Preparation of a genipin blue from egg protein and genipin

Article abstract of DOI:10.1080/14786419.2010.547859

Genipin blue is a pigment prepared from the reaction of genipin with amino acid. We describe herein a new method used to prepare genipin blue, watersoluble blue pigments, through the reaction of hen egg protein with genipin. The effects of reaction time, reaction temperature, the pH value of the solution and the mass ratio of the reactants on the preparation are studied. One part of genipin reacted with eight parts of hen egg protein (w/w) in water (pH value of reaction system 7.5) at 60°C for 96 h and gave blue pigments with the maximum colour value of 146.2. The blue pigments showed identical absorption activity in UV spectroscopy (λ_max=584 nm) to that of gardenia blue pigments, which were prepared from the reaction of genipin with amino acid.

Full text of DOI:10.1080/14786419.2010.547859

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Natural Product Research: Formerly
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Preparation of a genipin blue from egg
protein and genipin
Dan Yang a , Min Zhou a , Wanxing Wei a , Honglin Zhu a & Xiwang
Fan a
a School of Chemistry and Chemical Engineering , Guangxi
University , Nanning , China
Published online: 06 Sep 2011.
To cite this article: Dan Yang , Min Zhou , Wanxing Wei , Honglin Zhu & Xiwang Fan (2012)
Preparation of a genipin blue from egg protein and genipin, Natural Product Research: Formerly
Natural Product Letters, 26:8, 765-769, DOI: 10.1080/14786419.2010.547859
To link to this article: http://dx.doi.org/10.1080/14786419.2010.547859
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Natural Product Research
Vol. 26, No. 8, April 2012, 765–769
SHORT COMMUNICATION
Preparation of a genipin blue from egg protein and genipin
*
Dan Yang, Min Zhou, Wanxing Wei , Honglin Zhu and Xiwang Fan
School of Chemistry and Chemical Engineering, Guangxi University, Nanning, China
(Received 4 June 2010; final version received 7 December 2010)
Genipin blue is a pigment prepared from the reaction of genipin with amino
acid. We describe herein a new method used to prepare genipin blue, water-
soluble blue pigments, through the reaction of hen egg protein with genipin.
The effects of reaction time, reaction temperature, the pH value of the
solution and the mass ratio of the reactants on the preparation are studied.
One part of genipin reacted with eight parts of hen egg protein (w/w) in
water (pH value of reaction system 7.5) at 60ꢀC for 96 h and gave blue
pigments with the maximum colour value of 146.2. The blue pigments
showed identical absorption activity in UV spectroscopy (ꢀmax ¼584 nm) to
that of gardenia blue pigments, which were prepared from the reaction of
genipin with amino acid.
Keywords: genipin blue pigments; preparation; genipin; geniposide; hen
egg protein; amino acid
1. Introduction
Fruit of Gardenia jasminoides Ellis has been used as traditional medicine in China for
centuries. Blue pigments (genipin blue), prepared from the reaction of genipin (one
compound from hydrolysis of geniposide which was isolated from the fruit of
G. jasminoides Ellis) with amino acids, was used as a food additive in East Asia. For
its biodegradable and low cytotoxic properties, genipin has recently been investigated
as a cross-linking material in many biological applications (Bigi, Cojazzi,
Panzavolta, Roveri, & Rubin, 2002), just as bio-adhesive, wound dressing, and
bone substitutes (Muzzarelli, 2009). It has been widely used in a variety of medical
fields such as nerve regeneration and drug delivery (Koo, Lim, Jung, & Park, 2006;
Zhou, Zhao, & Zhang, 2009).
Recently, natural pigments have become increasingly desirable in food due to
their generally favourable health effects. Geniposide is the principal iridoid glucoside
in the fruit of G. jasminoides Ellis, and transformed into genipin by hydrolysing with
ꢁ-glycosidases (Fujikawa, Yokota, Koga, & Kumada, 1987; Xu et al., 2008). Genipin
then reacted with amino acids to generate natural blue pigments (ꢀmax ¼584 nm)
(Cho et al., 2006). The blue pigments from G. jasminoides are edible and currently
used as blue food pigments. The natural blue pigment is also used to dye a variety of
fabrics, including cotton, silk and wool (Lee, Lim, Bhoo, Park, & Hahn, 2003).
*Corresponding author. Email: wxwei@gxu.edu.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2010.547859
http://www.tandfonline.com

