Natural Food Pigments Research Articles

Hydroxysafflor yellow A, a natural food pigment, ameliorates atherosclerosis in ApoE−/− mice by inhibiting the SphK1/S1P/S1PR3 pathway

AbstractAtherosclerosis (AS) is the pathologic basis of many cardiovascular diseases (CVDs). Hydroxysafflor yellow A (HSYA) is a valuable natural food pigment that has been reported to have significant health‐promoting abilities. However, the anti‐AS efficacy and mechanisms of HSYA have not yet been characterized. Here, we found that treatment of apolipoprotein A (ApoE) knockout (ApoE−/−) mice with HSYA markedly ameliorated atherosclerosis evidenced by decreased levels of lipids, sphingosine‐1‐phosphate (S1P), inflammatory factors, oxidative stress, vascular endothelial permeability, and endothelial damage. Moreover, mechanistic studies revealed that HSYA treatment downregulated the expression of aortic sphingosine kinase 1 (SphK1), sphingosine‐1‐phosphate receptor 3 (S1PR3), Ras homolog family member A (RhoA), Rho‐associated coiled‐coil containing protein kinase (ROCK), and filamentous actin (F‐actin). The results of administration with HSYA reversed the effects of SphK1 agonist and S1PR3 agonist on oxidized low‐density lipoprotein (ox‐LDL)‐induced vascular endothelial cell migration ability and F‐actin expression, and decreased RhoA/ROCK protein expression further confirmed the conclusion that HSYA reduced vascular endothelial permeability by modulating the SphK1/S1P/S1PR3/RhoA/ROCK signaling pathway, thereby exerting anti‐atherosclerotic effects. Overall, this study indicated that HSYA might be a therapeutic candidate for the treatment of AS.

The effects of environmental factors on the synthesis of water-soluble Monascus red pigments via submerged fermentation: a review.

Monascus pigments (MPs) have been used as natural food pigments for many years. There is a high demand for Monascus red pigments (MRPs) to enhance color and for antibacterial and cancer prevention therapies in food and medicine. Most MRPs are not water soluble, and the yield of water-soluble MRPs is naturally low. On the other hand, water-soluble MRP is more cost effective for application in industrial mass production. Therefore, it is important to improve the yield of water-soluble MRPs. Environmental factors have a significant influence on the synthesis of water-soluble MRPs, which is crucial for the development of industrial production of water-soluble MRPs. This review introduces the biosynthetic pathways of water-soluble MRPs and summarizes the effects of environmental factors on the yield of water-soluble MRPs. Acetyl coenzyme A (acetyl-CoA) is a precursor for MPs synthesis. Carbon and nitrogen sources and the carbon/nitrogen ratio can impact MP production by regulating the metabolic pathway of acetyl-CoA. Optimization of fermentation conditions to change the morphology of Monascus can stimulate the synthesis of MPs. The appropriate choice of nitrogen sources and pH values can promote the synthesis of MRPs from MPs. Additives such as metal ions and non-ionic surfactants can affect the fluidity of Monascus cell membrane and promote the transformation of MRPs into water-soluble MRPs. This review will lay the foundation for the industrial production of water-soluble MRPs. © 2024 Society of Chemical Industry.

A critical review on the stability of natural food pigments and stabilization techniques

This comprehensive review article delves into the complex world of natural edible pigments, with a primary focus on their stability and the factors that influence them. The study primarily explores four classes of pigments: anthocyanins, betalains, chlorophylls and carotenoids by investigating both their intrinsic and extrinsic stability factors. The review examines factors affecting the stability of anthocyanins which act as intrinsic factors like their structure, intermolecular and intramolecular interactions, copigmentation, and self-association as well as extrinsic factors such as temperature, light exposure, metal ions, and enzymatic activities. The scrutiny extends to betalains which are nitrogen-based pigments, and delves into intrinsic factors like chemical composition and glycosylation, as well as extrinsic factors like temperature, light exposure, and oxygen levels affecting for their stability. Carotenoids are analyzed concerning their intrinsic and extrinsic stability factors. The article emphasizes the role of chemical structure, isomerization, and copigmentation as intrinsic factors and discusses how light, temperature, oxygen, and moisture levels influence carotenoid stability. The impacts of food processing methods on carotenoid preservation are explored by offering guidance on maximizing retention and nutritional value. Chlorophyll is examined for its sensitivity to external factors like light, temperature, oxygen exposure, pH, metal ions, enzymatic actions, and the food matrix composition. In conclusion, this review article provides a comprehensive exploration of the stability of natural edible pigments, highlighting the intricate interplay of intrinsic and extrinsic factors. In addition, it is important to note that all the references cited in this review article are within the past five years, ensuring the most up-to-date and relevant sources have been considered in the analysis.

