Sulfonation of Monascus pigments to produce water-soluble yellow pigments

Article abstract of DOI:10.1016/j.dyepig.2019.107965

Red Yeast Rice, a kind of Monascus pigments produced by fermentation of Monascus species on rice, is a traditional Chinese medicine and food colorant. Chemical modification of natural pigments is a common strategy to diversify the pigments to meet various demands. Herein, native Monascus pigments as well as some of their derivates were prepared. Sulfonation of Monascus pigments with conjugated double bonds adjacent to a carbonyl group was carried out to produce novel water-soluble yellow pigments (WSYPs). The chemical structure of novel WSYPs, i.e., an addition of H₂SO₃ to the double bond at the side-chain of Monascus pigments, was elucidated by MS and NMR analysis. The introduction of H₂SO₃ into Monascus pigments makes WSYPs exhibit yellow color and high water solubility. The yellow color, high water solubility, as well as relatively high stability in a wide pH range contribute the novel WSYPs as a potential food colorant.

Full text of DOI:10.1016/j.dyepig.2019.107965

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Sulfonation of Monascus pigments to produce water-soluble yellow pigments
Lujie Liu, Shu Wu, Wei Wang, Xuehong Zhang, Zhilong Wang
PII:
S0143-7208(19)32069-8
DOI:
https://doi.org/10.1016/j.dyepig.2019.107965
DYPI 107965
Reference:
To appear in: Dyes and Pigments
Received Date: 2 September 2019
Revised Date: 11 October 2019
Accepted Date: 12 October 2019
Please cite this article as: Liu L, Wu S, Wang W, Zhang X, Wang Z, Sulfonation of Monascus pigments
to produce water-soluble yellow pigments, Dyes and Pigments (2019), doi: https://doi.org/10.1016/
j.dyepig.2019.107965.
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Graf Abstract

