Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2017, 65, 9101-9109
Isolation and Characterization of a Novel, Highly Selective Astaxanthin-Producing Marine Bacterium Dalal Asker* Department of Food Science and Technology, Alexandria University, Aflaton Street, El-Shatby, 21545, Alexandria, Egypt Department of Materials Science and Engineering, University of Toronto, 184 College Street, Toronto, Ontario M5S 3E4, Canada S Supporting Information *
ABSTRACT: A high-throughput screening approach for astaxanthin-producing bacteria led to the discovery of a novel, highly selective astaxanthin-producing marine bacterium (strain N-5). Phylogenetic analysis based on partial 16S rRNA gene and phenotypic metabolic testing indicated it belongs to the genus Brevundimonas. Therefore, it was designated as Brevundimonas sp. strain N-5. To identify and quantify carotenoids produced by strain N-5, HPLC-DAD and HPLC-MS methods were used. The culture conditions including media, shaking, and time had significant effects on cell growth and carotenoids production including astaxanthin. The total carotenoids were ∼601.2 μg g−1 dry cells including a remarkable amount (364.6 μg g−1 dry cells) of optically pure astaxanthin (3S, 3′S) isomer, with high selectivity (∼60.6%) under medium aeration conditions. Notably, increasing the culture aeration enhanced astaxanthin production up to 85% of total carotenoids. This is the first report that describes a natural, highly selective astaxanthin-producing marine bacterium. KEYWORDS: astaxanthin, marine bacteria, 16S rDNA phylogeny, physiological characteristics, HPLC-DAD, HPLC-MS
■
INTRODUCTION Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) (3) (Figure 1A) is a naturally found lipid-soluble red pigment in aquatic
Astaxanthin protects against lipid peroxidation, oxidative damage of low-density lipoprotein-cholesterol7 as well as against UVA light-induced oxidative stress.8 In addition, astaxanthin has several biological activities such as anticancer,9 enhancing in vitro antibody production,10 anti-inflammatory effects,11,12 and antiaging agent.13 Therefore, astaxanthin is currently marketed as a nutraceutical4 and a medicinal ingredient against degenerative diseases such as cancer,14 skin-related illness, and heart disease.15 Owing to numerous health benefits, astaxanthin is ranked as the third most important carotenoid on the global market after β-carotene and lutein, with a predicted sales volume of 670 t valued at 1.1 billion US$ in 2020.16 Astaxanthin is mainly produced by solvent-based extraction from natural sources or by chemical synthesis. Synthetic astaxanthin is used primarily as a feed supplement and has not been approved by FDA as generally recognized as safe (GRAS) chemical in the U.S. and thus is not allowed as a functional food additive or medicinal ingredient.17 In contrast, natural astaxanthin is approved as GRAS and can be sold as a dietary supplement.18 Biotechnological production of astaxanthin using microbial sources was established using the green microalga Haematococcus pluvialis,19 yeast Phaf f ia rhodozyma20 and red carotenoidrich bacterium Paracoccus carotinifaciens.21 Because of the fastgrowing demand for natural astaxanthin, attempts to isolate astaxanthin-producing bacteria have resulted in several bacterial strains, most of which from marine water such as Paracoccus spp.,22−24 Brevundimonas sp. strain SD212,25 Erythrobacter sp.
Figure 1. Astaxanthin production by strain N-5. (a) Chemical structure of produced astaxanthin and its hydroxylated derivatives by strain N-5. (b) Orange-colonies produced by strain N-5. Colonies were grown on NA at 30 °C for 2 days.
animals such as fish (e.g., salmon) and crustaceans (e.g., lobster, crab, and shrimp), and it is responsible for their red attractive color. Astaxanthin has been approved as a feed colorant for animals and fish by the U.S. Food and Drug Administration (FDA).1 Since animals including fish cannot synthesize astaxanthin, it has been widely used in the aquaculture industry as a feed colorant for pigmentation of salmon and crustaceans.2 Astaxanthin is also approved as a food colorant (E161j) by European Commission and used in the food and beverage industries.3,4 The antioxidant activity of astaxanthin is higher than β-carotene (10 times) and vitamin E (500 times).5,6 © 2017 American Chemical Society
Received: Revised: Accepted: Published: 9101
August 1, 2017 September 13, 2017 September 18, 2017 September 18, 2017 DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry strain PC6,26 Halobacterium sp.,27 Altererythrobacter ishigakiensis,28 Sphingomicrobium astaxanthinifaciens,29 and Micrococcus sp. strain PAH83.30 Previously, we have isolated nonmarine astaxanthin-producing bacteria, Sphingomonas astaxanthinifaciens, from highly radioactive hot spring water.31 Sphingomonas faeni ISY is another nonmarine bacterium that has been isolated from cold storage.32 It is worth mentioning that all of these bacterial strains can produce astaxanthin as a minor carotenoid with low selectivity (less than 12%). In addition, except for Sphingomonas astaxanthinifaciens,31 detailed carotenoids composition has not been reported.33 In this paper, we describe taxonomic characteristics, carotenoids composition, and effect of culture conditions on the growth and carotenoids composition of an orange-pigmented bacterium (N-5) that was isolated using high-throughput screening. We found that strain N-5 selectively produces astaxanthin as its major carotenoid. To the best of our knowledge, this is the first report that describes a naturally highly selective source for astaxanthin production.
