CORRELATION BETWEEN CONDENSED TANNIN AND FIBER CONTENTS OF IRRADIATED POMEGRANATE SEED

Pomegranate seeds were exposed to electron and gamma irradiations at doses of 5, 10, 15 and 20 kGy to study the relationships between their condensed tannin, NDF, ADF contents, in vitro rumen methane production and protozoa population. Data were analyzed by using the GLM procedure. Orthogonal contrast showed that gamma irradiation significantly increased the NDF content of pomegranate seed as compared to control (P<0.05), however electron beam irradiation did not significantly affect NDF%. The condensed tannin content of the pomegranate seed was significantly decreased by gamma and electron beam irradiation compared to control (P<0.05). Our results showed that there was a negative correlation between the methane production and NDF content of the gamma irradiated pomegranate seed. This means that the methane production of the gamma irradiated pomegranate seed decreased with increasing NDF%. Therefore, it seems that, in addition to tannin, the reduction of the methane production was affected by the cell wall content. Generally, the cell wall content may be more important than tannins in limiting microbial and in vitro fermentation.


Introduction
The methane reduction strategies should improve ruminant production efficiency and mitigate global warming.The CH 4 produced from enteric fermentation of ruminants is not only related to environmental problems, but is also associated with energy losses and, hence reductions in their retention and use of energy.Typically 6-8%, but up to 12%, of the gross energy (GE) in feed is converted to CH 4 during microbial digestion in the rumen (Johnson and Johnson, 1995).Therefore, decreasing CH 4 production from ruminants is desirable for reducing greenhouse gas emissions and increasing utilisation of the digested energy.Many studies have shown that forages containing tannins reduce CH 4 emissions from ruminants (Woodward et al., 2001(Woodward et al., , 2002;;Hess et al., 2003;Animut et al., 2008;Kamra et al., 2006;Waghorn et al., 2002).Plant secondary metabolites (PSM) have been suggested as effective alternatives to antibiotics to suppress rumen methanogenesis through their antimicrobial activity (Makkar et al., 2007;Jayanegara et al., 2009).There has been an increased interest in the use of plants and plant extracts to mitigate enteric ruminal CH 4 emissions (Woodward et al., 2001;Waghorn et al., 2002).
The huge diversity in tannin structures may explain their variable effects on methanogenesis and rumen function with observed responses depending on the source, type and level of tannin (Mueller-Harvey, 2006;Patra et al., 2006;Waghorn and McNabb, 2003).Several studies indicate that condensed tannins have anti-methanogenic activity, either by direct inhibition of methanogens or indirectly through the inhibition of protozoa (Animut et al., 2008;Bhatta et al., 2009;Jayanegara et al., 2009).An even larger pool of plant sources of tannins exists, and these are often tropical shrub legumes such as pomegranate seeds.Based on their structure and chemical properties, tannins are divided into hydrolysable tannins and condensed tannins (CT, or proanthocyanidines, which have no carbohydrate core and are derived by condensation of flavonoid precursors or polymers of flavonoids; Baker, 1999).Due to the lower risk of toxicity, research has focused on condensed tannins (CT) rather than on hydrolysable tannins.
Tannins also reduce ruminal CH 4 production when included either as temperate legumes (Waghorn et al., 2002) or as purified tannin extracts (Roth et al., 2002).The higher extractability of these compounds in irradiated samples was observed (Behgar et al., 2011).Gamma ray and electron-beam irradiations have been proven to be successful in detannification and improvement of overall qualities of food and agricultural commodities (El-Niely, 2007;Shawrang et al., 2011).Irradiation processing has been used as a method to inactivate these antinutritional factors, alternations in cellular compounds and release of bound or insoluble phenolics especially at high doses of irradiation (El-Niely, 2007;Behgar et al., 2011).Generally, irradiation results in the degradation of tannin (Variyar et al., 1998) and a change in its molecular conformation (Topuz and Ozdemir, 2004).
There are no reports on potential differences in effects of activities of electron and gamma on condensed tannins of the pomegranate seed (PS) and the effects on CH 4 production and ciliated protozoal populations.The correlation between methane production and protozoa population and the antimethanogenic potential of condensed tannins of the pomegranate seed has not yet been explored.This study was carried out to determine the effect of condensed tannin, NDF and ADF contents of the pomegranate seed on the in vitro methane suppression and protozoa population in order to determine their correlation.