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Dyes and pigments
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Sulfonation of Monascus pigments to produce water-soluble
yellow pigments
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Lujie Liu1, Shu Wu1, Wei Wang 2, Xuehong Zhang2, Zhilong Wang1
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1 State Key Laboratory of Microbial Metabolism, and Engineering Research Center of Cell & Therapeutic Antibody,
Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
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State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong
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University, Shanghai 200240, China
Corresponding author: e-mail: zlwang@sjtu.edu.cn (Z. Wang)
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Abstract: Red Yeast Rice, a kind of Monascus pigments produced by fermentation of Monascus species on rice, is
a traditional Chinese medicine and food colorant. Chemical modification of natural pigments is a common strategy
to diversify the pigments to meet various demands. Herein, native Monascus pigments as well as some of their
derivates were prepared. Sulfonation of Monascus pigments with conjugated double bonds adjacent to a carbonyl
group was carried out to produce novel water-soluble yellow pigments (WSYPs). The chemical structure of novel
WSYPs, i.e., an addition of H2SO3 to the double bond at the side-chain of Monascus pigments, was elucidated by
MS and NMR analysis. The introduction of H2SO3 into Monascus pigments makes WSYPs exhibit yellow color
and high water solubility. The yellow color, high water solubility, as well as relatively high stability in a wide pH
range contribute the novel WSYPs as a potential food colorant.
Key words: Monascus pigments; Water-soluble yellow pigments; Sulfonation; Nucleophilic attack; Food colorant
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57 1. Introduction
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Red Yeast Rice, a kind of Monascus pigments produced by solid-state fermentation of Monascus species on
rice, is a traditional Chinese medicine and food colorant. It has been widely used as food colorant in China, Korea,
Japan and Southeast Asia for more than one thousand years [1-3]. By virtue of elaboration of the gene clusters of
Monascus species, the biosynthetic pathway of azaphilone core catalyzed by polyketide synthase has been detailed
[4, 5]. By means of further esterification of the azaphilone core with β-ketoacid, native Monascus pigments, such as
orange Monascus pigments (OMPs) (1, 2) and yellow Monascus pigments (YMPs) (3, 4) (Fig.1A), are
biosynthesized [6]. However, the native Monascus pigments are unsuitable for utilizing directly as food colorant
owing to their low water solubility [7]. Furthermore, the embryo-toxicity and teratogenicity of OMPs have also
been reported [8].
Location of Fig.1
It has been reported that OMPs can be chemically modified by reduction [9], oxidation [10, 11] and amination
[12-18]. Among them, amination of compound 2 into 5 or 6 (Fig.1B) for production of water-soluble red pigments
(WSRPs) has been studied extensively. As shown in Fig.1, cascade reactions, i.e., biosynthesis of OMPs and further
chemical modification of OMPs by amination reaction, occur naturally during microbial fermentation. During the
course of microbial culture, primary amines, such as amino acids and glucosamine [19] as well as ammonia, are
produced by microbial metabolism. Thus, the major colorant components of Red Yeast Rice are WSRPs with
various primary amines [1, 6, 20, 21]. Due to the excellent stability and high water solubility of WSRPs, Chinese
government has issued the standard for commercial production of WSRPs (Red Monascus Pigment®, GB
5009.150-2016). The Red Monascus Pigment® has been utilized widely as a food additive, especially for
substitution of nitrite to enhance meat color [22].
It is reported that OMPs as well as WSRPs can be further chemically modified by sodium borohydride to
produce yellow pigments [9, 23]. Alternatively, yellow pigments, which are produced by sulfonation of commercial
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Red Monascus Pigment [24], have also been approved as commercial food colorant by Chinese government
(Yellow Monascus Pigment®, GB 1886.66-2015). Yellow Monascus Pigment® is being produced by several
biotechnology companies in China [6]. In spite of the practical application of Yellow Monascus Pigment®, the
chemical reaction about sulfonation of Monascus pigments remains largely blurry. There are few works about the
identification of chemical components in Yellow Monascus Pigment® due to the troublesome separation procedure.
Till now, there is only one report about an isolation of the relatively hydrophobic components in Yellow Monascus
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Pigment® by preparative TLC and further deduction of the corresponding chemical structure by LC-MS [24].
In the present work, sulfonation of identified Monascus pigments to produce water-soluble yellow pigments
(WSYPs) was carried out and the chemical structure of the novel WSYPs was managed to elucidate. Firstly, native
Monascus pigments (Fig.1A) were isolated after microbial fermentation. The native OMPs was chemically
modified by amination reaction and then reduction in an aqueous sodium hydroxide solution (Fig.1B). Then,
sulfonation of Monascus pigments (including native ones and their derivates as shown in Fig.1) was carried out and
the chemical structure of novel WSYPs was elucidated by UPLC-MS and NMR. Finally, the color characters of the
novel WSYPs as a potential food colorant, such as color, water solubility, and stability, were further checked.
94 2. Materials and methods
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2.1. Chemicals
Acetonitrile (HPLC grade), was purchased from Avantor (US). Water was purified using a Mili-Q Ultra system
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(Millipore, US). Chloroform-d, methanol-d4 and dimethyl sulfoxide-d6 (DMSO-d6) were purchased from
Adamas-beta (Shanghai, China). Sodium dithionite was purchased from TiTan (Shanghai, China). Other agents
were chemical grade.
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2.2. Identification of native Monascus pigments
Native Monascus pigments were produced by submerged culture of Monascus ruber (ESI S1). Crude native
Monascus pigments (Fig.1A) were isolated from the fermentation broth (ESI S2). The crude OMPs and YMPs were
further purified by a preparative HPLC system (Shimadzu LC-6AD series equipped with an SPD-20
spectrophotometer using Shimadzu PRC-ODS EV0233 column). The sample injection volume was 1 ml. Isocratic
elution (acetonitrile/water, 70:30, V/V) with a flow rate of 10 ml/min was employed and OMPs were detected at the
wavelength 470 nm while and YMPs at 390 nm. Four kinds of native Monascus pigments (Fig.1A) were obtained.
The purified monascorubrin (2) and ankaflavin (4) were further identified by mass spectrometer (MS) and NMR
analysis.
Monascorubrin (2): orange needle solid; UV-vis (acetonitrile) λmax 243, 286, 468 nm; HRMS Anal. Calcd for
C23H27O5: 383.1858[M+H+]; found, 383.1863(ESI S3). Ankaflavin (4): yellow needle solid; UV-vis (acetonitrile)
λmax 224, 386 nm; HRMS Anal. Calcd for C23H31O5: 387.2171[M+H] +; found, 387.2170 (ESI S4). The HRMS data
are consistent with the literature data [25]. The corresponding data of HNMR and 13CNMR analysis were
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presented in table 1 and table 2, respectively. The results are consistent with the data in literatures [9, 26, 27].
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Location of table 1 & 2
2.3. Chemical modification of monascorubrin
OMPs include two chemical entities: rubropunctatin (1) and monascorubrin (2), which have similar structural
character and physicochemical property [18, 27]. According to Fig.1B, monascorubrin (2) was chosen as target
reactant for study. Monascorubrin (2) (0.1 g, 0.26 mmol) in ethanol (35 mL) and primary amine ((NH4)2SO4 or
monosodium glutamate; 0.52 mmol) in water (pH 7, 15 mL) were mixed, which was stirred at 30 oC for 72 h until
monascorubrin was consumed completely (monitored by thin-layer chromatography (TLC), developing solvent:
chloroform/methanol/water=56/10/0.9). The reaction mixture was concentrated by a rotary evaporator.
After evaporation of solvent, the solid residue including (NH4)2SO4 was redissolved by ethyl acetate/water
(1/1, V/V; 50 mL). The mixture was transferred into a 150 mL separating funnel and an ethyl acetate layer
containing monascorubramine (5) was then separated. The ethyl acetate layer was concentrated in a rotary
evaporator under vacuum to afford the crude product. Recrystallization of the crude pigment from ethanol yielded
red needles 5 (91 mg). The purified monascorubramine (5) was further identified by MS and NMR analysis.
Monascorubramine (5): Red needle solid; UV-vis (acetonitrile) λmax 300, 415, 531 nm; HRMS Anal. Calcd for
C23H26NO4, 380.1862 [M-H]-; found, 380.1853 (ESI S5). The corresponding data of 1HNMR and 13CNMR analysis
were presented in table 1 and table 2, respectively. The results are consistent with the data in literatures [26, 27].
Similarly, the solid residue including monosodium glutamate was redissolved by ethyl acetate/water (1/1, v/v;
50 mL). The pH of water phase was adjusted to 10 by NaOH aqueous solution (1 M), and then the water phase was
separated. The water phase was added ethyl acetate (30 mL) again, of which the pH was adjusted to 2.5. The target
compound was partitioned into the ethyl acetate phase under this condition. Then, ethyl acetate phase was fetched
and the solvent was removed to get the crude compound 6 (82 mg). Compound 6 was further identified by MS
analysis.
Compound 6: red amorphous solid; UV-vis (acetonitrile) λmax 425, 521nm; HRMS Anal. Calcd for C23H26NO4,
512.2284 [M+H]+; found, 512.2286 (ESI S6). The results are consistent with the data in literature [19].
Monascorubramine (5) (50 mg) in ethanol (20 mL) and NaOH aqueous solution (1 M, 20 mL) were mixed,
which was stirred at 40 oC for 10 h. The reaction mixture was concentrated by a rotary evaporator. The solid residue
was redissolved by ethyl acetate/water (1/1, V/V; 50 mL). The mixture was transferred into a 250 mL separating
funnel and an ethyl acetate layer was then separated. The ethyl acetate layer was concentrated in a rotary evaporator
under vacuum to afford the crude product, which was separated by preparative TLC (GF254, 200 * 200* 0.9 mm,
Huanghai, China) using chloroform/methanol/water (56/10/0.9, V/V/V) as the developing solvent to give the
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product 7 (45 mg). The purified compound 7 was further identified by MS and NMR analysis.
Compound 7: red amorphous solid; UV-vis (acetonitrile) λmax 238, 299, 477 nm; HRMS Anal. Calcd for C23H30NO4,
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384.2175 [M+H]+; found, 384.2179 (ESI S7). The corresponding data of HNMR and 13CNMR analysis were
presented in table 1 and table 2, respectively. The reduction of 5 to 7 in the aqueous NaOH solution is confirmed by
NMR elucidation in the first time.
2.4. Sulfonation of identified Monascus pigments
Sulfonation of identified Monascus pigments (2, 4-7) followed a similar procedure for sulfonation of Red
Monascus Pigment® [24]. Briefly, sodium dithionite (0.2 mmol) was added into a single Monascus pigment (2, 4-7,
0.1mmol) ethanol aqueous solution (50 %, V/V; pH 7; 20 mL), respectively. The ethanol aqueous solution was
stirred and refluxed at 70 oC for 6 h. The solvent was evaporated to give crude product, which was separated by
preparative TLC using chloroform/methanol (5/1, V/V) as developing solvent. No sulfonation reaction of
ankaflavin (4) was observed under the same condition. According to Fig.2, compounds 2, 5-7 produced the
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corresponding WSYPs 8-11. The corresponding HRMS data are deposited (ESI S8-S11). The HNMR and
13CNMR analysis of WSYP 11 were presented in table 1 and table 2, respectively. The chemical structures of
WSYP 8-11 are reported for the first time.
Location of Fig.2
2.5. Color and stability of WSYPs
One milliliter of WSYP aqueous solution (pH 7; 1 g/L) was diluted by an aqueous solution (pH 7) to
absorbance at the corresponding maximum absorbance wavelength approximately 1 absorbance unit (AU).
Ultraviolet-visible (UV-vis) spectrum of the WSYP solution was recorded on a Shimadzu UV-2600 spectrometer.
Each WSYPs (8-11) exhibited their own character absorbance in an aqueous solution (pH=7), such as compound 8
was corresponded to the maximum absorbance wavelength 500 nm; 9 to 470 nm; 10 to 470 nm; 11 to 455 nm. The
influence of pH on color of WSYP aqueous solutions was examined as following: 2 ml WSYP aqueous solution
(pH 7; 1 g/L) was diluted with 8 ml of Teorell Stenhagen buffer solution (pH=2-12, 0.2 M boric acid, 0.05 M citric
acid, and 0.1 M tri-sodium orthophosphate; adjustment of pH with 1 M hydrochloric acid or 1 M sodium hydroxide
solution). The change of absorbance at the corresponding maximum absorbance wavelength was regarded as the
influence of pH on color of WSYP in aqueous solutions. In order to examine the influence of pH on the stability of
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WSYPs, those samples were further incubated at 30 C. For a certain period of time, 3 ml of sample was fetched
and the absorbance at the maximum absorbance wavelength was recorded. The decrease of absorbance was
regarded as the stability of WSYP in the buffer solutions.
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2.6. Analysis methods
Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis was performed on a Water
ACQUITY UPLC system. An Acquity BEH C18 (100 mm×2.1 mm i.d., 1.7µm, Waters, Milford, USA) analytical
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column was used. The column was maintained at 50 C and eluted with water (A)/acetonitrile (B) solvent system.
The gradient eluting program was started from 10 % B, and changed to 50 % B within 5 min, then changed to 85 %
B within 2 min, and changed to 100 % B within 2 min, then maintained 100 % B within 2 min, and changed to 10 %
B within 0.5 min, and at last by equilibration at 10 % B for 2.5 min at a flow rate of 0.4 ml/min. MS was performed
in positive ion model otherwise specified. High resolution mass spectrum (HRMS) analysis was carried out via
Masslynx 4.1 software (Waters MS Technologies, Manchester, UK).
1H Nuclear Magnetic Resonance (NMR) and 13C NMR spectra were obtained on a Avance Ⅲ 400 MHz
spectrometer (Bruker, Germany) with Tetramethylsilane (TMS) as the internal standard. All chemical shifts were
given in ppm and coupling constants were given in Hz.
186 3. Results and discussion
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3.1. Structural elucidation of WSYPs
Fig.1B shows the famous amination reaction of 2 to 5. MS analysis of m/z 383.1863 [M+H] + is consistent
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with the formula C23H27O5 of 2 while m/z 380.1853 [M-H] with formula C23H26NO4 of 5 (ESI S3 & S5). For
structure elucidation and signal assignment, H and 13C spectra of 2, 4, 5, 7 and 11 were recorded. The complete
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assignments for H and C signals were further given in Tables 1 and table 2, respectively. There is no significant
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difference between 2 and 5, where the H spectrum of N-H in 5 is undetectable owing to the hydrogen with high
activity (table 1). The compound 5 was further reduced to produce 7 in an aqueous sodium hydroxide solution
(Fig.1B). UPLC-MS/MS measurement reveals the difference between compound 5 and 7 is 2H (molecular mass
2.0163) (ESI S5 vs S7), which hints the possible reduction of 5 to 7. Both the appearance of peak at 4.83 ppm (1H
spectrum of 7 in table 1) and the disappearance of peak at 195.30 ppm (13C spectrum of 5 in table 2) reveal the
replacement of the carbonyl at C-3 in compound 5 by a hydroxyl moiety in compound 7. Similar structure with a
hydroxyl moiety at C-3, i.e., reduction of OMPs as well as WSRPs by sodium borohydride, is also reported [9, 28].
According to Fig.2, various Monascus pigments are sulfonated into the corresponding WSYPs. As shown in
Fig.3A, UPLC-MS/MS measurement reveals the difference between compound 7 and WSYP 11 is H2SO3
(molecular mass 81.9725) (ESI S7 vs S11). Furthermore, the corresponding fragments of 7 (m/z 366.2048,
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C23H28NO3 , loss of a water; m/z 338.2104, C22H28NO2 , further loss of a carbonyl group ) are also found in the
fragments of 11 (m/z 448.1789, C23H30NO6S+, loss of a water, m/z 420.1835, C22H30NO5S+, further loss of a
carbonyl group). This result further confirms that the difference between compound 7 and WSYP 11 is H2SO3. The
1H and 13C spectra are applied to identify the position of H2SO3 in WSYP 11 (Table 1 & 2). The spectrograms of 7
and 11 are significant differences at C-10 and C-11 position. Signals of the vinyl protons (6.38 ppm at position
C-10 and 6.79 ppm at C-11) in the 1H spectrum of 7 disappear while proton signals (1.96 and 2.11 ppm at position
C-10 and 3.72 ppm at C-11) in the 1H spectrum of 11 appear. This information indicates sulfonation reaction occurs
at the double bond between C-10 and C-11 of compound 7. Proton vicinal coupling constant analysis further
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confirms that HSO3 connects to the position C-11 of WSYP 11. On the other hand, peak of 136.36 ppm at the
position C-10 and that of 132.53 ppm at C-11 in 13C spectrum of 7 are also replaced by peaks of 56.49 ppm and
60.70 ppm in 13C spectrum of 11, respectively. The chemical structure of novel WSYP 11, i.e., addition of HSO3 at
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C-11 of Monascus pigments by sulfonation reaction, is reported for the first time. Although a chemical with the
same molecular weight as compound 11 is also reported, other chemical structure is deduced due to the absence of
NMR datum [24]. UPLC-MS analysis indicates that the fragment of H2SO3 group is also added into the
corresponding Monascus pigments for formation of WSYP 8-10 (Fig.3B). On the contrary, no sulfonation reaction
occurs to ankaflavin (4).
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3.2. Mechanism for sulfonation of Monascus pigments
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Dithionite anion S2O4 can be decomposed into bisulfate (HSO3 ) and thiosulfate (S2O3 ) in an aqueous
solution [29]. The conjugated double bonds of Monascus pigments (2, 5, 6 and 7) are adjacent to a carbonyl group
(Red color in Fig.2). Those Monascus pigments may be tautomerized to the corresponding isomer I. The isomer I
involves conjugated double bonds adjacent to a hydroxyl group (Green color in Fig.2). Bucherer reaction, i.e.,
addition of a nucleophilic bisulfate anion at meta-C in α-naphthol to form the corresponding sulfonated product,
shows that conjugated double bonds adjacent to a hydroxyl group in α-naphthol is the key to sulfonation reaction
(ESI S12) [30]. Following the same mechanism of Bucherer reaction, the reaction mechanism for sulfonation of
Monascus pigments is proposed (Fig.2). The isomer I gives Ⅱ via an electrophilic addition of a proton at C-12. Ⅱ
tautomerizes to form the resonance-stabilized proton adduct Ⅲ. A nucleophilic bisulfate anion adds to the proton
adduct Ⅲ at C-11 to obtain Ⅳ and then tautomerizes to the corresponding lower-energy form (8-11). This
mechanism indicates conjugated double bonds adjacent to a carbonyl group is necessary for sulfonation of
Monascus pigments. This principle is consistent with the facts that sulfonation reaction occurs for monascorubrin
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(2), WSRPs 5 & 6, as well as reduced product 7 while not for ankaflavin (4).
3.3. Color and pH stability of WSYPs
Sulfonation reaction introduces H2SO3 into the double bond between C-10 and C-11 of Monascus pigments
(Fig.2). The reduction of π-conjugated system in WSYPs leads to blue shift in ultraviolet-visible absorbance
spectrum. The visible spectrum of every WSYPs as well as the picture of the corresponding pigment aqueous
solution was presented (Fig.4A). Each WSYP has the corresponding maximum absorbance wavelength as well as
extinction coefficient (8, UV (H2O) λmax (log ε) 500 (3.9); 9, UV (H2O) λmax (log ε) 470 (3.6); 10, UV (H2O) λmax
(log ε) 470 (3.7); and 11, UV (H2O) λmax (log ε) 470 (3.6)). Most of WSYPs in aqueous solution exhibit
orange-yellow while WSYP 11 exhibits bright yellow in an aqueous solution (pH=7). Compounds 9 and 11, as well
as their reactants 5 and 7, show very different colors and UV spectra, which may be attributed to the different
conjugation lengths in the corresponding π-conjugated system. The influence of pH on color of WSYPs was
examined by recording the absorbance at the corresponding maximum absorbance wavelength in different pH
buffer solutions (Fig.4B). WSYP 10 and 11 maintained the original color within a wide pH range. The color of
WSYP 8 aqueous solution was relatively stable at pH 4-11 while got yellow color at pH below 3. The strong
influence of pH on color of WSYP 9 aqueous solution was exhibited as a marked change of the absorbance in the
buffer solutions. The influence of pH on stability of these WSYPs was further examined (Fig.4C). After incubation
for 1 day, most of WSYPs (except for 9) were stable within a wide pH range. This trend about the influence of pH
on WSYP stability was further confirmed by the prolonged incubation time for 2 days (ESI S13). The results
indicate that the color as well as the stability of WSYPs is strongly dependent on their chemical structure, such as X
or Y group as shown in Fig.2. WSYPs (8-11) also exhibit good thermal stability in an aqueous solutions because the
WSYPs are obtained by reflux reaction at 70 oC during the preparative process.
Location of Fig.4
254 4. Conclusions
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Monascus pigments with conjugated double bonds adjacent to a carbonyl group can be sulfonated to produce
novel WSYPs by addition of H2SO3 at the double bond between C-10 and C-11 of Monascus pigments. The
introduction of H2SO3 into Monascus pigments makes the novel WSYPs exhibit yellow color, high water solubility,
as well as relatively high stability in aqueous solution within a wide pH range. All those characters should
contribute WSYPs as a potential novel food colorant. Structure diversity of WSYPs and the corresponding toxicity
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test are the future project in our lab.
Acknowledgment
This work was financially supported by Natural Science Foundation of Shanghai (No. 16ZR1416700).
References
1. Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, Zhang D, Cooper R, Chang M. Constituents of red yeast rice, a traditional
Chinese food and medicine. Journal of Agriculture and Food Chemistry 2000; 48: 5220-5225.
2. Chen W, He Y, Zhou Y, Shao Y, Feng Y, Li M, Chen F. Edible filamentous fungi from the species Monascus:
early traditional fermentations, modern molecular biology, and future genomics. Comprehension Review
of Food Science and Food Safety 2015; 14: 555-567.
3. Dufossé L. Red colourants from filamentous fungi: are they ready for the food industry? Journal of Food
Composition and Analysis 2018; 69: 156-161.
4. Balakrishnan B, Karki S, Chiu SH, Kim HJ, Suh JW, Nam B, Yoon YM, Chen CC, Kwon HJ. Genetic
localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster. Applied
Microbiology and Biotechnology 2013; 97: 6337-6345.
5. Chen W, Chen R, Liu Q, He Y, He K, Ding X, Kang L, Guo X, Xie N, Zhou Y, Lu Y, Cox RJ, Molnár I, Li M,
Shao Y, Chen F. Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi. Chemical
Science 2017; 8: 4917-4925.
6. Liu L, Zhao J, Huang Y, Xin Q, Wang Z. Diversifying of chemical structure of native Monascus pigments.
Frontier in Microbiology 2018; 9: 3143. DOI: 10.3389/fmicb. 2018. 03143.
7. Vendruscolo F, Schmidell W, de Oliveira D, Ninow JL. Kinetic of orange pigment production from Monascus
ruber on submerged fermentation. Bioprocess and Biosystem Engineering 2017; 40:115-121.
8. Martinková L, Zlová PJ, Ceselý D. Biological activity of polyketide pigments produced by the fungus
Monascus. Journal of Applied Microbiology1995; 79 (6): 609-616.
9. Martin EJS, Kurek PR, Schumacher EF, Rohrbach RP. Reduced Monascus pigment derivatives as yellow food
colorants. U.S. Patent 562 880; 1990.
10. El-Baz AF, El-Batal AI, Abomosalam FM, Tayel AA, Shetaia YM, Yang S-T. Extracellular biosynthesis of
anti-Candida silver nanoparticles using Monascus purpureus. Journal of Basic Microbiology 2016;
56:531-540.
11. Koli SH, Mohite BV, Borase HP, Patil SV. Monascus pigments mediated rapid green synthesis and
characterization of gold nanoparticles with possible mechanism. Journal of Cluster Science 2017; 28:
10

