# Novel Production Method for Plant Polyphenol from Livestock Excrement Using Subcritical Water Reaction

Authors: Mayu Yamamoto; Yasuhiro Futamura; Kouki Fujioka; Kenji Yamamoto
Journal: International Journal of Chemical Engineering
 (2008)

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## Abstract

Plant polyphenol, including vanillin, is often used as the intermediate materials of the medicines and vanilla flavoring. In agriculture generally vanillin is produced from vanilla plant and in industry from lignin of disposed wood pulp. We have recently developed a method for the production of plant polyphenol with the excrement as a natural resource of lignin, of the herbivorous animals, by using the subcritical water. The method for using the subcritical water is superior to that of the supercritical water because in the latter complete decomposition occurs. We have successfully produced the vanillin, protocatechuic acid, vanillic acid, and syringic acid in products. Our method is simpler and more efficient not only because it requires the shorter treatment time but also because it releases less amount of carbon dioxide into the atmosphere.


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## Body

## 1. Introduction

Livestock excrement in Japan
alone has been attained up to 40 000 tons
per year, which recently has been one of the national major excrement
management problems.
Almost all farms are located close to residential areas in Japan, which enhances the hazard of
water and soil pollution by the bad smell and by the onset of the potential infectious diseases. Therefore, we have to develop the method of disposing the excrement efficiently and safely.
In order to regulate the pollution, the Japanese government passed the law, “Law concerning the appropriate treatment and promotion of utilization of livestock manure” (universally applied on November 1st, 2004) [1]. The law requires the
standardization of management method. It is required to build covered and walled-off structures for the disposal of solid excrement with the concrete floors or other nonpermeable materials. As for the disposal of liquid
excrement and slurry, it is required to build tanks made of concrete or nonpermeable material.
According to the law the livestock excrement should not be piled up in open
air, which usually leads to a
long time on making the composts.There  has been another method to develop the electric power station with
supercritical water reactor to treat the livestock excrements in a company
Miyazaki Biomass Recycle (Miyazaki, Japan),
Shizuoka University (Shizuoka, Japan),
and the Japan Aerospace Exploration Agency (JAXA) [2–4], the former with chicken excrement and
the latter two with cattle excrement. In this case, however, the livestock
excrement as a natural resource decomposes into carbon dioxide gas (CO2)
and water (H2O).It is known that the livestock excrement contains a large
quantity of indigestible
dietary fibers of lignin. Lignin is a biopolymer in which hydroxyphenylpropane
units such as trans-p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol are connected with ether and
carbon-carbon linkages in a helical structure [5–10]. Nakahara reported extraction of the plant polyphenol
from wood pulp using supercritical water [11]. Adschiri and colleagues also reported extraction of the plant polyphenol by
solvothermal reaction [12–15]. The plant polyphenol including vanillin is very useful for intermediate material of the medicines and food flavor. Vanillin is currently extracted from vanilla plant
conventionally. Japan imported 260 tons and 340 tons of vanillin per year in 2004 and 2005,
respectively [16]. And
also Freudenberg [17] reported that
vanillin was produced from lignin by the alkali-nitrobenzene method.We here report the extraction of vanillin for the purpose of making the
resource out of livestock excrement into useful plant polyphenol for a wide
variety of industries.


## 2. Materials and Methods

The excrement of herbivorous animals, including cattle,
goats, and horses (University of Miyazaki) and a carnivorous animal, tigers (Ueno Zoological Gardens,
Tokyo, Japan), was used in our study. One-gram excrement and 4 mLH2O
were taken into the reactor, a stainless tube (SUS316) with the capacity of 5 mL. Then the reactor was heated up to
the temperatures (and pressures) of 100°C, 150°C (0.6 MPa), 200°C (1.5 MPa), 250°C (3.9 MPa),
and 300°C (8.3 MPa)
each in the batch
system. After treated for 60 minutes, the reactor was cooled down.We analyzed the products in an aqueous solution with a high
performance liquid chromatography. (HPLC) with a 600E System Controller, a 996
Photodiode Array Detector, and a 10190 HTR-B Temperature Control (Waters Co., MA, USA). A reversed-phase column (Shimadzu
Shim-pack CLC-ODS;ϕ=6.0 mm; column length, 150 mm; Kyoto, Japan)
was used at the temperature of 50°C. An aqueous solution of 2902.95 mL H2O,
31.5 mL methanol, and 5.55 mL propan-2-ol, 60 mL acetic acid, 8.17 g sodium
acetate was used as a mobile phase. The flow rate of
the mobile phase was 0.7 mL/min and the UV detector showed the
absorbance at 280 nm. We conducted the quantitative analyses of the plant polyphenol
concentration using the HPLC of the samples with the area of each peak. We also used a liquid
chromatography/mass spectrometer system (LC/MS) to reconfirm each peak obtained in HPLC analysis. The column (Tosoh TSK-GEL ODS-80; ϕ = 4.6 mm; column length 150 mm; Tokyo, Japan)
was applied for LC/MS. The temperature of column was 40°C and other conditions
were the same as described in the study of HPLC except that ammonium acetate
was used as a volatile salt instead of sodium acetate for the mobile phase.