766
D. Yang et al.
Figure 1. Preparation of genipin blue.
One new water-soluble genipin blue, prepared from genipin with hen egg protein
alternating amino acids, was reported briefly in our previous studies (Wei & Yang,
2009). Herein, we report the effects of reaction conditions on preparation of the
pigment (Figure 1).
2. Results and discussion
2.1. Effects of temperature and time on preparation of blue pigments
One part of genipin (which was prepared according to our previous studies (Wei &
Yang, 2009), purity 99.97% by HPLC) and eight parts of hen egg protein (which was
prepared from hen egg from market) were dissolved in water (pH value of reaction
system 7.5), stirred and made to react for 144 h at 30ꢀC, 40ꢀC, 50ꢀC, 60ꢀC and 70ꢀC,
respectively. Wavelengths at maximal absorbance (ꢀmax) of products were scanned
and the value was 584 nm. The absorbance of the products was detected in ꢀmax
interval of 24 h. Effects of temperature and time on the reaction showed (Figure 2)
that all products reached their maximum absorption after 96 h at different
temperature values, and product from a reaction at 70ꢀC gave the maximum
absorption in UV among different temperature values. The reaction at 70ꢀC was
rapid (reaction at 70ꢀC yielded blue product after 48 h, at 60ꢀC after 72 h and at 40ꢀC
and 50ꢀC after 96 h) and yielded sediment. Therefore, the optimal condition for the
reaction was determined (at 60ꢀC for 96 hours).
2.2. Effects of pH value on preparation of blue pigments
The reactions (one part of genipin and eight parts of egg protein) were carried out in
a pH value range 3.0–12.0 [(a) formic acid/NaOH buffer 3.0–4.0, (b) NaAc/HAc
buffer 5.0–6.0, (c) distilled water, (d) NH3/NH4Cl buffer 9.0 and (e) Borate/NaOH
buffer 12.0] at 60ꢀC for 96 h. Results showed (Table 1) that reaction did not occur in
acidic medium. The ꢀmax value of products was 584 nm, and the colour of products
was mauve or dark red under basic condition. It was due to saponification of genipin
in alkaline medium to release genipinic acid, and then to react with protein to give
other than blue pigments (Moritome, Kishi, & Fujii, 1999). Only the reaction in
distilled water yielded blue pigments with a ꢀmax of 584 nm.

Natural Product Research
767
Figure 2. Effect of temperature and time on blue pigment production rates.
Table 1. Effects of pH on reactions of genipin with egg protein.
Buffer system
A
B
C
D
E
pH
ꢀmax (nm)
Colour
3
–
5
–
7
584
Blue
9
596
Mauve
12
598
Dark red
White
Brown
2.3. Effect of the mass ratio of reactants on blue pigments production
Reactions were carried out in distilled water at 60ꢀC for 96 h in different mass ratios
of genipin and egg protein. Results showed that one part of genipin and eight parts
of egg protein gave the maximum absorption value (Figure 3) in ꢀmax ¼584 nm.
2.4. The colour value of blue products
Investigations show that effects of time, temperature, pH value of solution and the
mass ratio of reactants on the reaction gave an optimal preparation process.
Different reaction conditions led to different colour value (Table 2) of products.
584 nm
1 cm
Absorbance (A) was calculated as colour value E
Equation (1):
(1%) in the following
584 nm
1cm
E
ð1%Þ ¼ A ꢁ 10=m
ð1Þ
where m was the mass of blue pigment in gram.
When reaction was carried out at a ratio of 1 : 8 (w/w) of genipin and egg protein
at 60ꢀC for 96 h in distilled water, the product gave the maximum colour value,
584 nm
1 cm
E
(1%)¼146.2.

768
D. Yang et al.
Figure 3. Effect of the mass ratio of genipin and egg protein on blue pigment production
rates.
Table 2. Colour value of blue products.
Reaction
temperature (ꢀC)
Genipin/egg
protein (w/w)
Product’s
colour value
30
40
50
60
70
60
60
60
60
60
60
1 : 8
1 : 8
1 : 8
1 : 8
1 : 8
1 : 1
1 : 3
1 : 5
54.6
86.9
102.4
146.2
118.7
76.3
98.7
118.7
128.6
104.6
82.4
1 : 10
1 : 12
1 : 16
3. Conclusion
Preparation of water-soluble blue pigments from the reaction of genipin with egg
protein (mass ratio 1 : 8, w/w) was successfully carried out in distilled water at 60ꢀC
for 96 h. The blue products and other blue pigments from the reaction of genipin
with amino acids have the same absorption activities in UV (ꢀmax ¼584 nm).
Supplementary material
Experimental details relating to this paper are available online, alongside Figures S1–
S18.

Natural Product Research
769
Acknowledgements
This work was partly supported by the National Natural Science Foundation of China (Grant
no. 30660216). The authors also thank Shanyun Bio-Chemical Science and Technology
Corporation for providing the plant material for this work.
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