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Biotechnological production of potent antioxidant phenolics and study og their antiprotozoal
capacity.
Aguilera Antonio1, Contreras Juan2, Mata Benito3, Rodríguez Raul2 & Aguilar Cristobal2*
1
Universidad Autónoma Agraria Antonio Narro (UAAAN), Saltillo, Coahuila, México
Universidad Autónoma de Coahuila (UAdeC), Saltillo, Coahuila, México.
3
Universidad Autónoma de Nuevo León (UANL), Monterrey, NL, México.
2
*Corresponding Author: Prof. Cristóbal Aguilar. Departamento de Ciencia y Tecnología de Alimentos de la Facultad de Ciencias Químicas
de la Universidad Autónoma de Coahuila,. Saltillo, Coahuila, México. Tel +52 844 4161238, Email. [email protected]
Abstract
The solid-state bioprocess for microbial production of potent antioxidants is an emerging promissory
biotechnology. Ellagic (EA) and gallic acids (GA) are two potent antioxidants with important
physiological and functional properties. In this study EA and GA were produced by solid state
fermentation (SSF) using pomegranate husk and coffee pulp as supports and nutrient sources in
independent experiments. Aspergillus niger GH1 was used to release EA and GA from ellagitannins
and gallotannins present into agro-industrial residues. SSF was kinetically monitored during 168 h.
Polyphenolic content was evaluated during the fermentation; EA and GA released were measured by
HPLC. Antiprotozoal activities of both phytochemicals were evaluated against Entamoeba histolytica,
Trichomonas vaginalis and Giardia lamblia. Obtained results of the SSF demonstrated high rates of
biodegradation of the hexahydroxydiphenic group from the ellagitannins. The highest EA
accumulation was reached at the 96 h of culture (12.3 mg per gram of substrate). A yield of 0.3 g EA
per g of substrate was obtained. Maximum accumulation of GA from coffee pulp was reached at 48h
with a yield of 0.32g per gran of substrate. Highest values of protozoarial inhibition were obtained
with EA and GA in comparison than the control (metronizadole). Fungal SSF of pomegranate husk
and coffee pulp is an excellent alternative bioprocess for bio treatment of pomegranate and coffee
residues to produce potent antiprotozoarial bioactives, ellagic acid and gallic acid.
Keywords: solid state culture, polyphenols, ellagic acid, gallic acid
1. Introduction
Human diseases caused by parasitic protozoa such as Entamoeba histolytica, Trichomonas vaginalis
and Giardia lamblia occur with high prevalence worldwide [1, 2]. Among the human parasitoses,
amebiasis is the second greatest cause of death globally, malaria being the first. All of these parasitosis
are widely distributed in the world, but affect mainly developing countries [3-5]. Amebiasis is
characterized by destruction of the organs and tissues invaded, and its major clinical manifestations
are dysentery and hepatic abscesses [1, 6]. Trichomoniasis is a common cause of vaginitis, urethritis
and prostatitis [4] and has been linked to sterility problems, low birth weights and preterm delivery
[7]. The most frequent manifestations of G. lamblia infection are in the human and animal small
intestine [8, 9] and it is a common cause of urticaria, angioedema [10, 11] and atopic dermatitis [12,
13]. Metronidazole is one of the most efficacious medications for the treatment of amebiasis,
trichomoniasis and giardiasis [14]. Nevertheless, 2.5% to 5% of the causal agents of these diseases, E.
histolytica, T. vaginalis and G. lamblia respectively, display some level of resistance to metronidazole
[15, 16]. This represents a serious problem of public health , for this reason it is necessary to find new
bioactives with high efficacy to inhibit these microorganisms.
Ellagic acid (EA) and gallic acid (GA) are naturally occurring phenolic compounds widely distributed
in plants, the importance of this compound is due its diverse properties reported as potent antioxidant,
anti-inflammatory, anti’-tumoral, anti-microbial, anti-viral and anti-proliferative capacities (AguileraCarbo et al. 2007). Major ellagitannins source are wood oak (Quercus sp), chesnut (Castanea sp) and
myrobalan (Terminalia chebula) and some fruits like strawberry, raspberry, blueberry, cranberry,
pecan and walnut.(Clifford and Scalbert, 2000). Chemically, ellagitannins consist of glucose esterified
with hexahydroxidiphenic acid, gallic acid and their derivates (Shi et al., 2005). For industrial EA and
GA production from tannins, the acidic hydrolysis is the common method, however, it is an expensive
and low-yield procedure (Saavedra et al. 2005). Recently, several studies on biotechnological
production of EA and GA from several plant materials have been published (Huang et al. 2007a, b, c,
d; Robledo et. al. 2008; Aguilera-Carbo et al., 2008; Aguilar et al., 2008).
