoidine assembly line is first offloaded and cyclized by the thioesterase domain to form (S)-2, which is then dehydrogenated by the oxidation (Ox) domain and finally dimerized to yield 3. Based on this, we developed an IdgS-derived enzyme biocatalyst, IdgS-Ox* R539A, for preparing enantiomerically pure (S)-2. As a proof of concept, one-pot chemoenzymatic synthesis of 1 was achieved by combining the biocatalytic and chemical approaches.

Preparation and Characterization of Water-Insoluble Gardenia Blue Pigment.

Based on molecular simulations, the synthetic route of water-insoluble gardenia blue pigment was prepared by the reaction of genipin and L-Phenylalanine methyl ester hydrochloride. A highly purified pigment was obtained after extraction by chloroform and purification by silica gel column chromatography, and the value of color is up to 288. A study on the structural characteristics of the pigment was implemented with a scanning electron microscope, ultraviolet-visible spectrophotometer, Fourier transform infrared spectrometer, X-ray photoelectron spectrometer, and quatropde-time of flight mass spectrometer. The results showed that the surface of the pigment was largely smooth and spherical; The λmax was 607 nm, and the main functional groups include O-C=O, C=O, C-N, C=C, OH, and benzene ring; We detrained six different molecular weight and chemical structures of pigments and speculated the particular structures and formation mechanisms of three kinds of pigment, whose molecular weights are 690.1156, 720.1226, and 708.1246 Da, respectively. The pigment was only able to be dissolved in ethanol, methanol, acetone, ethyl acetate, and other strong polar organic solvents, but was not able to be dissolved in water, ethyl ether, petroleum ether, and other weak polar organic solvents. In terms of light and thermal stabilities, water-insoluble gardenia blue pigment is significantly better than water-soluble gardenia blue pigment (p < 0.05). When it is under direct light for 7 days or incubated at 80-120 °C for 24 h, the pigment residual rates were 74.90, 95.26, 88.27, and 87.72%, respectively.

Electrochromic Properties and Electrochemical Behavior of Marennine, a Bioactive Blue-Green Pigment Produced by the Marine Diatom Haslea ostrearia.

Marennine has long been known as the unique peculiar pigment responsible for the natural greening of oysters. It is specifically produced by the marine diatom Haslea ostrearia and it is a natural blue molecule indeed promising for food industry because of the rarity of such non-toxic, blue-colored pigments. In the search for its still not defined molecular structure, investigation of the color changes with the redox state has been carried out combining different approaches. Reducing and oxidizing chemicals have been added to purified marennine solutions and a stable blue-green color has been confirmed for the oxidized state, while a yellow color corresponded to the reduced unstable state. Raman spectroscopy has been used to monitor changes in the Raman spectra corresponding to the different colored states, and cyclic voltammetry has allowed the detection of a redox system in which protons and electrons are exchanged. These findings show that marennine is a suitable stable blue pigment for use in food applications and help in the elucidation of the chromophore structure.

Vaccinium bracteatum Thunb. as a promising resource of bioactive compounds with health benefits: An updated review.

Vaccinium bracteatum Thunb.(VBT) is well-known for many physiological and bioactivities in some ancient Chinese pharmacopeias and modern researches. The health benefits are related to the presence of various nutritional and bioactive compounds. This review aims to demonstrate an updated overview of VBT in respect of botanical characters, nutritional and bioactive composition, main biological activities, and current applications. Various studies have emphasized at promising health benefits of VBT against hyperglycemia, oxidative stress, inflammation, depressive disorder, and retinal damage. However, the applications of VBT are limited to some native traditional foods and Chinese medicine. The novel beneficial efficacy and applications are still needed to be investigated. In conclusion, more research is necessary to overcome these gaps between the in-depth insights of health benefits and potential industrial applications. This review will contribute in future research for developing the functional foods derived from VBT.

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese.

New cases of blue cheese discoloration has led to recent research to identify the causal agent and factors that favor blue pigment appearing. Nonetheless, very few reports have described the source of contamination and the measurements to eradicate the microbiological source on cheese farms by determining the relation between blue discoloration on fresh cheese and the Pseudomonas fluorescens group. Thus, 60 samples from a cheese farm (cheese, equipment surfaces, tap water, and raw and pasteurized milk) were analyzed by phenotypical, MALDI-TOF, 16S rRNA sequencing and pulsed-field gel electrophoresis tests to determine the causal agent. The results obtained by pulsed-field gel electrophoresis with restriction enzymes XbaI and SpeI confirmed tap water as the initial contaminated source. The above-mentioned result was essential to avoid Pseudomonas contamination due to the most residual microorganisms being inactivated through a new disinfection program.

Spectroscopic, mineral, and antioxidant characteristics of blue colored powders prepared from cornflower aqueous extracts.

The present study aimed to prepare blue colored powders from an aqueous extract of cornflower petals. Low temperature (4 °C) aqueous extraction (1:20) and microencapsulation by freeze-drying were performed. A mixture of stabilizers (maltodextrin, guar gum, and lecithin) in a proportion of 10% to the amount of extract was used. The results indicated that the addition of 2% and 4% guar gum to maltodextrin (8-6%) significantly increased the efficiency of the process, but 4% guar gum caused the formation of amorphous particles; therefore, 2% guar gum addition was found to be the most optimal. The FT-IR and FT-Raman band characteristics for guar gum, lecithin, and maltodextrin dominated over those for anthocyanins contained in the powders made from cornflower petals. The blue powders had total phenolic content of 19.5-26.6 mg GAE/g DW. The antioxidant activity of the prepared powders measured by ABTS, CHEL, OH, and RED was high.