290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
2719-2732.
12. Wong H-C, Koehler PE. Production of red water-soluble Monascus pigments. Journal of Food Science 1983; 48:
1200-1203.
13. Lin TF, Yakushijin K, Buchi GH, Demain AL. Formation of water-soluble Monascus red pigments by biological
and semi-synthetic processes. Journal of Industrial Microbiology 1992; 9: 173-179.
14. Jung H, Kim C, Kim K, Shin C. Color characteristics of Monascus pigments derived by fermentation with
various amino acids. Journal of Agriculture and Food Chemistry 2003; 51: 1302-1306.
15. Wei W-G, Yao Z-J. Synthesis studies toward chloroazaphilone and vinylogous γ-pyridones: two common
natural product core structures. Journal of Organic Chemistry 2005; 70: 4585-4590.
16. Choe D, Lee J, Woo S, Shin CS. Evaluation of the amine derivatives of Monascus pigment with anti-obesity
activities. Food Chemistry 2012; 134:315-323.
17. Xiong X, Zhang X, Wu Z, Wang Z. Coupled aminophilic reaction and directed metabolic channeling to red
Monascus pigments by extractive fermentation in nonionic surfactant micelle aqueous solution. Process
Biochemistry 2015; 50: 180-187.
18. Jia L, Tu X, He K, Wang C, Yin S, Zhou Y, Chen W. Monascorubrin and rubropunctatin: preparation and
reaction
characteristics
with
amines.
Dyes
and
Pigments
2019;
doi:
https://doi.org/10.1016/j.dyepig.2019.107629
19. Hajjaj H, Klaébé A, Loret MO, Tzédakis T, Goma G, Blanc PJ. Production and identification of
N-glucosylrubropunctamine and N-glucosylmonascorubramine from Monascus ruber and occurrence of
electron donor-acceptor complexes in these red pigments. Applied and Environmental Microbiology 1997;
63(7):2671-2678.
20. Feng Y, Shao Y, Chen F. Monascus pigments. Applied Microbiology and Biotechnology 2012; 96: 1421-1440.
21. Dufossé L, Galaup P, Yaron A, Arad SM, Blanc P, Murthy KNC, Ravishankar GA. Microorganisms and
microalgae as sources of pigments for food use: a scientific oddity or an industrial reality? Trends in Food
Science and Technology 2005; 16: 389 -406.
22. Fabre CE, Santerre AL, Loret MO, Barerian R, Pareilleux A, Goma G, Blanc PJ. Production and food
applications of the red pigments of Monascus ruber. Journal of Food Science 1993; 58(5):1099-1103.
23. Zheng YQ, Xin YW, Guo YH. Cytotoxicity of Monascus pigments and their derivatives to human cancer cells.
Journal of Agriculture and Food Chemistry 2010; 58: 9523-9528.
24. Yang H, Li J, Wang Y, Gan C. Identification of water-soluble Monascus yellow pigments using
11