## 3. Results and Discussion

Today there are two methods of production of vanilla. A conventional way is to extract it directly from the vanilla plant. The vanilla plants are native to Mexico, but people in Madagascar
were the first to produce for commercial uses. The flavoring comes from the
seedpod and the bean of the vanilla plants. The process of making vanilla into
the spice is a very long and complicated method. Therefore, they could not yield a large quantity of vanilla. The
other method is obtaining the vanilla from wood pulp lignin disposed in
industry. Adschiri and colleagues not only extracted the lignin under supercritical water and succeeded in
the production of the plant polyphenol but they also identified many other entities than that of vanillin. However, it takes much time and energy to
refine the plant polyphenol.In order to turn the waste into useful products, we have
devised a simple method for vanillin production with the lignin
from the livestock excrements.We have shown here in Figure1(b) an HPLC profile of the products in the solution, which
smells like coffee and cocoa powder, obtained from the cattle excrement at 200°C reaction. Following the HPLC profile of the standard
compounds (Figure  1(a)),
we can identify the following peaks: the peak of protocatechuic acid at 10
minutes, vanillic acid at 32 minutes, syringic acid at 55 minutes, and vanillin
at 56 minutes. We can find the LC peaks 1 to 4 at the same retention time as
those of standard compounds. For the confirmation of each substance we have
conducted the liquid chromatography/mass spectrometer for the further study. Figure 2 shows the mass
spectra obtained from the LC peaks 1 to 4 indicated in Figure  1(b).UV chromatograms of HPLC of (a) the standard reagents
and (b) the cattle excrement treated
at200°C. The chromatograms were obtained from the LC/MS system with
the reversed-phase column (Tosoh TSK-GEL ODS-80; ϕ = 4.6 mm; column length 150 mm). The
temperature of column was 40°C. The flow rate was 0.7 mL/min. The UV detector
showed the absorbance at 280 nm.
(a)(b)The mass spectra of the HPLC peaks of the cattle excrement treated at200°C.
Mass scan was performed in negative ion detection mode after
electrospray ionization. Theoretical ratios of mass to charge (m/z) of deprotonated ions ([M-H]-)
of protocatechuic
acid, vanillic acid, syringic acid and vanillin are 153.02, 167.03, 197.04, and
151.04, respectively.
(a)
LC peak 1(b)
LC peak 2(c)
LC peak 3(d)
LC peak 4We can identify the single major peak of ratio of mass to
charge (m/z) with protocatechuic acid
in Figure  2(a).
It is also shown in Figures 2(c) and  2(d) with syringic acid and vanillin. On the other hand,
we can identify two major peaks of m/z,
which was shown in Figure 2(b); one is that of vanillic acid and the other remains
unidentified.We carried out the experiment with the different subcritical
water reaction
temperature for the purpose of the dependency of the products yields.
Figure3 shows the
results of the quantitative analyses of each plant polyphenol. Our desired plant polyphenol is not found in the
condition of 300°C, which suggests that the
temperature dependency on the product yields exists. Protocatechuic acid was
obtained at 200°C. Vanillic acid was isolated from the excrement mostly at
150°C and its yield decreased at 200°C. Syringic acid was obtained in the range
of 150°C to 200°C. Although we identified vanillin at 200°C, at 250°C it yielded
more but no vanillin was found when the reaction temperature was carried out 300°C. The yield of vanillin at
250°C was the maximum. The total amount of vanillic acid and vanillin reached almost constant at 50 μg by 1 g of excrement between temperatures in the range of 150°C to 250°C. It is considered that
vanillic acid was converted to vanillin via redox reaction under subcritical
water. Further study is required to clarify this point.Quantitative HPLC analysis of polyphenols extracted
from the cattle excrement at 150, 200, 250 and300°C. (a) protocatechuic acid, (b) vanillic
acid, (c) syringic acid, and (d) vanillin. The circles show the yield of plant polyphenol per 1 g of
excrement. A reversed-phase
column (Shimadzu Shim-Pack CLC-ODS; ϕ = 6.0 mm; column length, 150 mm) was used at