■
3.0−10.0) was evaluated in NB broth adjusted with HCl or NaOH. Oxidase activity was tested by spreading the bacterial cells on a cytochrome oxidase strip (BioChemika, Fluka) and observing its color change. Other enzymatic activities, growth on carbohydrates, acid production from carbohydrates, nitrate reduction, and the production of H2S, indole, and acetoin were examined using the commercial systems API 20NE. In addition, the Biolog system (Biolog Inc., Hayward, CA) was used for biochemical identification of strain N-5 based on the evaluation of carbon source utilization, where 95 substrates were tested by using the GN2 microplate system according to the manufacturer’s instructions. Strain N-5 was precultured briefly in NB broth at 30 °C for 2 days. Cells were scraped from the surface of the agar plate, suspended in GN/GP-IF inoculating fluid and adjusted to a final OD600 of 0.7. A volume of 150 μL of the cell suspension was inoculated in each of the 96 wells of the Biolog GN2 microplate. The microplate was incubated at 30 °C for 16 and 24 h. After this period, the presence of purple color was observed, indicating the use of carbon source by the cells. Biolog’s MicroLog 2.4.2 software was used to identify the bacterium from its carbon substrate oxidation pattern.35 16S rDNA-Based Phylogenetic Analysis. Genomic DNA of strain N-5 was extracted by using DNeasy Tissue Kit (Qiagen Sciences, Valencia, CA) as recommended by the manufacturer. The MicroSeq 500 rDNA Bacterial Sequencing Kit (PE Biosystems, Foster City, CA) was used to amplify and sequence approximately the first 500 bp of the 16S rRNA gene, according to the manufacturer’s instructions. The detailed PCR method is described in the Supporting Information Materials and Methods. The nucleotide sequence of 16S rDNA gene was determined directly by using a BigDye Terminator v3 1 cycle sequencing kit on an ABI 3100 automated DNA sequencer (Applied Biosystems, CA). The 16S rRNA gene sequence obtained was compared with those available in the GenBank, EMBL, and DDBJ databases with the gapped BLASTN 2.0.5 program (http://www.ncbi. nlm.nih.gov/BLAST/). A neighbor-joining phylogenetic tree36 was constructed using the ClustalW37 and NJ plot38 programs and bootstrapping.39 Effect of Culture Conditions on Growth and Carotenoids Production. For routine work, strain N-5 was grown on NA at 30 °C for 48 h. For the optimization of growth and production of total and individual carotenoids, several growth conditions were tested including medium type, agitation rate, and medium supplementation with glucose (1%). The detailed culture conditions are described in the Supporting Information Materials and Methods. Extraction of Carotenoids. Harvested cells in 96-well deep plates were suspended in 300 μL of dimethyl sulfoxide (DMSO) and agitated on a microplate shaker (100 rpm) at 50 °C in the dark until the cells were bleached. Then, the DMSO extracts were briefly mixed with 150 μL of methanol and centrifuged twice at 5 000g for 5 min. The resultant supernatants (200 μL) were transferred into 96-well assay plates for carotenoids analysis and quantification. High-Performance Liquid Chromatography (HPLC) Analysis. Carotenoid extracts in 96-well assay plates were first screened for polar carotenoids production (e.g., astaxanthin) using HPLC method A (Agilent 1100, Agilent Technologies, Palo Alto, CA). In method A, HPLC was coupled with diode array detection (HPLC-DAD) and used to identify both polar and nonpolar carotenoids in very short time (10 min); each 10 μL of the DMSO−methanol extract was applied onto a CAPCELLPAC C18 column (35 mm × 4.6 mm, 5 μm particle size; column temperature 35 °C) (Shiseido, Tokyo, Japan) using an automatic injection system for 96-well plate and eluted with a mixture of solvents containing acetonitrile−methanol−2-propanol, (85:10:5, v/v/v) at a flow rate of 1 mL min−1. Strain N-5 was selected and its individual carotenoids were identified and quantified using three HPLC methods (B−D). Method B, HPLC-DAD was used to separate and identify the polar carotenoids; each 10 μL sample was also applied onto the C18 column and eluted with acetonitrile−water (90:10, v/v) at 1.0 mL min−1. Method C was carried out on a LC/MS system, in which HPLC was coupled with a linear trap quadrupole (LTQ) mass spectrometer (Thermo Electron, San Jose, CA) to determine the molecular weight of carotenoids; each 10 μL sample was applied onto a CAPCELLPAK C18 column (35 mm × 4.6 mm, 5 μm
MATERIALS AND METHODS
Chemicals and Reagents. Chemicals were purchased from Kokusan (Tokyo, Japan), unless indicated otherwise. All commercial media were purchased from Difco Laboratories (Detroit, MI) including nutrient broth (NB) (contained in a liter: beef extract, 3 g; peptone, 5 g; NaCl, 19.9 g; and other sea salts, 11.73 g; final pH, 7.6. Marine broth (MB), 2216 (contained in a liter: yeast extract, 1 g; peptone, 5 g; NaCl, 19.9 g; and other sea salts, 11.73 g; final pH, 7.6). Caulobacter broth CB3 (contained in a liter: yeast extract, 3 g; peptone, 6 g; and MgSO4·7H2O, 0.6 g; final pH, 7.6. Yeast malt (YM) broth (contained in a liter: yeast extract, 3 g; malt extract, 3 g; peptone, 5 g; dextrose,10 g; final pH, 5.6). In the case of NBG medium, NB medium was supplemented with 1% glucose. Nutrient (NA) and marine agar (MA) 2216 were solidified by adding 1.5% agar. Isolation of an Astaxanthin-Producing Bacterium. Marine water samples were collected from Shimoda Port, Shizuoka Prefecture on the Pacific coast of Middle Japan. After serial dilutions with saline buffer, 0.1 mL aliquots were spread on NA and incubated at 30 °C for up to 2−6 days. Single colored colonies (yellow, orange, pink, and red) grown on NA medium were transferred into 96-well plates containing NB medium using a colony picker QPix2 (Genetix, Hampshire, U.K.), then incubated at 30 °C for 2 d. Once grown, colonies were replicated into 96-well deep plates containing 2 mL of NB and then incubated with shaking (HiGro shaker, Gene Machines) at 400 rpm and 30 °C for 2 