320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
HPLC-PAD-LSD, high-resolution ESI-MS, and MS-MS. Food Chemistry 2018; 245: 536 -541.
25. Zheng YQ, Xin YW, Guo YH. Study on the fingerprint profile of Monascus products with HPLC-FD, PAD and
MS. Food Chemistry 2009; 113: 705-711.
26. Sweeny JG, Estrada-Valdes MC, Iacobucci GA, Sato H, Sakamura S. Protoprotection of the red pigments of
Monascus anka in aqueous media by 1, 4, 6-trihydroxynaphthalene. Journal of Agriculture and Food
Chemistry1981; 29(6): 1189-1193.
27. Akihisa T, Tokuda H, Yasukawa K, Ukiya M, Kiyota A, Sakamoto N, Suzuki T, Tanabe N, Nishino H.
Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice
(red-mold rice) and their chemopreventive effects. Journal of Agriculture and Food Chemistry 2005; 53:
562-565.
28. Hsu Y-W, Hsu L-C, Liang Y-H, Kuo Y-H, Pan T-M. New bioactive orange pigments with yellow fluorescence
from Monascus-fermented Dioscorea. Journal of Agriculture and Food Chemistry 2011; 59: 4512-4518.
29. Werbel LM, Capps DB, Elslager EF, Paearlman W, Short FW, Weinstein EA, Worth DF. N-mono- and N,
N-dialkyl-N’-1-naphthylalkylenediamines. Journal of Medicinal Chemistry 1963; 6:637-646.
30. Seeboth H. The Bucherer reaction and the preparative use of its intermediate products. Angewandte Chemie
International Edition1967; 6 (4): 307-317.
12