the temperature of 50°C. An aqueous solution of 2902.95 mL H2O, 31.5 mL methanol, and 5.55 mL propan-2-ol, 60 mL acetic acid, 8.17 g sodium acetate
was used as a mobile phase. UV absorbance was detected at 280 nm.
(a)
Protocatechuic acid(b)
Vanillic acid(c)
Synringic acid(d)
VanillinWe proceeded to further experiments with the other animals to
identify production of vanillic acid and vanillin. We analyzed the products
after the treatment with HPLC. Figure4 shows the HPLC profiles of the products in the
solution obtained from the cattle, goat, horse, and tiger excrement under the
condition of 200°C. We found peaks of vanillic acid and vanillin around 35 and
55 minutes,
respectively, obtained from the products of the excrement cattle, goats, and
horses: herbivorous animals. However, we could not find the products of the
excrement of the tigers: carnivorous animal. Herbivorous animals take in the
plants containing high content of lignin. The microbes in digestive organs digest the plant
fiber. Most of the lignin contained in the plant fiber, however, cannot be digested
by the microbes and other digestive enzyme. The lignin is difficult to digest for the herbivorous
animals including the three animals we analyzed [18–24]. In the case of carnivorous animals,
on the contrary, they take in mainly flesh meat that the digestive enzyme in
their digestive organs hydrolyzed into the amino acid. We therefore reasoned that the excrement of the herbivorous
animals is superior as a lignin source. Our experimental results are in
agreement with the hypothesis as observed from the
different amounts of the lignin in excrements between the herbivorous and carnivorous animals.UV Chromatograms of (a) cattle, (b) goat, (c) horse,
and (d) tiger excrement treated at200°C. We used HPLC system with the reversed-phase column (Shimadzu
Shim-Pack CLC-ODS; ϕ = 6.0 mm; column length, 150 mm) at the
temperature of 50°C. The mobile phase is the same as that of LC/MS; ammonium
acetate was used instead of sodium acetate. The UV detector showed the
absorbance at 280 nm.
(a)
Cattle(b)
Gout(c)
Horse(d)
TigerAlthough  we have shown here that a
valuable product could be extracted from excrements of the animals, they can be
used as other resources.
The biomasses are recyclable organic resources derived from
plants and animals:
livestock excrement, wood chip, and the chaff, for example. The livestock
excrement has been attained up to 50 million tons per year in Japan. The problem of oil shortage
in the near future is widely anticipated. We could avoid it by using the abundant excrement as a
carbon resource. Furthermore, the suitability, as food, of the
excrement products obtained in our method is now being tested on animals.It is becoming increasingly apparent that the use of enormously large quantity of fossil fuel leads to the global
warming. The more the consumption of it leads to the higher the concentration ofCO2 in the atmosphere. The carbon given from the biomass, however, is
originally fixed by the plant photosynthesis from the atmosphere. Our system
does not release all the carbon atoms in the excrement into the atmosphere as
CO2 but helps to keep the carbon atoms in the molecule of the plant
polyphenol. Furthermore, agriculture not only provides us food, but also
supports the biomass as natural resources. If we consider the shortage of the oil in the
coming years, we need to find other ways to obtain materials extracted from oil at the present, rather than the conventional industrial method.
Recycling is one way to overcome this problem. The usage of the biomass for industrial materials
will be an alternative way for this purpose [25, 26]. Our way of the production of
polyphenol will provide alternative ways; actually, we have already succeeded in producing a kind of phenol-plastic
from the polyphenol obtained from the livestock excrement by subcritical water
reaction (data not shown).Some compounds of the polyphenol are hard to synthesize, which leads us to develop a more effective and larger-scaled extraction system. The agriculture in this sense
will also play a significantly important role in a sustainable society in the near
future, implying the pathway, if we could develop the mass production system of
polyphenol by using this method.



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*Source: 603957-2008-09-14.xml*