Sample preparation and irradiation treatments
Pomegranate in normal conditions has the following elements: juice 62%, skin 24% and seeds 14%.In production line, the skins and the seeds load separately.Therefore, it is possible for us to load and ship one or both of them as commercial goods to the buyers.Traditionally, the grains are used in husbandry and some feeding farms.The extracted oil from these grains contains: The pomegranate seed (PS) was obtained from the Neyriz Green Farm pomegranate juice factory, in Fars, Iran, during the pomegranate harvest season and was air dried before it was used in this study.Irradiations of samples were done in Radiation Application Research School, Nuclear Science and Technology Research Institute.Gamma-ray (GR) irradiation was completed by using a cobalt-60 irradiator at 20 • C. Three-paper packages of samples were irradiated with total doses of 5, 10, 15 and 20 kGy in the presence of air.After irradiation and prior to sealing the plastic bags, samples were allowed to air equilibrate for 2 h.
Three polyethylene packages of samples were exposed to 10 MeV electron beam (EB) of a Rhodotron accelerator model Belgium), with various doses (5, 10, 15 and 20 kGy).All irradiations were performed at room temperature, with 4 mA beam of 10 MeV electrons.Regarding the low thickness of the sample packages, single-sided irradiation was used.The required doses were delivered to the samples by adjusting the conveyer speed when each of the sample batches passed under the beam.Condensed tannins (CT) were determined according to Galyean M. L. (1997) procedure and results were expressed as catechin equivalents (mg of CE/g of dry sample).

Protozoal population
Ruminal fluid from three Sanjabi fistulated rams (2-3-year old and ranged in weight from 40 to 50 kg) fed at maintenance requirements (two times a day, a diet of lucerne hay and whole barley, 70:30, DM basis) was diluted anaerobically in the anaerobic dilution solution.According to Dehority (1998), the media were placed in a culture tube containing 10 mL of medium and substrate added.The tube was closed anaerobically and incubated in a 39°C incubator.Whole serum bottle contents for protozoal counts were preserved by diluting with an equal volume of formalin solution (185 ml formaldehyde/l distilled water).Total numbers and generic composition of ciliate protozoa were determined according to the procedures described by Dehority (1984Dehority ( , 1993)).Protozoa were counted at a magnification of 100X with a 0.5-mm-square counting grid mounted into the eyepiece.A total of 50 grids evenly spaced over the entire chamber were counted.The chamber was then turned 180", another 50 grids were counted, and the two counts were averaged.Dilutions giving counts between 100 and 150 cells per 50 grids were the most satisfactory for counting.Where required, further dilutions were made with 30% glycerol.The protozoa numbers were calculated according to Kamra et al. (1991).

Measurement of methane production
For measuring methane production, after 24 hours of pomegranate seed incubation, 2 mL of NaOH (10 M) were introduced to estimate methane production following the method by Fievez et al. (2005).NaOH (10 M) was introduced into the incubated contents, thereby avoiding gas escape.Mixing of the contents with NaOH allowed absorption of CO 2 , with the gas volume remaining in the syringe considered to be CH 4 (Demeyer et al., 1988).Data were obtained on volume of gas and methane (CH 4 ) produced.Net methane and gas productions were calculated by the differences of the methane and gas in the test syringe and the corresponding blank; the methane concentration was determined according to Jayanegara et al. (2009):

Methane concentration = Statistical analyses
Data were analyzed by analysis of variance using the general linear model procedure.All statistical analysis was carried out using SAS software (SAS v. 9.1; Statistical Analysis System).Comparison of irradiation groups and control and between ionizing radiations (gamma and electron) was conducted by orthogonal comparison.The least significant difference (LSD) was used to compare and estimate the differences between irradiation treatment dose and the un-irradiated pomegranate seed (control).
Correlation between condensed tannin and fiber contents of irradiated pomegranate seed 347