Potential and Trends Processing of Shrimp Industry by-Products in Food: A Review

The shrimp processing industry represents the largest fisheries sector globally due to its high demand and market value. Generally, the only shrimp parts that are often produced in factories are in the form of headless and peeled shrimp (shrimp without skin and head), about 88.5%. During processing, by-products are produced and require proper treatment. Shrimp by-product production has increased dramatically in recent years, leading to waste collection, disposal and pollution problems. The trend of utilisation of shrimp by-products needs to be carried out because these by-products have the potential to produce innovative products that have added value and are sustainable. By-products such as shrimp heads and shells contain proteins, minerals, fats, amino acids and bioactive compound components that can be used as additives and raw materials. The purpose of this paper is to examine the potential utilization of shrimp processing industry by-products. By converting these by-products into value-added products such as bioplastic, flavouring, natural food pigments, shrimp oil, and protein hydrolysates. Processing using various conventional and enzymatic extraction methods has the potential to reduce by-products. Utilisation of shrimp by-products can provide an attractive alternative to reduce dependence on synthetic products in the food industry, while providing the added benefit of more efficient and environmentally friendly by-product management.

A review on mechanisms and impacts of cold plasma treatment as a non-thermal technology on food pigments.

Food characteristics like appearance and color, which are delicate parameters during food processing, are important determinants of product acceptance because of the growing trend toward more diverse and healthier diets worldwide, as well as the increase in population and its effects on food consumption. Cold plasma (CP), as a novel technology, has marked a new trend in agriculture and food processing due to the various advantages of meeting both the physicochemical and nutritional characteristics of food products with minimal changes in physical, chemical, nutritional, and sensorial properties. CP processing has a positive impact on food quality, including the preservation of natural food pigments. This article describes the influence of CP on natural food pigments and color changes in vegetables and fruits. Attributes of natural pigments, such as carotenoids, chlorophyll, anthocyanin, betalain, and myoglobin, are presented. In addition, the characteristics and mechanisms of CP processes were studied, and the effect of CP on mentioned pigments was investigated in recent literature, showing that the use of CP technology led to better preservation of pigments, improving their preservation and extraction yield. While certain modest and undesirable changes in color are documented, overall, the exposure of most food items to CP resulted in minor loss and even beneficial influence on color. More study is needed since not all elements of CP treatment are currently understood. The negative and positive effects of CP on natural food pigments in various products are discussed in this review.

Pulsed electric field assisted extraction of natural food pigments and colorings from plant matrices.

Coloring compounds are widely applied to manufacturing foods and beverages. The worldwide food market is replacing artificial colorants with natural alternatives, given the increased consumer demand for natural products. However, these substitutes are still an issue due to their high production cost and low chemical and physical stability. Furthermore, natural pigments are highly sensitive to processes applied in conventional extraction techniques, such as thermal, mechanical, and chemical stresses. In this regard, pulsed electric field (PEF) technology has emerged as a promising non-thermal alternative for recovering and producing natural colorings from food matrices. Its action mechanism on cell structures through the electroporation effect is a smart alternative to overcoming the challenging issues associated with producing natural colorants. In this scenario, this review provides an overview of the PEF assisted extraction of natural pigments and colorants, such as anthocyanins (red-blue-purple), betalains (red), carotenoids (yellow-orange-red), and chlorophylls (green) from plant sources. Moreover, the potential and limitations of this emerging technology to integrate the extraction process of natural colorants were discussed.

Optimization of Monascus purpureus for Natural Food Pigments Production on Potato Wastes and Their Application in Ice Lolly.