days. Cells were harvested by centrifugation (Sorvall Legend, Kendro Scientific, Asheville, NC) at 3500g for 10 min at 4 °C. Carotenoids were extracted and analyzed by HPLC. Strain N-5 was selected for its ability to produce astaxanthin and further purified by repeatedly streaking a single colony on the NA plate. The purified pigmented isolate was stored on the NA agar slant for routine work and Microbank (long-term storage) and stored at 4 °C and −20 °C, respectively. Brevundimonas sp. SD-212 (NBRC 101024) was purchased from NBRC (NITE Biological Resource Center, Chiba, Japan) and cultivated on MB and MA media. Morphological, Physiological, and Biochemical Characterization of Strain N-5. Strain N-5 was cultured on the NA plate at 30 °C for up to 2 days. Cell morphology, colonial characteristics, and physiological properties were tested by the conventional methods used for bacterial systematics.34 Gram staining was performed according to the method described by Smibert and Krieg.34 Cells were observed under the light microscope, while motility was observed using the hanging drop method by incubating cells in NB. Anaerobic growth was assessed on the NA using a GasPak anaerobic system (BBL). Catalase activity was tested.34 To determine the optimal growth temperature, the strain was cultivated on NA at 4, 16, 20, 30, 37, 40, 45, and 50 °C. NaCl tolerance was examined using NB broth supplemented with different concentrations of NaCl. Growth at various pH values (pH 9102
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry particle size; column temperature 35 °C) (Shiseido). A mobile phase gradient of solvent A (water) and solvent B (acetonitrile) was used. The gradient elution conditions were as follows: solvent B was increased from 30% to 100% (v/v) in 10 min at a flow rate of 1 mL min−1 at 35 °C and the detection wavelength was set at 480 nm. Mass detector ESI of m/z 300−1200 was used in positive mode. Astaxanthin was identified by retention time, absorption spectrum, and molecular weight. Commercial β-carotene and astaxanthin (Sigma, St. Louis, MO) were used as standards. Extract of Brevundimonas sp. SD-212 (NBRC 101024) was used as a reference for 2,2′-dihydroxyastaxanthin (2,3,2′,3′-tetrahydroxy-β,β-carotene-4,4′-dione).25 Purification and Identification of trans-Astaxanthin. Carotenoid extract from strain N-5 was further tested for carotenoid profiles using a chiral HPLC as described in refs 40 and 41. Strain N-5 was cultured in 100 mL of NB at 30 °C for 2 days. Cells were harvested by centrifugation and lyophilized. The pigment was extracted from the dried bacterial cells with 5 mL of ethyl acetate and concentrated to dryness. The pigments were redissolved in methanol and applied onto a silica gel 60 (Merck & Co., Ltd.) column with 20 mm in diameter × 200 mm in height. Individual carotenoids were eluted using hexane−acetone (60:40, v/v) as a mobile phase, at 1.5 mL min−1 flow rate. Five fractions of 5 mL each were collected, concentrated to dryness, redissolved in methanol, and analyzed using HPLC method C. Astaxanthin fraction was analyzed using HPLC method D. The stereochemistry of astaxanthin at the C-3 and C-3′ positions was identified according to Abu-Lafi and Turujman.40 Purified astaxanthin was chromatographed in a Sumichiral OA-2000 column (4.0 mm × 250 mm, Sumitomo Chemicals) with n-heptane− methylene chloride−isopropanol (70:25:5, v/v/v) at a flow rate of 1.0 mL min−1 and detected at 480 nm.
Table 1. Physiological Characteristics of Brevundimonas Strain N-5 characteristic
result
Morphology: cell shape cell size (μm)
rods 0.6−0.7 × 2.0−2.5
Biochemical properties: oxidase, catalase, β-glucosidase, esculin hydrolysis glucose acidification, O−F test, indole production, arginine, dihydrolase, urease, gelatin hydrolysis, arginine dihydrolase, lysine decarboxylase, tryptophan deaminase Assimilation of carbon source (12): glucose, maltose, malate arabinose, mannose, mannitol, N-acetyl-glucosamine, gluconate, caprate, adipate, citrate, phenylacetic acid
+ −
+ −
different carbon sources (95 substrates) by strain N-5. Results showed that strain N-5 is capable of utilizing 29 out of the total 95 as sole carbon sources (Table S1). Strain N-5 was identified as Brevundimonas vesicularis with high similarity of 0.83 and 100% probability, according to the Biolog identification system (carbon source utilization pattern).35 Phylogenetic Characterization. MicroSeq provides rapid, clear-cut identification of bacterial isolates on genus and species levels.42 Using MicroSeq500, a partial 16S rRNA gene sequence (∼443 bp) of strain N-5 was obtained and compared with those available in GenBank/EMBL/DDBJ nucleotide sequence database. It was revealed that the novel strain belongs to the family Caulobacteraceae, subclass α-Proteobacteria, and class Proteobacteria. The closest relatives were Brevundimonas spp. (97−99% similarity). A neighbor-joining phylogenetic tree (Figure 2) showed close clustering with the genus Brevundimonas spp. and a high homology rate of 99% with both of B. vesicularis LGM 2350T and B. nasdae GTC 1043T. Nevertheless, there were phenotypic differences between strain N-5 and the two species (Table S2). Strain N-5 differed from both B. vesicularis DSM 7226T and B. nasdae DSM 14572T by being negative for production of indole and acid from glucose and for assimilation of arabinose, gluconate, adipic acid, citrate, and phenyl acetate. Strain N-5 differed from B. vesicularis DSM 7226T by being negative for gelatin hydrolysis, yet positive for β-galactosidase activity. On the other hand, strain N-5 differed from B. nasdae DSM 14572T by having orange-pigmented colonies and being unable to assimilate capric and N-acetyl Dglucosamine. Orange pigmentation has never been given in the description of B. nasdae, which is cream-pigmented.43 Therefore, further identification using polymorphism approach (e.g., chemotaxonomy and DNA−DNA hybridization) was required to determine the species name of strain N-5, and it was thus designated as Brevundimonas sp. strain N-5. Identification of Astaxanthin. The pigment extract of strain N-5 (NB medium, 30 °C, shaking 150 rpm, 2 days) was analyzed and identified using four complementary highperformance liquid chromatography (HPLC) methods (A, B, C, and D).44 Method A (HPLC-DAD) is a rapid method (10 min) for detecting nonpolar (e.g., β-carotene) and polar (e.g., astaxanthin) carotenoids.44 As shown (Figure 3A), the carotenoid produced by N-5 separated into two overlapping peaks (Rt = 0.81 and 1.23 min) similar to that of synthetic