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359
Table 1. 1H NMR Assignments of 2, 4, 5, 7 and 11 (δ, ppm; J, Hz) a
Position
1
2
4
5
7
11
(chloroform-d)
7.86 (s)
(chloroform-d)
5.01 (d, 12.6)
4.68 (d, 12.6)
(chloroform-d)
9.27 (s)
(methanol-d4)
(DMSO-d6)
7.79 (s)
8.25 (s)
3
5
6
4.83 (s)
7.00 (s)
4.55 (s)
6.79 (s)
2.43 (dd, 17.6, 11.6)
3.20 (dt, 4.2, 13.2)
2.97 (dt, 7.4, 18.1)
5.25 (s)
6.88 (s)
6.77 (s)
6.76 (s)
8
6.14 (s)
6.65 (s)
6.43 (s)
10
6.04 (d, 14.3)
5.88 (dd, 1.6, 15.4) 6.36 (d, 15.9)
6.38 (d, 15.9)
2.11 (dd, 1.2,12.2 )
1.96 (dd, 7.9,15.8)
3.72 (m)
11
12
13
15
17
18
23
N
6.57 (m)
6.49 (m)
7.04 (m)
6.79 (m)
1.94 (d, 7.0, 1.7) 1.84 (dd, 7.0, 1.4)
2.04 (d, 6.0)
1.80 (s)
1.99 (dd, 6.8, 1.6 ) 1.08 (d, 13.5)
1.70 (s)
1.42 (s)
1.21 (s)
1.45(s)
3.69 (d, 13.3)
2.62 (m)
2.92 (m)
2.87 (m)
1. 65 (m)
0.85 (t, 6.8)
*
2.80 (m)
1.60 (m)
0.90 (t, 6.9)
*
2. 58 (m)
1.59 (m)
1.58 (m)
0.86 (t, 7.1)
0.84 (t, 6.8)
0.81 (t, 6.8)
5.29 (s)
360
361
362
363
a s, singlet; d, doublet; t, triplet; m, multiplet.
*: Undetectable peak of the active hydrogen in 1HNMR spectrum due to its volatility.
13

