a,*
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi, 214036 Jiangsu, China
b
Zhejiang Hangzhou Xinfu Pharmaceutical Co., Ltd., Hangzhou, 311300 Zhejiang, China
Received 16 November 2005; received in revised form 7 March 2006; accepted 8 March 2006
Available online 16 June 2006
Abstract
A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic
acid were compared, and the highest yield reached 2.2 g l 1 of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l 1. The ltrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the ltrate was bioconverted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l 1 when 5 g l 1 of glucose and 25 g of
HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that d13CPDB of vanillin prepared
was much dierent from chemically synthesized vanillin.
2006 Elsevier Ltd. All rights reserved.
Keywords: Waste residue of rice bran oil; Vanillin; Aspergillus niger; Pycnoporus cinnabarinus
1. Introduction
Vanillin (3-methoxy-4-hydroxybenzaldehyde) is one of
the most universally used avors in foods, fragrances,
beverages and pharmaceuticals (Priefert et al., 2001). It
was rstly isolated from vanilla beans in 1816 and its
structure was determined in 1874. Today, the world consumption of vanillin is estimated to be 12,000 t per year.
Approximately 50 t is in the form of natural vanillin
extracted from vanilla pods, with chemically synthetic
vanillin providing the remainder (Li and Rosazza, 2000).
However, under current US and European legislation,
the chemically synthesized avor chemicals could not be
used for natural avors (Muheim and Lerch, 1999).
Therefore, the increasing consumer request for natural
products created many biotechnological processes to produce natural vanillin (Lesage-Meessen et al., 1999).
Corresponding author. Tel.: +86 510 5865133; fax: +86 510 5808498.
E-mail address: zhengpu@sytu.edu.cn (Pu. Zheng).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.03.028
Microbial transformation from natural substrates, including phenolic stibenes (Yoshimoto et al., 1990), lignin
(Guiraud et al., 1999), isoeugenol (Li et al., 2005), eugenol
(Walton et al., 2000), ferulic acid (Lesage-Meessen et al.,
1996; Sun, 2002), vanillic acid (Stentelaire et al., 2000),
aromatic amino acid (Diaz et al., 2001), sugar beet pulp
(Bonnin et al., 2000, 2001), wheat straw (Klinke et al.,
2002), biomass slurry fuel (Kartal et al., 2004) were investigated, and ferulic acid from raw material was considered
as a suitable precursor for vanillin.
Ferulic acid is an extremely abundant hydroxycinnamic
acid in plant cell wall, which exists in the plant in its free
form, covalently linked to biopolymers, such as polysaccharide compounds (Ishii and Tadashi, 1997), triterpene
alcohols and plant sterols (Rosazza et al., 1995). It occurs
in common agricultural waste residues such as cereal bran,
sugar beet pulp, and was therefore chosen as raw material
for transformation to vanillin by fungi. The biotechnological process to produce vanillin from agro by-products had
been investigated. 105 mg l 1 and 767 mg l 1 of vanillin
were obtained in the two-step bioconversion combining
1116
Heated treatment
(135 oC, 5 h)
Cool
Precipitated
(-Sitosterol ect.)
medicine materials
Fig. 1. Preparation of ferulic acid from waste residue of rice bran oil.
1117
-1
13
2.0
1.5
1.0
0.5
0.0
0
12
24
36
Time (h)
Vanillic acid
60
72
Ferulic acid
2.5
2.0
1.5
1.0
0.5
0.0
0
20
40
Ferulic acid
Based on the above results, further experiment was conducted in order to obtain a mass of vanillic acid enriched
broth of A. niger CGMCC0774. The fermentation of A.
niger and bioconversion process converting ferulic acid to
vanillic acid was scaled-up to 25 l level. Under of the optimum fermentation conditions, A. niger CGMCC0774 was
grown for 42 h to produce the enzyme with ferulic acid
48
Fig. 2. The conversion time course when the initial ferulic acid concentration was 2.0 g l 1. The bioconversion was carried out at 37 C, with
shaking speed of 150 rpm.
2.6. The
60
80
100
Time (h)
Vanillic acid
120
140
Vanillin
1118
0
4
6
8
10
12
Residual in
conversion medium
(g l 1)
Vanillin
Vanillyl
alcohol
Vanillin
Vanillyl
alcohol
0.63
0.29
0.20
0.10
0.04
0.03
0.32
0.15
0.12
0.12
0.12
0.12
0
0.79
0.86
0.92
1.05
1.06
0
<0.01
<0.01
<0.01
Table 2
Eect of vanillic acid concentration on vanillin production
Vanillic acid
(g l 1)
HZ802 resin
(g)
Vanillin
(g l 1)
Molar conversion
yield (%)
2
3
4
5
10
15
20
25
1.1
1.7
2.2
2.5
60.8
62.6
60.7
55.2
The bioconversion was carried out at 35 C, pH 5.0, with shaking speed of
120 rpm. HZ802 resin was added after12 h and conversion continued 54 h.
Table 3
The 13C isotope characteristic of various vanillin
d13CPDB (&)
Vanillin
Chemically synthesized vanillin from guaiacol
Rhovanil natural from ferulic acid
Vanillin from residue of rice bran oil
26.88
36.67
36.11
The determination was carried out with DeltaPlusXL (Bristol, the UK).
13
In order to obtain the natural label, the vanillin product was authenticated by the 13C isotope analysis. Isotopic
mass spectrometry reected vanillin from dierent sources
(agricultural or petrochemicals). The 13C isotope of the
crystallized vanillin produced from waste residue of rice
bran oil was identical with Rhovanil Natural (Rhodia
Co. France), and dierent from chemically synthetic vanillin. The result was shown in Table 3.
4. Discussion
Considering the increasingly requirement for natural avors in the food industry, this work was focused on production of vanillin from a natural raw materials by use
of microorganisms. Waste residue of rice bran oil is a very
cheap and abundant by-product of the rice rening industry in China. In this study, we attempt to use waste residue
of rice bran oil as ferulic acid source for vanillin production
at the rst time. With preliminary treatment, free ferulic
acid in ferulic acid enriched fraction released from waste
residue of rice bran oil was converted to vanillic acid by
A. niger CGMCC0774, and then the vanillic acid enriched
broth was further converted to vanillin by P. cinnabarinus
CGMCC1115 cells. Under the optimum condition of
ferulic acid consecutive bioconversion by A. niger and
P. cinnabarinus, the maximum concentration of vanillin
reached 2.8 g l 1 with a molar yield of 61.9% at 72 h. Based
1119