Our group, previously reported the first findings on fungal EA and GA production through SSF
(Aguilera-Carbó et al., 2008; Robledo et al. 2008) demonstrating that the pomegranate husk residue is
an excellent alternative for EA production. Also, a biodegradation process of ET’s for EA production
has been proposed (Aguilera-Carbo et al. 2007). SSF is one of the most attractive alternative to
management of agro industrial by-products, in this case the residues of pomegranate husk contain an
interesting profile of nutrients such as large amounts of insoluble carbohydrates, small amount of
protein, minerals and some remaining juice and other soluble substances favoring a rapid microbial
growth. These properties can be approached for the production of high value-added metabolites.
In this study we evaluated the EA and GA production by Aspergillus niger GH1 using a pomegranate
husk and coffee pulp residues as support of SSF and their antiprotozoal activities.
2 Materials and methods
Powder of pomegranate husk and coffee pulp. The samples of pomegranate fruits were collected from
a rustic orchat in Sabinas, Coahuila, México. The pomegranate fruits were cleaned with water and
separated in husk and seed, the husk were dried in a funnel dryer, at 60 ºC for 48 h. Coffee pulp was
acquired from a coffee producer in Veracruz, Mexico. Similar conditions of dehydration were
employed. The dried material of both sources was pulverized and sifted to a 30 mesh particle size in
an industrial homogenizer (5 L, model LP12 Series 600-182, JR Maquinaria para mercado S.A. de
C.V., México) and stored in dark and dry conditions. The material obtained was called powder of
pomegranate husk (PPH) and coffee pulp powder (CPP).
Microorganism strain. Aspergillus niger GH1, was provided by Food Research Department collection
(Universidad Autónoma de Coahuila, in Saltillo, Coahuila, México). This was previously isolated
from a native plant of a semidesert zone, and selected to grow in high tannin concentrations (CruzHernandez et al., 2005).
Propagation, composition media and culture conditions. The mycophill agar medium was used for
propagation of fungal inoculum. The culture broth for SSF were PPH and CPP media composed with
(gL-1) NaNO3 6.0, KH2PO4 2.4, MgSO4 1.2, KCl 1.2 and inoculated with 2x107 spores per gram of
support (PPH or CPP). The fermentation was carry out in tray reactors under moisture of 70 % at 30
ºC by seven days. SSF was analyzed in triplicates.
Pre treatment of material fermented for the determination. Fermented PPH/CPP was resuspended with
water (30 mL) and shacked in a RIVAL immersion blender (model IB901 MX) during two cycles of
30 s, the material was transfered at 50 mL conic tubes and these were immersed in a vibrating sonic
bath for 30 min (Bransonic, Model 2510R-MTH, Branson Corp, CT, USA). The material was
centrifuged at 6000 rpm by 30 min, decanted and the liquid fraction was recovered (Aguilera-Carbo et
al. 2007a).
Analytical methods. The biomass content was indirectly evaluated by spectrophotometry using the
glucosamine content determination (Boone-Villa et al., 2008). Substrate consumption (total
polyphenols content) was evaluated using the methodology reported by Makkar (1993).
Sample Preparation of ellagic acid/gallic acid determination and quantification by HPLC. Fermented
CPP was pressed and filtered to get a liquor and the GA was directly quantified in the HPLC. The
fermented PPH was resuspended with 30 mL of ethanol and homogenized in a submerged blender by
two cycles of 30 s, the material was transfered at 50 mL conic tubes and immersed into a vibrating
sonic bath by 30 min, the mixed material, an aliquot of 1.5 mL was transferred into an eppendorff tube
and centrifuged at 6000 rpm (3600g) by 20 min. The supernatant was decanted and the precipitate was
resuspended in ethanol. The sample was immersed into a vibrating sonic bath again for 30 min and the
solution was transferred into clean test tubes and filled up to a 5 mL with ethanol and reimmersed into
vibrating sonic bath for 2 h. The suspended material was filtered through 0.45 m nylon membrane
and injected into HPLC. The EA recovered was quantified by HPLC method previously reported by
our group (Aguilera-Carbo et al., 2008a), The HPLC Varian Pro Star systems with a photodiode array
detector (PDA Pro Star 330) was used Separation was carried out with a Prodigy ODS column (5 m;
250 x 4.6 mm, Phenomenex) and temperature of 25°C. A gradient profile of mobile phase, consisting
of acetonitrile (solvent A) and 0.3% acetic acid in water (v/v) (solvent B), 7-20% A (0-7 min), 20-30%
B (7-12 min), 30% B (12-18 min), 30-60% B (18-20 min), 60-100% B (20-23 min), 100% B (23-30
min) y 7% B (30-31)and 7 min for baseline stabilization was applied at a flow rate of 0.6 mL/min.