364
365
366
367
368
369
370
371
372
373
374
Table 2 13C NMR Assignments of 2, 4, 5, 7 and 11 (δ, ppm)
Position
2
4
5
7
11
(chloroform-d) (chloroform-d) (chloroform-d) (methanol-d4) (DMSO-d6)
1
153.00
109.77
191.00
85.97
63.74
135.37
189.86
103.29
28.91
42.97
150.92
83.19
160.45
113.87
124.36
17.70
18.44
169.62
54.82
202.50
42.97
23.05
29.38
28.98
31.59
138.37
100.95
195.30
86.77
123.00
128.70
71.89
124.50
130.11
61.66
72.73
174.82
118.51
139.61
84.84
171.45
56.49
60.70
19.03
15.59
174.92
96.42
209.08
53.57
29.16
31.75
29.47
35.57
2
3
4
113.78
167.56
97.09
5
169.41
113.40
141.82
104.35
156.61
116.48
136.59
18.96
153.64
98.53
6
7
140.83
86.77
146.82
82.52
8
9
147.55
123.28
117.53
19.25
153.61
136.36
132.53
17.72
10
11
12
13
14
15
16
17
18
19
20
21
28.52
29.19
17.54
171.83
122.55
197.60
41.85
174.24
116.20
196.32
40.46
174.50
98.49
197.45
39.24
23.91
24.89
25.30
29.38
29.66
29.29
29.38
29.55
28.92
31.92
31.80
31.61
14