During potato chips manufacturing, large amounts of wastewater and potato powder wastes are produced. The wastewater obtained at washing after cutting the peeled potatoes into slices was analyzed, and a large quantity of organic compounds and minerals such as starch (1.69%), protein (1.5%), total carbohydrate (4.94%), reducing sugar (0.01%), ash (0.14%), crude fat (0.11%), Ca (28 mg/L), Mg (245 mg/L), Fe (45.5 mg/L), and Zn (6.5 mg/L) were recorded; these wastes could be considered as valuable by-products if used as a fermentation medium to increase the value of the subsequent products and to exceed the cost of reprocessing. In this study, we used wastewater and potato powder wastes as a growth medium for pigment and biomass production by Monascus purpureus (Went NRRL 1992). The response surface methodology was used to optimize total pigment and fungal biomass production. The influence of potato powder waste concentration, fermentation period, and peptone concentration on total pigment and biomass production was investigated using the Box-Behnken design method with 3-factors and 3-levels. The optimal production parameters were potato powder waste concentration of 7.81%, fermentation period of 12.82 days, and peptone concentration of 2.87%, which produced a maximum total pigment of 29.86 AU/ml that include, respectively, a maximum biomass weight of 0.126 g/ml and the yield of pigment of 236.98 AU/g biomass. The pigments produced were used as coloring agents for ice lolly. This study has revealed that the ice lolly preparations supplemented with these pigments received high acceptability. Finally, we recommend using wastewater and potato powder wastes for pigment and biomass production, which could reduce the cost of the pigment production process on an industrial scale in the future.

Nanoencapsulation of Red Bell Pepper Carotenoids: Comparison of Encapsulating Agents in an Emulsion Based System

Red bell pepper extract rich in carotenoids was (RBPE) encapsulated with four different encapsulating agents: calcium caseinate (ECC), bovine gelatin (EBG), whey proteins isolate (EWPI), and concentrate (EWPC), aiming to investigate the most effective material to coat and enable the water dispersibility of pigments. Formulations were obtained by the oil in water (O/W) emulsification technique, followed by freeze-drying. Samples were analyzed by encapsulation efficiency, high-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), atomic force microscopy (AFM), thermogravimetric analysis (TGA), dispersion stability, and CIELab. Nanoformulations showed a carotenoid encapsulation efficiency of 54.0% (ECC), 57.6% (EWPI), 56.6 % (EWPC), 64.0 % (EBG). Recovered carotenoid profiles from nanoformulations showed similarity to the RBPE, indicating the efficiency of the encapsulation process. Average particle sizes of approximately 109 nm (ECC), 71 nm (EWPI), 64 nm (EWPC), and 173 nm (EBG) were obtained. AFM revealed that all formulations exhibited spherical forms and a heterogeneous distribution profile. Regarding TGA, formulations presented similar thermal behaviors to and lower decomposition speeds than RBPE, suggesting improved thermal stability. Powder formulations were easily dispersed in water (8 mg/mL) and presented intense color and stability to sedimentation for 48 h. Results indicated that all formulations and the chosen technique efficiently increased carotenoid dispersibility in water, indicating their potential to be applied as natural food pigments.

Greener technologies in agri-food wastes valorization for plant pigments: Step towards circular economy

The need for food has drastically increased with the world population's unprecedented growth, which consequently raised agricultural production. The increased agricultural and food production as a whole has resulted in the generation of enormous amounts of food waste, which is now posing a threat to both the environment and humanity. To address these issues, it is now crucial to persuade people to use promising green technologies to manage and valorize agri-food waste into valuable food additives. The recovery of value-added pigments from agri-food waste as natural food colorants is one of the new business prospects being created by approaches influenced by the circular economy model that have been continuously growing globally. These natural pigments are expected to substantially impact the development of functional foods and offer a wealth of bio-therapeutic potential. The production of naturally safe food pigments from the agri-food waste offers a greener approach within circular economy concept while avoiding the use of synthetic-petro-based colourants from the food chain. This also promotes the incorporation of natural food pigments obtained through sustainable resources. This state-of-the-art graphical review focused on the recent advances in green valorization technologies of agri-food waste to exploit natural plant pigments and their relation to sustainable food production and green circular economy.