The sample injection was ofL. A wavelength of 254 nm was used.
Mathematical models
Biomass production was adjusted with the Velhurst-Pearl logistic equation, originally developed for
population growth.

dX
X 
  M 1 
X
dt
X
max 

(1)
where X is biomass density (g per L, per cm2 or per kg), μM the maximum specific growth rate (h−1)
and XM the equilibrium level of X for which, dX/dt = 0 for X > 0. Solution of the above equation can
be written as follows:
X
X max
 X  X 0  M t
e
1   max
X0


(2)
where X0 is the initial condition for X. Eq. (2) is useful to fit experimental data by Eq. (1), finding the
least value of the sum of squared errors as a function of parameters, X0, XM and μM.
Substrate consumption was modeled using a two-term expression proposed by Viniegra-Gonzalez et
al. (2003) as follows:

dS
1 dX

 mX
dt YX / S dt
(3)
where S is the substrate concentration (g per L, per cm2 or per kg), YX/S the biomass yield coefficient (g
X/g S) and m the maintenance coefficient (g S/g X h). Solution of Eq. (3) can be obtained as a function
of X as follows:
 X  X 0   X max  m   X max  X 0 
  
 ln 
S (t )  S 0  


  X max  X 
 YX / S  
(4)
where S0 is the initial condition for substrate level, S. Eq. (4) helps to test the importance of the
maintenance coefficient, m, because a state plot of S(t) vs. X(t) will yield a straight line with slope,
1/YX/S, whenever m is negligible. Otherwise, a logarithmic correction will appear with coefficient,
mXM/μM.
Kinetics of product formation can be modeled using the equation as proposed by Aguilar et al. (2001)
as follows:
dP
dX
 YP / X
 kX
dt
dt
(5)
where P is the product concentration, YP/X the product yield in terms of biomass (units of product per
unit of biomass) and k the secondary coefficient of product formation or destruction. Eq. (5) is similar
to Eq. (3), but here the coefficient k can be negative, zero, or positive, since product formation or
destruction is not necessarily related to growth. Again it is possible to solve Eq. (5) as a function of
biomass
P(t )  P0  YP / X ( X  X 0 ) 
kX M
M
 X  X0 
ln  M

 XM  X 
(6)
The specific growth rate (
was from the straight line by minimal square regression. The specific production rate of enzyme, qP,
was defined as follows:
qP   M  YP / X
(7)
The specific substrate uptake rate, qS, was defined as follows:
qS   M YX / S
(8)
YP/X and YX/S were estimated from the linear correlation between the ellagic acid/gallic acid and
biomass concentration, and biomass and pomegranate husk powder/coffee pulp, respectively.
Basic model for metabolite productivity
Productivity for fermentation systems can be expressed in different ways. In this paper it is chosen to
define productivity, Γ, for every t>0 as P(t)/t, within the overall culture medium. For example, if the
porosity and the liquid content of a given SSF are known, productivity, in terms of reaction volume,
can be corrected by corresponding proportional factors. Also, if antioxidants are accumulated to the
medium and leached out at the end of the fermentation, final productivity can be estimated by taking
into account the dilution factor. However, in all cases, the initial figure, related to microbial
physiology is productivity defined as follows:
P
max  maximum of  
t 
(9)
That is, for a given fermentation curve, Γmax, will be the maximum of the ratio between the product
level per liquid broth volume, P, added to the system and divided by the fermentation time, t. In most
cases, Γmax will be evaluated at the peak of metabolic activities production, but, this is not always the
case because of the time factor involved and the asymptotic nature of end fermentation points.
Ref), which helps to identify the major
physiological factors involved in productivity of a given experimental system that in some cases can
be corrected by otter two parameters.