Results and Discussion
Table 1 shows the effects of electron beam and gamma ray irradiations of the pomegranate seed on the fiber content, condensed tannin content and methane production.Orthogonal comparison showed the significant effects of irradiation on the condensed tannin content of the pomegranate seed (P<0.01).Gamma radiation maybe affected the NDF and condensed tannin content of the pomegranate seed and its methane production significantly.The effects of gamma and electron radiations between low and upper dose of irradiation on pomegranate seed condensed tannin were significant (P<0.01) and there was a difference between gamma irradiation and electron irradiation (P<0.05).The levels of the NDF, ADF, condensed tannin, methane production of the unirradiated and irradiated pomegranate seed are shown in Table 2.The NDF contents of the pomegranate seed at 10 kGy of gamma and electron beam irradiations were 68.30 and 70.28% respectively, which significantly increased compared to control (P<0.05).The methane production at doses of 10, 15 kGy of gamma irradiated and 15 kGy of the electron irradiated pomegranate seed was decreased significantly as compared to control (P<0.05).Irradiation treatments significantly decreased condensed tannin (P<0.01).As shown in Table 2, decreases in condensed tannin were dose dependent, and it means that higher doses of gamma and electron beam radiation caused higher reduction of condensed tannin content.Orthogonal contrast showed that gamma irradiation significantly increased the NDF content of the pomegranate seed as compared to control (P<0.05).Electron beam irradiation did not affect the NDF content.Generally, irradiation treatment has an effect on the structure of cellulose present in agricultural cellulosic raw materials (Dela Rosa et al., 1983).There is some difference in the effects of irradiation on the fiber content of feeds.Electron beam irradiation in high doses (50, 100 and 150 kGy) decreased NDF and ADF contents of soybean and cotton seed meal (Tahan et al., 2012).But Ebrahimi-Mahmoudabad and Taghinejad-Roudbaneh (2011) reported that electron beam irradiation at doses of 15, 30 and 45 kGy had no significant effect on the NDF content of whole cotton seed, soybean and canola seeds.Ebrahimi et al. (2009) found that gamma radiation had no effect on the chemical composition and fiber content of feeds.In our study, irradiation at a dose of 10 kGy significantly increased the NDF content of PS as compared to control (P<0.05).Similarly, Khosravi et al. (2012) reported that electron beam irradiation increased the ADF content of the pomegranate seed, and did not affect the NDF content.Tahan et al. (2012) revealed that the differences could be due to different doses and kinds of irradiation and laboratory circumstances in cell wall analysis.In addition, free radical formation, depolymerization (chain-scission) or cross-linking of cellulose and glucose chain (Polvi and Nordlund, 2014;Khan et al., 2006;Pekel et al., 2004) had important influences.The efficiency of these types Correlation between condensed tannin and fiber contents of irradiated pomegranate seed 349 of reactions depends mainly on the polymer structure and radiation dose (Charlesby, 1981).Five genera of protozoa (Diplodinium, Entodinium, Dasytricha, Isotricha and Ophryoscolex) were observed (Tables 3 and 4).Total population and five genera of protozoa were not affected by irradiation of the pomegranate seed.There were some differences between the effect of gamma radiation and electron radiation on the number of Diplodinium and Ophryoscolex.The number of Diplodinium and Ophryoscolex at low dose (5 and 10 kGy) of GR was significantly higher than control (P<0.05).
The condensed tannin content of the pomegranate seed was significantly decreased by gamma and electron beam irradiations compared to control (P<0.01).There was a difference between the effects of gamma and electron radiations on reduction of condensed tannin (P<0.05).Decreases in condensed tannin were dose dependent, where the higher doses of gamma and electron beam caused higher condensed tannin reduction.Reduction of tannin by irradiation in this study was consistent with some previous studies using gamma irradiation (El-Niely, 2007;Behgar et al., 2011;De Toledo et al., 2007) and electron beam irradiation (Bhat and Sridhar, 2008;Shawrang et al., 2011).Generally, irradiation resulted in the degradation of tannin (Variyar et al., 1998) and a change in its molecular conformation (Topuz and Ozdemir, 2004).Gamma irradiation and electron beam irradiation have been proven to be successful in detanninification and improvement of overall qualities of food and agricultural commodities (El-Niely, 2007;Shawrang et al., 2011).The mechanism of gamma action on tannin has been related to generation of the hydroxyl and superoxide anion radicals (Riley, 1994) which indiscriminately attack neighboring molecules, but the mode of electron beam action on tannins has not been demonstrated.In this study, the decrease of methane production in the gamma irradiated pomegranate seed was significant compared to control (P<0.05).But, the electron beam irradiation did not have a significant effect on reduction of CH 4 production.It could be due to increasing the NDF content of the gamma irradiated pomegranate seed as compared to control.The electron beam irradiation did not affect the NDF content.Our results showed that there was a negative correlation between the methane production and NDF content of the gamma irradiated pomegranate seed.Generally, the negative correlation of the cell wall content and condensed tannins with gas production parameters was reported by several studies (Kamalak et al., 2004;Ndlovu and Nherera, 1997;Nsahlai et al., 1994).But, there are some differences in the effect of irradiation on chemical composition and gas characteristics.Pellikaan et al. (2011) reported that the addition of PEG (Poly Ethylene Glycol) to chestnut, tara and myrabolan tannins only caused a modest increase in gas and CH 4 production, whereas PEG inclusion with tea tannins decreased CH 4 and gas production.Larbi et al. (1998) reported a weak relationship between condensed tannins and gas production of tree leaves during wet and dry seasons in West Africa.A possible reason could be differences in the nature of tannins between browse species (Jackson et al., 1996).Some authors have suggested that the molecular weight of condensed tannins has a direct effect on CH 4 production with the impact more pronounced at higher molecular weight (Huang et al., 2011).In addition, cell wall contents may be more important than tannins in limiting in vitro fermentation (Ndlovu and Nherera, 1997).Therefore, it seems that, in addition to tannin, the reduction of CH 4 production was affected by the cell wall content.
The correlation coefficients between experimental parameters and methane production of the pomegranate seed are presented in Table 5.There was a negative correlation (r= -0.67, P<0.05) between the methane production and NDF content of the gamma irradiated pomegranate seed.The correlation coefficients between the NDF content and electron beam irradiated PS were not significant.There were no correlations between NDF, ADF and condensed tannin contents of the irradiated pomegranate seed and protozoa population.The total population and five genera of protozoa were not affected by irradiation of the pomegranate seed.There were some differences between the effect of gamma radiation and electron radiation on the number of Diplodinium and Ophryoscolex.The number of Diplodinium and Ophryoscolex at low dose (5 and 10 kGy) of GR was significantly higher than control (P<0.05).There were no correlations between NDF, ADF, condensed tannin contents of a pomegranate seed and protozoa population (Table 6).Research on the influence of plant secondary metabolites (PSM) on ruminal protozoa population was not consistent, i.e., no effect (Benchaar et al., 2008), decreases (Hess et al., 2004;Nasri and Ben Salem, 2012) or increases (Raghuvansi et al., 2007).Such discrepancies may be due to the diet type, animal variability, sampling methods (Yanez Ruiz et al., 2004), level and type of plant metabolites (Patra and Saxena, 2011) and the variability in the adaptation of the protozoa to plant secondary metabolites, or the previous experience of the animal to plant secondary metabolites (Abreu et al., 2004;Wallace et al., 2002).