Azaphilone alkaloids: prospective source of natural food pigments

Azaphilone, biosynthesized by polyketide synthase, is a class of fungal metabolites. In this review, after brief introduction of the natural azaphilone diversity, we in detail discussed azaphilic addition reaction involving conversion of natural azaphilone into the corresponding azaphilone alkaloid. Then, setting red Monascus pigments (a traditional food colorant in China) as example, we presented a new strategy, i.e., interfacing azaphilic addition reaction with living microbial metabolism in a one-pot process, to produce azaphilone alkaloid with a specified amine residue (red Monascus pigments) during submerged culture. Benefit from the red Monascus pigments with a specified amine residue, the influence of primary amine on characteristics of the food colorant was highlighted. Finally, the progress for screening of alternative azaphilone alkaloids (production from interfacing azaphilic addition reaction with submerged culture of Talaromyces sp. or Penicillium sp.) as natural food colorant was reviewed. KEY POINTS: • Azaphilic addition reaction of natural azaphilone is biocompatible • Red Monascus pigment is a classic example of azaphilone alkaloids • Azaphilone alkaloids are alterative natural food colorant.

A Review of Phycobiliproteins of Cyanobacteria: Structure, Function and Industrial Applications in Food and Pharmaceutical Industries

Phycobiliproteins are accessory photosynthetic pigments with a tetrapyrrole structure derived from bacterial strains that are organized on a thylakoid membrane inside a structure called phycobilisomes. Phycobiliproteins have been extensively commercialized in the manufacture of fluorescent probes for clinical and immunological analysis, in addition to their ability to dye with proven antioxidant and medicinal properties. Phycocyanin, Phycoerythrin and Allophycocyanin are the main types of phycobiliproteins that are widely used as useful food supplements today; however, in Iran, the true value of this natural pigment with bioactive properties has not been realized. Todays, the use of artificial colors and antioxidants in food product has led to an increase in cancer in many people. Therefore, awareness of the presence of natural food pigments of natural origin is of particular important. On the other hand, since so far no review article on extraction, separation and purification as well as evaluation of the biological activity of the pigment Phycoerythrin and Phycocyanin, has been published in Iran, so such review articles can pave the way for the introduction of natural edible pigments from cyanobacteria are considered to be usable in the food industry. Therefore, the purpose of this review article is to introduce the structure, function, biosynthesis and different methods of extraction of Phycobiliproteins in industrial dimensions along with different applications of Phycocyanin in food and pharmaceutical industries.

Effect of nonthermal treatments on selected natural food pigments and color changes in plant material.

In recent years, traditional high-temperature food processing is continuously being replaced by nonthermal processes. Nonthermal processes have a positive effect on food quality, including color and maintaining natural food pigments. Thus, this article describes the influence of nonthermal, new, and traditional treatments on natural food pigments and color changes in plant materials. Characteristics of natural pigments, such as anthocyanins, betalains, carotenoids, chlorophylls, and so forth available in the plant tissue, are shortly presented. Also, the characteristics and mechanism of nonthermal processes such as pulsed electric field, ultrasound, high hydrostatic pressure, pulsed light, cold plasma, supercritical fluid extraction, and lactic acid fermentation are described. Furthermore, the disadvantages of these processes are mentioned. Each treatment is evaluated in terms of its effects on all types of natural food pigments, and the possible applications are discussed. Analysis of the latest literature showed that the use of nonthermal technologies resulted in better preservation of pigments contained in the plant tissue and improved yield of extraction. However, it is important to select the appropriate processing parameters and to optimize this process in relation to a specific type of raw material.

Production and application of natural food pigments by Monascus ruber using potato chips manufacturing wastes

This work was established for using potato chips manufacturing wastes as a substrate for the production of food colorant pigments by Monascus ruber Went AUMC 5705, then it was utilized individually for coloring some food products (drops sweets). The effects of different environmental and nutritional factors were studied in solid state fermentation (SSF). The maximum concentrations of red, orange, and yellow pigments produced by M. ruber were 147.3, 179.6 and 221.7 U/g of dry fermented materials, respectively. The optimum conditions for pigments production were determined to be 50%, 6.5, 3 % and 15 days, for the initial moisture content, pH, (NH4)2SO4 , and incubation period, respectively. Moreover, the optimum inoculum size, particle size of potato chips and fermentation temperature, were determined to be 81×104 spores/10g dry substrate, 1.5 mm and 30 °C under dark condition, respectively. Then, the pigments mixture was separated into red, orange and yellow water insoluble pigments that used as coloring agents of drops sweets flavoured with fruits odours. This study revealed that potato chips wastes are an innovative raw material for pigments production by M. ruber in SSF.