■
RESULTS AND DISCUSSION Isolation, Morphological, and Biochemical Characterization. A high-throughput screening of cartenoid-producing microorganisms resulted in 25 astaxanthin-producing bacterial strains from marine water samples that were collected from the Pacific coast of Japan (unpublished results). Among the isolates, a novel, highly selective astaxanthin-producing marine bacterium, labeled strain (N-5), was isolated based on its ability to produce orange pigment on NA plate after 2 days of cultivation at 30 °C. Strain N-5 formed colonies that were medium in size (1.0−2.0 mm in diameter), circular, shiny, and orange (Figure 1B). Unless otherwise specified, all characteristics described hereafter are those of the cells of N-5 grown on NA at 30 °C for 2 days. Cells were Gram-negative and motile rods (0.6−0.7 μm in diameter and 2.0−2.5 μm in length), which grew singly or in pairs and chains (Figure S1). Spores were not observed. The physiological characteristics of N-5 are summarized in Table 1. Strain N-5 was a strictly aerobic chemoheterotroph. The temperature range for the growth of strain N-5 was between 20 and 40 °C (25−37 °C, optimum). No growth occurred at 45 °C. The pH range for growth was between 4 and 10 (6−8, optimum). The NaCl range for growth was between 0 and 5%, with optimum growth at 2% NaCl. The strain exhibited positive reactions for catalase, oxidase, βglucosidase, and esculin hydrolysis. Strain N-5 was negative in glucose acidification, O−F test, indole production, arginine dihydrolase, urease, gelatin hydrolysis, lysine decarboxylase, and tryptophan deaminase. In assimilation tests using API 20 NE among 12 carbon sources tested, growth of strain N-5 was observed only in the presence of glucose, maltose, and malate (Table 1). In addition, biochemical identification and metabolic characterization of strain N-5 were performed using the Biolog identification system.35 Since strain N-5 was found to be a Gram-negative and oxidase positive, GN2 microplate was used. Table S1 shows a summary of the Biolog results of using 9103
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry
Figure 2. Phylogenetic tree based on the partial (420-bp) 16S rRNA gene sequence of strain N-5 and related taxa. The tree was constructed by the neighbor-joining method. Bootstrap values, expressed as a percentage of 1000 replicates, that are >50% are shown at branch points. GenBank accession numbers are shown in parentheses. Asticcacaulis biprosthecium DSM 4723T (AJ247193) was used as the outgroup. Bar, 0.01 substitutions per nucleotide position.
2), and astaxanthin (peak 3). The characteristics of these individual carotenoids were consistent with those previously reported.25 The chemical structures of carotenoids produced by strain N-5 are illustrated in Figure 1A. The total carotenoids produced by strain N-5 was 601.2 μg g−1 dry cells, which includes a remarkable amount of astaxanthin (364.6 μg g−1 dry cells) with high selectivity (∼60.6% of total carotenoids). Strain N-5 produced a medium amount (174.1 μg g−1 dry cells or ∼29.0%) of 2-hydroxyastaxanthin and a low amount (62.5 μg g−1 dry cells or ∼10.4%) of 2,2′-dihydroxyastaxanthin. Previously, two strains that belong to genus Brevundimonas spp. have been reported to produce astaxanthin25 or its hydroxylated derivatives.45 A marine Brevundimonas sp. SD-212 was reported to produce seven carotenoid compounds (2hydroxyastaxanthin, 2-hydroxyadonixanthin, erythroxanthin, 2,2′-dihydroxyastaxanthin, 2,2′-dihydroxyadonixanthin, astaxanthin, and adonixanthin), with low selectivity of astaxanthin (9.9% in total carotenoid and 1.5 mg from a 27 L culture).25 However, when SD-212 was grown in a small culture volume (20 mL of MB), only five compounds (2,2′-dihydroxyastaxanthin, 2-dihydroxyastaxanthin, 2,2′-dihydroxyadonixanthin, 2,2′ dihydroxyzeaxanthin (nostoxanthin), 2-hydroxyadonixanthin, and adonixanthin) were identified using our HPLC methods A, B and C (unpublished results). While the characteristics of these carotenoids were consistent with those
astaxanthin (Asx-S) standard. The absorption spectra of these peaks were characterized by a symmetrical single peak (478 nm, λmax), which is identical to that of the Asx-S (Figure 3B). Nonpolar carotenoids were not detected. These results indicate that strain N-5 produces astaxanthin and its hydroxylated derivatives. To further identify the individual carotenoids produced by strain N-5, its carotenoid extract was further analyzed by HPLC methods B (HPLC-DAD) and C (HPLCMS),44 which resulted in the detection of three peaks (Figures 3C and 4C). Using HPLC method B, the carotenoid profile of strain N-5 was characterized by two minor peaks (1 and 2) at Rt 0.91 and 1.16 min, respectively (Figure 3C), and a major peak (3) at Rt 2.15 min (similar to Asx-S). The three compounds also had the same absorption spectra (478 nm, λmax) (Figure 3D), identical to that of Asx-S. However, compounds 1 and 2 eluted earlier than Asx-S, indicating they are more polar. Using HPLC-MS (method C), the same three peaks appeared at 16.66, 20.85, and 24.61 min, respectively (Figure 4C). In addition, Rt of peak 3 was also identical with that of Asx-S (Figure 4A). LC-MS m/z of the peaks (1, 2) were 629 [M+ + 1] and 613 [M+ + 1], respectively. LC-MS m/z of the peak 3 was 597 [M+ + 1], which is identical to Asx-S (Figure 4B,D). On the basis of HPLC elution times, absorption spectra, and molecular weights, we suggest that the three carotenoids are 2,2′ dihydroxyastaxanthin (peak 1), 2 hydroxyastaxanthin (peak 9104