R ef   M YP / X X max
(10)
Antiprotozoal activity assays
E. histolytica strain HM-1:IMSS, T. vaginalis strain GT-13 and G. lamblia strain 0989:IMSS were
used in this study. E. histolytica and T. vaginalis were grown in a medium named PEHPS, which is
and a acronym of its main components written in Spanish, casein peptone, liver and pancreas extract
pancreas and bovine serum [30] and G. lamblia in TYI-S-33 supplemented with bile [31]. All three
species were subcultured three times each week. Parasites used in the assays to determine drug
susceptibility were harvested when cultures had reached the middle of their respective logarithmic
growth phase.
Stock solutions
EA and GA was recovered from fermented material according to protocol reported by (Aguilera et al,
2009) The concentration of both was adjusted at 1 mg/mL. Metronidazole was used as antiprotozoal
control. ET’s and metronidazole were dissolved in double distilled water, and EA was dissolved in
dimethyl sulfoxide (DMSO). All stock solutions were stored at –20 °C until used. Immediately before
the assays, serial two-fold dilutions of the stock solutions were made in basal PEHP medium (without
serum). Fifty microliters of each solution was put into 1 mL glass screw-capped cylindrical vials with
a conical interior (vial micro storage Cat. No. 2070-00001, Bellco Biotechnology, Bellco Glass Inc.,
Vineland, NJ, USA). All vials were filled with 950 μl of a freshly prepared parasite suspension in
PEHP medium plus 10% bovine serum, with E. histolytica, T. vaginalis and G. lamblia at
concentrations of 2 × 104, 1 × 105 and 2 × 105 trophozoites/mL respectively. All vials were incubated
at 36 °C. Those vials containing E. histolytica were incubated for 72 h and those with G. lamblia or T.
vaginalis for 24 h. The vials were then chilled in ice water for 20 min, and the number of trophozoites
per milliliter in each tube was counted using a hemocytometer. The percentage of growth inhibition
with respect to untreated controls was then determined. The 50% inhibitory concentration (IC50) of
each drug was calculated by probit analysis [32]. Each drug was assayed in triplicate three times with
each protozoan species and the mean and 95% confidence limits calculated.
Data analysis
Kinetic study of EA/GA production was evaluated by triplicates and the kinetic parameters were
calculated. Antiprotozoal activity results were analyzed by ANOVA and a comparison of mean values
was carried out using a Tukey’s Test. IC50 values, were calculated and reported.
Results and discussion
In this study we evaluated the kinetic parameters of EA/GA production by Aspergillus niger GH1
using pomegranate husk/coffee pulp residues as supports of SSF. Antiprotozoal activity of EA and GA
produced and recovered were compared against ET’s and metronidazole.
Figure 1 shows the biomass production where it was observed a lag phase during the first 50 hours of
culture, After this time, the exponential growth was permanent reaching the maximal biomass
concentration (0.45gg-1) at 120 hours. A. niger GH1 grew faster and higher in comparison with the
same strain grown on creosote bush leaves (Aguilera-Carbo et al., 2008). In a similar study, Vatem
and Shetty (2002) reported the growth of Rhizopus oligosporus during the SSF of cranberry pomace
for antioxidant production, where the fungus reached its maximum growth after 240 h of culture.
Consumption of THP from PPH, to the first 24 hours we observed an increase on THP concentration
around 20%, can must to THP solubility on the culture media, More than 40 % (0.061 gg-1) of THP
were consumed at the 48 hours, while the following days polyphenols concentration remained
constant, this behavior could be due to the THP degradation and the generation and consumption of
polyphenols monomers. Figure 1 shows that the A. niger GH1 strain is capable to degrade the THP
from pomegranate husk powder, in a great manner mainly due to the material chemical composition.
THP content reported in pomegranate husk are monomeric ET’s, such punicalagin, vascalagin and
some glycosides of ellagic acid (with hexoses, pentoses, ramnoses, etc.) which are potential sources of
EA (Seeram et al. 2005).
The results for EA accumulation are showed in figure 1 where it is possible to observe that during the
fermentation time the maximal ellagic acid concentration was reached at 96 hours with an amount of
12.3 mgg-1 in comparison with 1.5 mgg-1 EA present at initial time (EA free present in pomegranate
husk powder). These results suggest that the ET’s fraction from THP, are degraded by enzymes from
A. niger GH1 strain.
After 96 hours of fermentation it was observed a decrement in the ellagic acid concentration, have it in
end of the culture 2 mgg-1.This behavior appear may be caused by interaction of EA with proteins,
some metals or well, to be consumed by the fungus, but does not exist reports about the ellagic acid
consumption by microorganism, It is important to consider that EA has been mentioned as inhibitor of
microbial growth (Aguilera’Carbo et al. 2007). Values found in the present research for EA production
are higher than those reported by Vattem and Shetty (2003) using Lentinus edodes fermenting
-1
cranberry pomace (320
in extracts with ethanol at time of fermentation of 120 hours in SSF).