The Stability, Microstructure, and Microrheological Properties of Monascus Pigment Double Emulsions Stabilized by Polyglycerol Polyricinoleate and Soybean Protein Isolate.

Monascus pigment is a natural food pigment and is commonly used for coloring and as antiseptic of cured meat products, confectionery, cakes, and beverages. However, Monascus pigment is sensitive to environmental conditions. The main aim of this study was to investigate the effect of polyglycerol polyricinoleate (PGPR) and soy protein isolate (SPI) on the particle size, zeta potential, physical stability, microstructure, and microrheological properties of Monascus pigment double emulsions. The effects of ionic strength, heating, and freeze thawing treatment on the stabilities of Monascus pigment double emulsions were also characterized. It was found that the optimum PGPR and SPI concentrations for fabricating Monascus pigment double emulsion were 3.6 and 3.0 wt%, respectively. The fabricated Monascus pigment double emulsion was composed of fine particles with narrow and uniform size distributions. Microrheological property results suggested that the elastic characteristic of the Monascus pigment double emulsion was dominated with increasing PGPR and SPI contents. It was mainly due to the increased collision and interaction between the droplets during the movement resulting in force increasing. Monascus pigment double emulsions with <5 mM CaCl2 prevented calcium to destroy the physical stability of emulsions, while Monascus pigment double emulsions with more than 10 mM CaCl2 formed creaming. After freeze thawing treatment, creaming occurred in Monascus pigment double emulsion. However, it was stable against heating treatment due to heating leading to a dense network structure. It could be contributed to the practical applications of Monascus pigment double emulsions in food products.

Safe natural bio-pigment production by Monascus purpureus using mixed carbon sources with cytotoxicity evaluation on root tips of Allium cepa L.

By increasing the undesirable side effects of synthetic food pigments on human health, using safe natural food pigment become an urgent issue. Incorporate corn starch with oils conducted a high impact on red pigment production by Monascus purpureus. Fortification the medium with sesame oil raised the pigment production by 80% and the dry mass by 63% compared with free oil medium. Response surface methodology maximizes the production with 114.6% (12.8 A500) using medium constituents (g/l); Sesame oil 5; Corn starch 30; Yeast extract 1.5; KH2PO4 2.5 and MgSO4.7H2O 0.1. After evaluating red pigment stability in three common food components, citric acid showed a great effect on residual stability percentage compared with ascorbic and salicylic acid which decrease slightly the residual stability percentage at light and dark conditions. The mitotic index of red pigment was lower than the negative control at all tested concentrations. Different types of mitotic chromosomal abnormalities e.g. lagging chromosome, chromosomal bridge, chromosome and chromatin fragments, outside chromosome, chromosomal stickiness and micro nuclei were recorded. Insignificant increase in total mitotic aberrations percentage in all tested root tips treated with all concentrations of red pigment (1.23, 1.58, 1.63, 2.32 and 2.40%) compared with negative control (0.91%). There was a significant increase in total aberrations percentage after treatment with all concentrations (10, 15, 20 and 25%) of positive control (2.93, 3.00, 3.55 and 6.53 respectively) except (5%) which was insignificant (2.71%). From the previous data, this red pigment can be used as an alternative safe pigment in the food industry.

Noninvasive and Safe Cell Viability Assay for Breast Cancer MCF-7 Cells Using Natural Food Pigment.

A dye exclusion test (DET) was performed to determine the viability of human breast cancer cells MCF-7, using natural food pigments as compared with trypan blue (TB), a typical synthetic dye for DET known to exhibit teratogenicity and cytotoxicity. We demonstrated that Monascus pigment (MP) is noninvasive to living cells and can effectively stain only dead cells. This study is the first verification of the applicability of MP to cancer cells. The appropriate MP concentration was 0.4% (0.02% as the concentration of pure MP) and all the dead cells were stained within 10 min. We found that the cell proliferation or the reduced nicotinamide adenine dinucleotide (NADH) activity of living cells was maintained over 48 h. Although 0.1% TB did not show an increase in dead cells, a marked decrease in NADH activity was confirmed. In addition, even when MP coexisted with cisplatin, staining of dead cells was maintained for 47 h, indicating stability to drugs (reagents). The cost of MP is estimated to be about 1/10 of TB. The fact that MP can be used as a cell viability determination reagent for Euglena and Paramecium, as shown in preceding papers, and also for MCF-7, as shown in this paper, indicates the possibility of application in more cells of different species.