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry
first fraction (1) was eluted at 16.66 min and gave a [M+ + 1] ion peak at m/z 629, corresponding to 2,2′-dihydroxyastaxanthin. A second fraction (2) was eluted at 20.85 min and gave a [M+ + 1] ion peak at m/z 613, corresponding to 2′hydroxyastaxanthin. Finally, a third fraction (3) that represents astaxanthin was eluted at 24.61 min and gave a [M+ + 1] ion peak at m/z 597. Chiral HPLC (method D) was used to analyze the total carotenoids extract, purified astaxanthin (peak 3), and synthetic astaxanthin (AsxS) (Figure S2). While, in nature, two astaxanthin isomers were reported to be synthesized by biological sources including (3S, 3′S)- and (3R, 3′R)astaxanthin, synthetic astaxanthin (AsxS) exhibited three astaxanthin enantiomers (3R, 3′S, 3R, 3′R, and 3S, 3′S) (Figure S2A,B).40 Total carotenoids extract (Figure S2A) and purified astaxanthin (Figure S2B) produced by strain N-5 showed optically pure (3S, 3′S)-chirality (single stereochemistry at the C-3 and C-3′ positions), which was identified as 3S, 3′S-astaxanthin. This isomer and its esterified form are produced from wild Atlantic salmon and Haematococcus, respectively.46 A second natural astaxanthin isomer (3R, 3′R)astaxanthin is produced by the yeast Xanthophyllomyces dendrorhous and Antarctic krill Euphausia superba.47 Effect of Culture Conditions and Media Composition. Initially, the astaxanthin and carotenoids production of strain N-5 were quantified after growth in NB broth under medium aeration (i.e., 20 mL of broth medium in a 150 mL volume Erlenmeyer flask with shaking at 150 rpm for 2 days). Under these conditions, strain N-5 produced total carotenoids of 601.2 μg g−1 dry cells including a remarkable amount of astaxanthin (364.6 μg g−1 dry cells). Since aeration affects both bacterial cell growth and carotenoids production,48 we thus studied the cell growth of strain N-5 and its carotenoids production under low, medium, and high aeration conditions. As expected, both cell growth and carotenoids production increased with increasing aeration from low to medium. However, high aeration rate impacted growth negatively (Figure 5A). The highest and lowest degrees of pigmentation were observed under high and low aeration conditions, respectively (Figure 5B). Unexpectedly, the highest astaxanthin
Figure 3. Carotenoids produced by strain N-5. (A, C) HPLC elution profiles (λmax 480 nm) using (A) method A and (C) method B. (B, D) UV−vis spectra of each chromatogram peak. Cells were grown in NB for 2 days at 30 °C with shaking. Carotenoid fractions analyzed by reverse-phase liquid chromatography using methods A and B (see Materials and Methods) were identified as 2,2′-dihydroxyastaxanthin (peak 1), 2-hydroxyastaxanthin (peak 2), and astaxanthin (peak 3).
previously reported,25 the astaxanthin peak was not detected. Another soil bacterium (Brevundimonas vesicularis strain DC263) was reported to produce two hydroxylated carotenoids (2,2′-dihydroxyastaxanthin and 2,2′-dihydroxyadonixanthin) as its major carotenoids.45 In addition, all bacterial strains that were previously reported produced astaxanthin as a minor component and exhibited low productivity.22−29,31,32,45 Therefore, we believe this is the first report of a highly productive and selective bacterial source of natural astaxanthin. Separation and Purification of trans-Astaxanthin. Astaxanthin and its hydroxylated derivatives were purified from the carotenoids extract of N-5 using an open chromatography column of silica gel 60 coupled with a mobile phase of hexane−acetone (7:3, v/v). Using HPLC method C, a
Figure 4. HPLC-MS analysis of carotenoid extract from strain N-5. (A, C) HPLC elution profiles (λmax 480 nm) using method C of synthetic astaxanthin (A) and carotenoids produced by strain N-5 (C). ESI mass spectra of (B) synthetic astaxanthin and (C) astaxanthin produced by strain N-5 (peak 3). 9105
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry
We showed that strain N-5 can utilize commonly used carbon sources such as glucose, maltose, malate, hydrocarbons, alcohols, amino acids, and organic acids (Table 1 and Table S1). It can also utilize amino acids (e.g., D-alanine, asparagine, proline, serine), peptone, beef extract, yeast extract, and casamino acid. Therefore, strain N-5 can be cultured for astaxanthin production using commercial media. In NB broth with shaking at 120 rpm at 30 °C, cells of strain N-5 followed an exponential growth phase (3−18 h), reached a stationary phase (30−48 h) and then slowly decreased after reaching 48 h (Figure S3A). Carotenoids production and growth followed a similar pattern of increasing until the maximum at 2 days and then decreasing with increased time up to 5 days (Figure S3B,C). To enhance the growth and astaxanthin production under medium aeration condition in NB medium, we supplemented the medium with 1% glucose (NBG) (Figure S3B,C). Incubation of cultures for up to 5 days decreased both growth and carotenoids production (Figure S3B,C). Moreover, the addition of 1% glucose decreased cell growth but increased total carotenoids production (from 601.2 to 1483.3 μg g−1 dry cells) and degree of pigmentation (Figure 6A,B and Table 2). Figure 5. Effect of aeration on growth and carotenoids production strain N-5. (A) Growth (OD660) and carotenoids production (OD480), (B) degree of pigmentation, (C) HPLC elution profiles (λmax 480 nm) using HPLC method B. (D) % of individual carotenoid of the total carotenoids. Data and error bars represent the average and standard deviation of three measurements, respectively.
accumulation (85%) was obtained under high aeration conditions (Figure 5C,D), while the lowest astaxanthin (22.4%) was obtained under low aeration conditions. Table 2 Figure 6. Effect of culture media on (A) growth (OD660) and carotenoids production (OD480) and (B) degree of pigmentation of strain N-5. Data and error bars represent the average and standard deviation of three measurements, respectively.