22
23
22.83
14.30
22.54
14.01
22.64
14.12
22.22
13.02
22.56
14.43
375
376
377
378
379
380
381
List of figures
382
383
384
385
Fig.1 Preparation of Monascus pigments
A: Native Monascus pigments. OMPs including two entities rubropunctatin (1) and monascorubrin (2) while YMPs
including monascin (3) and ankaflavin (4); B: Diversifying of monascorubrin by chemical modification. One of
OMPs, monascorubrin (2), is chemical modification into WSRPs (5, 6) by amination reaction, where
386
387
388
GLU=glutamic acid. The WSRP 5 is further reduced into 7 in 1 M NaOH aqueous solution
Fig.2 Proposed mechanism for sulfonation of Monascus pigments
Monascus pigments, including 2 and 5-7, having the characteristic structure of conjugated double bonds adjacent to
389
390
391
392
393
394
395
396
397
398
399
400
401
402
carbonyl group (Red color), can be tautomerized to give the corresponding isomer I. The isomer I, having the
characteristic structure of conjugated double bonds adjacent to a hydroxyl group (Green color), can be sulfated
following the same mechanism as that of Bucherer reaction (ESI S12).
Fig. 3 UPLC-MS/MS analysis of sulfonated product WSYPs
A: Fragments involving in compound 7 and 11 (ESI S7 vs S11); B: The fragment H2SO3 involving in WSYPs 8-10
(ESI S3vs S8, S5 vs S9 and S6 vs S10), where HRMS analysis is performed in negative ion model for compound 5
while in positive ion model for the others.
Fig. 4 Colorant characters of WSYPs
A: Visible spectra of WSYP in aqueous solutions (pH=7) (inserted the pictures of WSYP in the aqueous solution).
B: Influence of pH on the absorbance of WSYPs (measured at the corresponding maximum absorbance wavelength
as shown in Fig.4A). C: Stability of WSYPs in an aqueous solution at different pH values (incubation in 30 oC for
1 day).
15