An update on the new achievements in the nanocapsulation of anthocyanins

Natural food pigments are commonly utilized for the improvement of the qualitative properties of foods and/or inhibit the development of chronic and degenerative diseases. Several studies have documented the beneficial health effects of natural food pigments, such as anthocyanins, chlorophylls, and carotenoids. These effects mainly depend on the stability, bioactivity, and bioavailability of these pigments. Various techniques have been used to encapsulate natural pigments. Anthocyanins are a member of flavonoid groups, which are responsible for attractive food colors. Due to the positive surface charge of anthocyanin molecules, they absorb light and gain color. The micro- and nano-encapsulation of ingredients using natural polymers are important techniques to improve their stability, solubility, and bioavailability. This review study aimed to elaborate on the recent advancement in the encapsulation of anthocyanin as an attractive natural pigment using five techniques, including coacervation, spray drying, liposomal system, electrospraying, and microwave-assisted encapsulation methods.

Portable colorimetric detection of copper ion in drinking water via red beet pigment and smartphone

The concentration of copper ion (Cu2+) in drinking water is closely related to the water quality and human health. Therefore, it is necessary to develop a novel, efficient and selective method for the detection of Cu2+ in drinking water. In this work, natural food pigment—red beet pigment was selected as a colorimetric indicator for the detection of Cu2+, which possessed a bright color, excellent water solubility and safety. Under alkaline condition, red beet pigment reacted selectively with Cu2+ by redox reaction and chelation, resulting in a color change from purple to orange-red. After analyzing the blue (B) value of the red beet pigment solution, it could be found that there was a good linear relationship between B value and the concentration of Cu2+ in the range of 4–20 μM, with a detection limit of 0.84 μM. In addition, a smart phone application based on Android system was developed for visual detection of Cu2+ by identifying B value of the colorimetric probe solution. The feasibility of this method was successfully verified in the detection of Cu2+ in drinking water by the standard-addition method, reflecting its great potential of practical application in environment monitoring and food security controlling. The method provides a new insight for rapid detection of heavy metal ions using nature pigments.

Stabilization of Black Rice (Oryza Sativa, L. Indica) Anthocyanins Using Plant Extracts for Copigmentation and Maltodextrin for Encapsulation.

Black rice (Oryza sativa L. indica) is one of the cereal commodities considered to be a source of anthocyanin. One of the obstacles of using anthocyanins as natural food colorants is their low stability. The objective of this study was to determine the effect of natural plants extracts on anthocyanin copigmentation and to study the stability of the encapsulated copigmented anthocyanin during storage under different temperatures and light exposure. Dried bilimbi leaves (BL) and sow thistle leaves (STL) were extracted using ethanol to obtain the crude flavonoid extract that was subsequently purified using solid phase extraction. Both crude (CBL, CSTL) and purified (PBL, PSTL) extract were used as copigment agents with the molar ratio of 1:0.1 to 1:10 at pH 1 and pH 3. The copigmentation effect was evaluated by measuring the wavelength and absorbance shifts at wavelengths of 500 to 600 nm. The stability of the dried anthocyanin powder was evaluated under different temperatures and light exposure for up to 4 weeks. Anthocyanin was successfully copigmented with BL and STL extracts showing a wavelength shift and absorbance increase at pH 1 and pH 3. Purification of BL enhanced the hyperchromic effect more than that of the crude form, in contrast to the STL. Meanwhile, the retention of absorbance (%) for encapsulated copigmented anthocyanin samples was within 74% to 83% at the end of the storage period. PRACTICAL APPLICATION: Natural food pigment such as anthocyanin obtained from plant extract usually shows very limited stability under various processing conditions. This study shows that color stability of anthocyanin extracted from black rice can be improved by copigmentation with flavonoids extracted from particular plants. Encapsulation of the copigmented anthocyanin could further enhance its color stability. The availability of encapsulated and copigmented anthocyanins with improved color stability is expected to provide more alternatives for especially the beverage industry to choose suitable natural colorant for their products.

Noninvasive and safe cell viability assay for Euglena gracilis using natural food pigment.

Noninvasive and safe cell viability assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to find Monascus pigment (MP) or anthocyanin pigment (AP) works as a good viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell viability assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of E. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, viability of cells in culture can be monitored with them over a long period.