Table 2. Effect of Medium Composition and Aeration on Production of Carotenoids, Astaxanthin and Its Hydroxylated Derivatives by Strain N-5a carotenoid production (μg g−1 dry cells) factor
TC
2HO-Asx
HO-Asx
Asx
Medium NB NBG MB YM
601.2 ± 60 1283.3 ± 88 641.9 ± 50 302.5 ± 55
62.5 50.5 171.1 249.8
174.3 366.3 239.7 6.4
364.3 812.4 231.1 46.3
Aeration low medium high
752.3 ± 63 601.2 ± 60 955.4 ± 79
306.0 62.5 15.4
277.7 174.3 85.4
168.6 364.3 837.0
In addition, HPLC showed that astaxanthin increased slightly from 60.6 to 63.3%, 2,2′-dihydroxyastaxanthin decreased from 10.4% to 3.9%, and 2-hydroxyastaxanthin negligibly decreased by 0.5% (Figure 7A,B). In contrast, decreasing the concentration of glucose to 0.5% increased growth and carotenoids production (data not shown). Previous reports indicated a significant effect of glucose concentration on cell growth and carotenoid production.48 We also tested the effect of three commercial media (MB, YM, and CB3) under medium aeration. As shown in Figure 6A, all tested media supported the growth and carotenoids production of strain N-5. Growth was favored in MB broth compared to NB broth, but carotenoids production increased negligibly by 0.9%, which decreased the degree of pigmentation (Figure 6A,B and Table 2). HPLC showed that the percentage of astaxanthin decreased from 60.6% to 36%, while 2-hydroxyastaxanthin and 2,2′dihydroxyastaxanthin (hydroxylated astaxanthin compounds) increased from 29.0% and 10.4% to 37.3% and 25.7%, respectively (Figure 7A,B). For bacterial culture grown in YM broth, cell growth increased but the degree of pigmentation and carotenoids production decreased significantly (Figure 6A,B and Table 2). Furthermore, cells tended to accumulate hydroxylated astaxanthin compounds (84.7%) while the astaxanthin percentage dropped to 15.3% per total carotenoids (Figure 7A,B). Culturing strain N-5 in CB3 broth decreased
a
TC, total carotenoids; 2HO-Asx, 2,2′-dihydroxyastaxanthin; HO-Asx, 2 hydroxyastaxanthin; Asx, astaxanthin. Tested media: nutrient broth (NB), 1% glucose supplemented nutrient broth (NBG), marine broth (MB), yeast malt broth (YM). Cells were cultivated in NB medium with three different aeration conditions: Low (4 mL of NB broth in 18 tube, 120 rpm), medium (20 mL of NB broth in 150 mL flask, 150 rpm), and high (50 mL of NB broth in 500 mL baffled flask, 200 rpm).
lists the production of astaxanthin (837.0 μg g−1 dry cells) and total carotenoids (955.4 μg g−1 dry cells) under high aeration conditions. Under low aeration, remarkable amounts of hydroxylated astaxanthin compounds, 2-hydroxyastaxanthin (36.9%) and 2,2′-dihydroxyastaxanthin (40.7%), were produced (Figure 5C,D). 9106
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry
Figure 7. Effect of different culture media on (A) HPLC elution profiles of carotenoids produced by strain N-5 and (B) percentages of individual carotenoids. Data and error bars represent the average and standard deviation of three measurements, respectively.
■
cell growth, carotenoids production, and degree of pigmentation (Figure 6A,B). Table 2 summarizes the effect of media and aeration on total and individual carotenoids (μg g−1 dry cells). The total carotenoid production by strain N-5 ranged from 302.5 to 1283.3 μg g−1 dry cells (Table 2). Strain N-5 was routinely subcultured on NA and NB at 30 °C for 2 days. The strain stably produced astaxanthin with high-selectivity for at least 1 year, with multiple subculturing. In summary, a novel and highly selective astaxanthinproducing marine bacterium was isolated and characterized using high-throughput screening approach. Biochemical (Biolog) and molecular (microseq) based methods indicated the novel strain belongs to the genus Brevundimonas and was designated as Brevundimonas sp. strain N-5. Using a comprehensive HPLC-DAD-MS analysis, individual carotenoids produced by strain N-5 were identified as astaxanthin, 2hydroxyastaxanthin, and 2,2′-dihydroxyastaxanthin, based on their retention time, photodiode-array absorbance spectroscopy, and mass spectrometry. Our results indicated that culture conditions, such as media type, shaking, and time, had great effects on cell growth, carotenoid production, and the ratio of astaxanthin to its hydroxylated derivatives. For instance, strain N-5 in NB broth with medium aeration produced a total carotenoid of ∼601.2 μg g−1 dry cells, including a remarkable amount of astaxanthin (364.6 μg g−1 dry cells) with high selectivity (∼60.6% of total carotenoids). In addition, the highest astaxanthin production was achieved by supplementing the culture with 1% glucose although cell growth decreased. Compared with other astaxanthin producers that require further purification of astaxanthin from its esters (e.g., Haematococcus pluvialis) or other carotenoids species (e.g., Paracoccus), strain N-5 is potentially a promising and highly selective biotechnological source for natural and pure 3S, 3′S astaxanthin. Patented Strain. Strain N-5 is protected by the Japan Patent No. 2005-170393, Reference No. X10504922 (owned by the Asahi Kasei Corporation) for production of astaxanthin. N-5 (FERM P-20516) was deposited to National Institute of Bioscience and Human-Technology (NIBHT).
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03556. Methods for 16S rDNA-based phylogenetic analysis and studying the effect of culture conditions on growth and carotenoids production; pattern of sole carbon source utilization by strain N-5 (Table S1); differential characteristics between strain N-5 and the type strains of the closely related species of Brevundimonas spp. (Table S2); light microscope micrograph of strain N-5 cells (Figure S1); chiral HPLC for separation and identification of all-trans-astaxanthin produced by strain N-5 (Figure S2); growth and production of carotenoids by strain N-5 in NB broth with or without glucose (Figure S3) (PDF) Accession Codes
The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequences of strain N-5 = (FERM P-20516) is LC310852.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-647-573-9778. E-mail:
[email protected]. ORCID
Dalal Asker: 0000-0002-2291-7579 Notes
The author declares no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the Asahi Kasei Corporation. Thanks to Koji Isaka (Asahi Kasei Corporation) for his help in HPLC analysis. Thanks to Tarek Awad (University of Toronto) for fruitful discussions and to Tarek Awad and Alaa Awad (University of Toronto) for critically proofreading the manuscript.