Conclusion
Orthogonal contrast showed that gamma irradiation significantly increased the NDF content of the pomegranate seed as compared to control (P<0.05),however electron beam irradiation did not significantly affect NDF%.Results showed that irradiation treatments decreased condensed tannin of the pomegranate seed.Generally, irradiation resulted in the degradation of tannin and a change in its molecular conformation.
The decrease of methane production in the gamma irradiated pomegranate seed was significant compared to control (P<0.05).But, electron beam irradiation did not have a significant effect on reduction of CH 4 production.It could be due to increasing of the NDF content of the gamma irradiated pomegranate seed as compared to control.However, electron beam irradiation did not affect the NDF content.Our results showed that there was a negative correlation between the methane production and NDF content of the gamma irradiated pomegranate seed.In addition, cell wall contents may be more important than tannins in limiting in vitro fermentation.Therefore, it seems that in addition to tannin, the reduction of CH 4 production was affected by the cell wall content.The total population and five genera of protozoa were not affected by irradiation of the pomegranate seed.Furthermore, in this study, it was also established that condensed tannin, NDF and ADF contents of the pomegranate seed and methanogenesis were not essentially related to the protozoa population.

Figure 1 .
Figure 1.Total gas volume and and methane (CH 4 ) production of pomegranate seeds before and after irradiation (GR: gamma ray, EB: electron beam).

Table 1 .
Orthogonal contrast (mean square) of NDF, ADF, condensed tannin content and methane production of the pomegranate seed between different doses of irradiation.

Table 2 .
Effects of irradiation on the NDF, ADF and condensed tannin content and on methane production.

Table 3 .
Orthogonal contrast of protozoa concentration after incubation of the irradiated pomegranate seed.

Table 4 .
Effects of the irradiated pomegranate seed on ruminal protozoa concentration after incubation.

Table 5 .
Correlation (r)between experimental parameters and methane production of the pomegranate seed.