403
404
405
406
407
408
409
410
411
412
413
Fig.1
17
19
6
21
18
20
16
O
10
12
8
1
15
5
4
7
9
11
O 14
2
O
3
O
13
O
414
415
1
2
416
417
418
3
4
419
420
A
421
422
423
B
424
425
16

426
427
428
429
430
431
432
433
434
435
Fig.2
436
437
438
439
440
441
442
443
444
445
446
17

447
448
449
450
451
452
453
454
455
456
457
458
Fig.3
H+
H+
O
O
O
O
H2SO3
O
O
S O
O
O
O
O
O
OH
O
O
+
2
C23H27O5
8
C23H29O8S+
M. W. = 465.1584
M. W. = 383.1863
H-
H+
O
O
O
O
H2SO3
NH
NH
S O
O
O
OH
O
O
-
C23H30NO7S+
M. W. = 464.1743
5
C23H26NO4
9
M. W. = 380.1853
H+
H+
O
O
H2SO3
O
O
N
N
SO3H
COOH
O
COOH
O
COOH
O
COOH
10 C28H36NO11S+
+
6
C28H34NO8
M. W. = 594.2012
M. W. = 512.2286
459
460
461
462
A
B
463
464
465
466
467
468
18

469
470
471
472
473
474
475
476
477
478
Fig.4
479
480
1.5
1.2
0.9
0.6
0.3
0
8
9
10
11
350
400
450
500
550
600
Wavelength (nm)
481
482
A
483
120
100
80
60
40
20
0
2.5
2
1.5
1
8
9
10
11
8
9
10
11
0.5
0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
pH
pH
484
19

485
B
C
20

Highlights
ò
ò
ò
ò
Sulfonation of Monascus pigments with a conjugated double bonds adjacent to a carbonyl group
Production of WSYPs) by addition of H2SO3 at the double bond of Monascus pigments
WSYPs exhibiting yellow color, high solubility in water as well as pH stability
Dependence of the colorant character of WSYPs on the structure of Monascus pigments

Declare
The authors declare no competing financial interest