■
ABREVIATIONS USED GRAS, generally recognized as safe; NB, nutrient broth; MB, marine broth; CB3, Caulobacter broth; YM, yeast malt; NBG, NB was supplemented by 1% glucose; NA and MA, nutrient 9107
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry
(19) Bubrick, P. Production of astaxanthin from Haematococcus. Bioresour. Technol. 1991, 38, 237−239. (20) Miller, M. W.; Yoneyama, M.; Soneda, M. Phaf f ia, a new yeast genus in the Deuteromycotina (Blastomycetes). Int. J. Syst. Bacteriol. 1976, 26, 286−291. (21) Bories, G.; Brantom, P.; de Barberà, J. B.; Chesson, A.; Cocconcelli, P.; Debski, B.; Dierick, N.; Franklin, A.; Gropp, J.; Halle, I. Safety and efficacy of Panaferd-AX (red carotenoid-rich bacterium (Paracoccus carotinifaciens) as feed additive for salmon and trout. EFSA J. 2007, 546, 1−30. (22) Lee, J. H.; Kim, Y. S.; Choi, T. J.; Lee, W. J.; Kim, Y. T. Paracoccus haeundaensis sp. nov., a Gram-negative, halophilic, astaxanthin-producing bacterium. Int. J. Syst. Evol. Microbiol. 2004, 54 (5), 1699−1702. (23) Tsubokura, A.; Yoneda, H.; Mizuta, H. Paracoccus carotinifaciens sp. nov., a new aerobic gram-negative astaxanthin-producing bacterium. Int. J. Syst. Bacteriol. 1999, 49 (1), 277−282. (24) Yokoyama, A.; Izumida, H.; Miki, W. Production of astaxanthin and 4-ketozeaxanthin by the marine bacterium, Agrobacterium aurantiacum. Biosci., Biotechnol., Biochem. 1994, 58, 1842−1844. (25) Yokoyama, A.; Miki, W.; Izumida, H.; Shizuri, Y. New Trihydroxy-keto-carotenoids isolated from an astaxanthin-producing marine bacterium. Biosci., Biotechnol., Biochem. 1996, 60, 200−203. (26) Yokoyama, A.; Izumida, H.; Shizuri, Y. New carotenoid sulfates isolated from a marine bacterium. Biosci., Biotechnol., Biochem. 1996, 60 (11), 1877−1878. (27) Calo, P.; Miguel, T. D.; Sieiro, C.; Velazquez, J. B.; Villa, T. G. Ketocarotenoids in halobacteria: 3-hydroxy-echinenone and transastaxanthin. J. Appl. Bacteriol. 1995, 79, 282−285. (28) Matsumoto, M.; Iwama, D.; Arakaki, A.; Tanaka, A.; Tanaka, T.; Miyashita, H.; Matsunaga, T. Altererythrobacter ishigakiensis sp. nov., an astaxanthin-producing bacterium isolated from a marine sediment. Int. J. Syst. Evol. Microbiol. 2011, 61 (12), 2956−2961. (29) Shahina, M.; Hameed, A.; Lin, S.-Y.; Hsu, Y.-H.; Liu, Y.-C.; Cheng, I.-C.; Lee, M.-R.; Lai, W.-A.; Lee, R.-J.; Young, C.-C. Sphingomicrobium astaxanthinifaciens sp. nov., an astaxanthin-producing glycolipid-rich bacterium isolated from surface seawater and emended description of the genus Sphingomicrobium. Int. J. Syst. Evol. Microbiol. 2013, 63 (9), 3415−3422. (30) Ibrahim, A. Production of carotenoids by a newly isolated marine Micrococcus sp. Biotechnology 2008, 7 (3), 469−474. (31) Asker, D.; Beppu, T.; Ueda, K. Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan. FEMS Microbiol. Lett. 2007, 273 (2), 140−148. (32) Mageswari, A.; Subramanian, P.; Srinivasan, R.; Karthikeyan, S.; Gothandam, K. M. Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures. Microbiol. Res. 2015, 179, 38−44. (33) Asker, D.; Awad, T. S.; Beppu, T.; Ueda, K. A novel radiotolerant astaxanthin-producing bacterium reveals a new astaxanthin derivative: astaxanthin dirhamnoside. Methods Mol. Biol. 2012, 892, 61−97. (34) Smibert, R. M.; Krieg, N. R. Phenotypic Characterization; American Society for Microbiology: Washington, DC, 1994; pp 607− 654. (35) Solit, R.; Biolog. MicroLog System, Release 4.0., User Guide; Biolog Inc.: Hayward, CA, 1999. (36) Saitou, N.; Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4 (4), 406−425. (37) Thompson, J. D.; Higgins, D. G.; Gibson, T. J.; CLUSTAL, W. improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22 (22), 4673−4680. (38) Perriere, G.; Gouy, M. WWW-query: an on-line retrieval system for biological sequence banks. Biochimie 1996, 78 (5), 364−369.