Interactions between L. monocytogenes and P. fluorescens in Dual-Species Biofilms under Simulated Dairy Processing Conditions.

In dairy processing environments, many bacterial species adhere and form biofilms on surfaces and equipment, leading to foodborne illness and food spoilage. Among them, Listeria monocytogenes and Pseudomonas spp. could be present in mixed-species biofilms. This study aimed to evaluate the interactions between L. monocytogenes and P. fluorescens in biofilms simulating dairy processing conditions, as well as the capability of P. fluorescens in co-culture to produce the blue pigment in a Ricotta-based model system. The biofilm-forming capability of single- and mixed-cultures was evaluated on polystyrene (PS) and stainless steel (SS) surfaces at 12 °C for 168 h. The biofilm biomass was measured, the planktonic and sessile cells and the carbohydrates in biofilms were quantified. The biofilms were also observed through Confocal Laser Scanning Microscopy analysis. Results showed that only P. fluorescens was able to form biofilms on PS. Moreover, in dual-species biofilms at the end of the incubation time (168 h at 12 °C), a lower biomass compared to P. fluorescens mono-species was observed on PS. On SS, the biofilm cell population of L. monocytogenes was higher in the dual-species than in mono-species, particularly after 48 h. Carbohydrates quantity in the dual-species system was higher than in mono-species and was revealed also at 168 h. The production of blue pigment by P. fluorescens was revealed both in single- and co-culture after 72 h of incubation (12 °C). This work highlights the interactions between the two species, under the experimental conditions studied in the present research, which can influence biofilm formation (biomass and sessile cells) but not the capability of P. fluorescens to produce blue pigment.

Comparison of blue discoloration in radish root among different varieties and blue pigment stability analysis.

The internal blue discoloration of radish root after harvest is a physiological phenomenon that decreases the radish quality. Internal blue discoloration in the roots of 16 varieties of Chinese radish along with the stability of blue pigment under different light, pH, and temperature conditions were investigated. Among the varieties LB05-244 and LB05-240 displayed the greatest degrees of discoloration, while the Piton and Dense radishes exhibited the lowest degrees of discoloration. The light and pH conditions along with the storage temperature affected the pigment stability. The degradation of blue pigment occurred faster under blue light than under green, red, and white light and darkness. Blue pigment degraded fastest at pH values of 13 and 1. The blue pigments in radish exhibited thermal instability, with complete degradation occurring in 5 or 10 min at 90 or 100 °C, respectively. In conclusion, variety affected the discoloration. The pigment exhibited light, pH and thermal instability.

Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production.

Indigoidine is a dark-blue natural pigment with application prospect and synthesized from glutamine (Gln) by series of indigoidine synthetases (IndCs). Indigoidine production can be improved by enhancing Gln pool via supplementing Gln directly or converting metabolism glutamate (Glu) to Gln by glutamine synthetase (GlnA). But, Gln is expensive, and excess Gln inhibits indigoidine production of the recombinant strain. Supplementing Glu instead of Gln may improve the productive and economic efficiency of indigoidine, but the local activities and positions of the indigoidine pathway enzymes GlnA, Sc-IndC, and the helper protein of Sc-IndC (IndB) should be well arranged. We identified the Streptomyces chromofuscus ATCC 49982 derived IndC (Sc-IndC) as an more efficient IndC compared to other IndCs applied for constructing indigoidine-producting strains, and designed series of protein scaffold complexes with architectures of PDZ, SH3, and GBD domains (PxSyG1) to arrange the pathway enzymes. The strain recruiting GlnA, Sc-IndC, and IndB on the PDZ, SH3, and GBD domains of scaffold P1S2G1, respectively, was the most efficient. In the strain, the GlnA supplied sufficient local Gln for Sc-IndC from Glu, and the generated Gln was immediately consumed by Sc-IndC to relieve cell growth inhibition caused by Gln. The optimum Glu concentration (6 g/L) for the strain was higher than those of the strains recruiting Sc-IndC on the GBD domain, which was away from the PDZ domain recruiting GlnA. The highest titer of indigoidine was 12 g/L, which was two folds of the control without scaffold (5.8 g/L). The titer is 5 g/L higher than the control without Glu supplemented (6.9 g/L), meaning that 97% of the supplemented Glu was transformed into indigoidine. The batch fermentation with the optimum strain in a 5-L reactor achieved an indigoidine titer of 14 g/L in 60 h. To our knowledge, this was the most efficient indigoidine productivity achieved so far. The optimization strategies by protein scaffold should be applicative to other pathways with complex substrate demands. KEY POINTS: •Protein scaffold systems were designed to arrange the indigoidine synthetic pathway. •The scaffold system improved supplement of Gln for indigoidine production from Glu. •The inhibition caused by excess Gln was relieved by proper designed scaffold. •The yield and titer of indigoidine was improved by arranging the pathway enzymes. Graphical abstract.