0.5
0.014
0.45
0.012
0.01
0.35
0.3
0.008
0.25
0.006
0.2
0.15
0.004
Ellagic acid (geaDS-1 )
Biomass (gxgDS-1)
0.4
0.1
0.002
0.05
0
0
0.14
THP (gTHP /gDS
-1
0.12
0.1
0.08
0.06
0.04
0.02
0
0
50
100
150
200
Time (h)
Figure 1. Mycelial growth (●), EA production (■) and THP consumption (▲).
Important advances in EA production from ellagitannins have been reported in submerged co-cultures
by Huang et al. (2007a, b, c). Recently, high EA yields (24%) were obtained after optimization of the
co-culture of A. oryzae with Trichoderma reesei using acorn cups extract containing up to 62%
ellagitannins as substrate (Huang et al., 2007c). However, in SSC the information is limited to those
studies reported by Vattem & Shetty 2002; Vattem & Shetty 2003, using cranberry pomace as support
and source of ellagitannins with very low EA yields. Huang et al. (2007c) suggested for the first time,
the presence of ellagitannin acyl hydrolase as the enzyme responsible of the EA accumulation, which
indicates that a new tannase is involved in the biodegradation ellagitannins. Also, they reported that
such enzyme had an synergistic activity with other enzymes as xylanase and cellulase to enhance the
EA accumulation. However, further studies are needed to define the catalytic role and properties of
this new EHA or ellagitannin acyl hydrolase detected. Aguilera-Carbo et al. (2008) reported that the
SSF of A. niger GH1 using creosote bush ellagitannins impregnated in polyurethane foam could
remarkably enhance EA accumulation.
The kinetic parameters estimated under evaluated conditions are showed in Table 1. It is important to
note that a yield of 0.024 grams of EA per gram of biomass produced can be reached, however the
experimental value was 6 times lower than that value. SSF of pomegranate husk can yield 0.323 grams
of EA per gram of substrate, but the experimental value was of 0.299. These kinetic parameters
demonstrated that it is necessary to optimize the SSF bioprocess to enhance the EA production.
Table 1 kinetics parameters on EA production in SSF
Parameter
Yx/s
Yp/x
Yp/s
Qs
Qp
P
-4
1.29x10-4
8.51x10-5
Experimentals
0.378
0.024
0.323
0.021
1.92x10
Calculated
0.357
0.0049
0.299
0.279
4.87x10-4
Table 2 shows the results obtained with the antiprotozoal assay. In all cases, the IC50 values
calculated were within the reported ranges for the respective parasite species and antiprotozoal drugs.
EA shows the highest capacity to inhibit the paratise T.vaginalis, while ET’s shows the highest
capacity to inhibit to G. lambila and E. histolytica. EA and ET’s antiprotozoal activity values were
significantly higher than those obtained with the metronidazole (control). also shows inhibited the
parasites
This study reports for first time that EA and ET’s have higher antiprotozoal activity than other drugs
reported in literature, including emetine, timidazole, secnidazole, ornidazole and dimetridazole.
Values of IC50 (
Bioactive
EA
GA
ET’s
Metronidazole
for inhibition of three important protozoaric organisms
Entamoeba histolytica Trichomonas vaginalis
Giardia lamblia
0.096 (0.037 – 0.155)
0.074 ( 0.036 – 0.112)
0.086 (0.030 – 0.141)
0.423 (0.315 – 0.532)
0.362 (0.239 – 0.413)
0.296 (0.228 – 0.364)
0,049 (0.038 – 0.060)
0,091 (0,080 – 0,102)
0,057 (0.049 – 0.065)
0,711 (0,618 – 0,803)
1.04 (0.882 – 1.198)
0.512 (0.445 – 0.579)
Conclusions
Biotechnological EA/GA production is a interesting field where is necessary more studies on
exploration of a novel sources, byproducts carry out of optimization in EA production. The kinetic
evaluation of EA/GA production by Aspergillus niger GH1 using a pomegranate husk or coffee pulp
residues as supports allow to define kinetic parameters involved under production if this metabolite
were calculated and can be considerate for the optimization of EA/GA in SSF.
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