and marine agar; HPLC, high-performance liquid chromatography; DAD, diode array detection; MS, mass spectrometry; TC, total carotenoids; 2HO-Asx, 2,2′-dihydroxyastaxanthin; HO-Asx, 2-hydroxyastaxanthin; Asx, astaxanthin; Asx-S, synthetic astaxanthin
■
REFERENCES
(1) U.S. Food and Drug Administration. Summary of Color Additives for Use in the United States in Foods, Drugs, Cosmetics, and Medical Devices, https://www.fda.gov/forindustry/coloradditives/ coloradditiveinventories/ucm115641.htm (accessed July 5, 2017). (2) Goodwin, T. Metabolism, nutrition, and function of carotenoids. Annu. Rev. Nutr. 1986, 6 (1), 273−297. (3) Scotter, M. J. Colour Additives for Foods and Beverages; Elsevier: 2015. (4) Belviranli, M.; Okudan, N. Well-known antioxidants and newcomers in sport nutrition: coenzyme Q10, quercetin, resveratrol, pterostilbene, pycnogenol and astaxanthin. In Antioxidants in Sport Nutrition; Lamprecht, M., Ed. CRC Press: Boca Raton, FL, 2015; Chapter 5. (5) Miki, W. Biological functions and activities of animal carotenoids. Pure Appl. Chem. 1991, 63 (1), 141−146. (6) Naguib, Y. M. Antioxidant activities of astaxanthin and related carotenoids. J. Agric. Food Chem. 2000, 48 (4), 1150−1154. (7) Iwamoto, T.; Hosoda, K.; Hirano, R.; Kurata, H.; Matsumoto, A.; Miki, W.; Kamiyama, M.; Itakura, H.; Yamamoto, S.; Kondo, K. Inhibition of low-density lipoprotein oxidation by astaxanthin. J. Atheroscler. Thromb. 2000, 7 (4), 216−22. (8) O’Connor, I.; O’Brien, N. Modulation of UVA light-induced oxidative stress by beta-carotene, lutein and astaxanthin in cultured fibroblasts. J. Dermatol. Sci. 1998, 16 (3), 226−230. (9) Chew, B.; Park, J.; Wong, M.; Wong, T. A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and astaxanthin in mice in vivo. Anticancer Res. 1998, 19 (3A), 1849−1853. (10) Jyonouchi, H.; Sun, S.; Gross, M. Effect of carotenoids on in vitro immunoglobulin production by human peripheral blood mononuclear cells: astaxanthin, a carotenoid without vitamin A activity, enhances in vitro immunoglobulin production in response to a T-dependent stimulant and antigen. Nutr. Cancer 1995, 23 (2), 171− 183. (11) Wang, X.; Willen, R.; Wadstrom, T. Astaxanthin-rich algal meal and vitamin C inhibit Helicobacter pylori infection in BALB/cA mice. Antimicrob. Agents Chemother. 2000, 44 (9), 2452−2457. (12) Ohgami, K.; Shiratori, K.; Kotake, S.; Nishida, T.; Mizuki, N.; Yazawa, K.; Ohno, S. Effects of astaxanthin on lipopolysaccharideinduced inflammation in vitro and in vivo. Invest. Ophthalmol. Visual Sci. 2003, 44 (6), 2694−2701. (13) Huangfu, J.; Liu, J.; Sun, Z.; Wang, M.; Jiang, Y.; Chen, Z.-Y.; Chen, F. Antiaging effects of astaxanthin-rich alga Haematococcus pluvialis on fruit flies under oxidative stress. J. Agric. Food Chem. 2013, 61 (32), 7800−7804. (14) Chew, B. P.; Park, J. S.; Wong, M. W.; Wong, T. S. A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and astaxanthin in mice in vivo. Anticancer Res. 1999, 19 (3A), 1849−1853. (15) Guerin, M.; Huntley, M. E.; Olaizola, M. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 2003, 21 (5), 210−216. (16) Global Astaxanthin Market - Sources. Technologies and Applications, http://www.reportlinker.com/p02788641/GlobalAstaxanthin-Market-Sources-Technologies-and-Applications.html (accessed July 4, 2017). (17) Tangerås, A.; Slinde, E. Coloring of salmonids in aquaculture: the yeast Phaf f ia rhodozyma as a source of astaxanthin. In Fisheries Processing; Springer, 1994; pp 391−431. (18) Astaxanthin wins full GRAS status, http://www.nutraingredientsusa.com/Regulation/Astaxanthin-wins-full-GRAS-status (accessed June 5, 2017). 9108
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
Article
Journal of Agricultural and Food Chemistry (39) Felsenstein, D.; Carney, W. P.; Iacoviello, V. R.; Hirsch, M. S. Phenotypic properties of atypical lymphocytes in cytomegalovirusinduced mononucleosis. J. Infect. Dis. 1985, 152 (1), 198−203. (40) Abu-Lafi, S.; Turujman, S. A chiral HPLC method for the simultaneous separation of configurational isomers of the predominant cis/trans forms of astaxanthin. Enantiomer 1996, 2 (1), 17−25. (41) Wang, C.; Armstrong, D. W.; Chang, C.-D. Rapid baseline separation of enantiomers and a mesoform of all-trans-astaxanthin, 13cis-astaxanthin, adonirubin, and adonixanthin in standards and commercial supplements. J. Chromatogr. A 2008, 1194 (2), 172−177. (42) Tang, Y.-W.; Ellis, N. M.; Hopkins, M. K.; Smith, D. H.; Dodge, D. E.; Persing, D. H. Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gramnegative bacilli. J. Clin. Microbiol. 1998, 36 (12), 3674−3679. (43) Li, Y.; Kawamura, Y.; Fujiwara, N.; Naka, T.; Liu, H.; Huang, X.; Kobayashi, K.; Ezaki, T. Sphingomonas yabuuchiae sp. nov. and Brevundimonas nasdae sp. nov., isolated from the Russian space laboratory Mir. Int. J. Syst. Evol. Microbiol. 2004, 54 (3), 819−825. (44) Asker, D.; Awad, T. S.; Beppu, T.; Ueda, K. Isolation, characterization, and diversity of novel radiotolerant carotenoidproducing bacteria. Methods Mol. Biol. 2012, 892, 21−60. (45) Tao, L.; Rouvière, P. E.; Cheng, Q. A carotenoid synthesis gene cluster from a non-marine Brevundimonas that synthesizes hydroxylated astaxanthin. Gene 2006, 379, 101−108. (46) Foss, P.; Renstrøm, B.; Liaaen-Jensen, S. Natural occurrence of enantiomeric and Meso astaxanthin 7-crustaceans including zooplankton. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol 1987, 86 (2), 313−314. (47) Hussein, G.; Sankawa, U.; Goto, H.; Matsumoto, K.; Watanabe, H. Astaxanthin, a carotenoid with potential in human health and nutrition. J. Nat. Prod. 2006, 69 (3), 443−449. (48) Taylor, R.; Davies, B. The influence of culture conditions on carotenogenesis in Streptococcus faecium UNH564P. J. Gen. Microbiol. 1976, 92 (2), 325−334.
9109
DOI: 10.1021/acs.jafc.7b03556 J. Agric. Food Chem. 2017, 65, 9101−9109
