Ionizing Radiation of Pomegranate (Punica granatum ...

Jordan Journal of Agricultural Sciences, Volume 12, No.2 2016

Ionizing Radiation of Pomegranate (Punica granatum L.) Peels Influences on in Vitro Gas Production and Dry Matter Digestibility M. Zarei1, F. Kafilzadeh1, P. Shawrang2

ABSTRACT The effect of irradiation on pomegranate (Punica granatum L.) peel (PP) was evaluated. Irradiation had no effect on chemical composition of PP. The results showed that gamma and electron beam (EB) irradiation significantly decreased (P0.05). The effect of irradiation on in vitro dry matter digestibility at doses of 10 and 15 kGy EB was not significant (P>0.05). Irradiation also had no effect on in vitro organic matter digestibility (P>0.05). Irradiation did not change (P>0.05) partitioning factor, gas volume at 24 hour and metabolizable energy. This study showed that ionizing radiation processing can be used as an efficient method in eliminates anti-nutritional factors without any adverse effects on nutritional value of pomegranate peel compared to other methods. Keywords: Condensed tannin, Digestibility, Irradiation, Pomegranate peel.

about 3% of the weight of the fruit, the juice about 30%

INTRODUCTION

of the fruit weight; and the peel which also include the Global production of trees and shrub foliage and

interior network membranes. These products have led to

as

production of high quantities of pomegranate byproduct

pomegranate (Punica granatum L.) has greatly increased

biomass. Pomegranate (Punica granatum L.) peel (PP) is

in recent years, due to recognition of the health-

important in animal production because they do not

promoting potential (antioxidant, antimicrobial, anti-

compete with human food. Jami et al. (2012) and

inflammatory, anticancer and other biological activities)

Shabtay et al. (2012) noted a significant increase in

of various components of this fruit to its human

the digestibility of dry matter, crude protein (CP), and

consumers (Aviram et al., 2008, Prakash and Prakash,

neutral detergent fiber, as well as milk and energy-

2011). The fruit composed of three parts: the seeds,

corrected milk yields in cows fed 4% PP extract

different

agro-industrial

by-products

such

supplement. Shabtay et al. (2008) demonstrated that PP 1

Department of Animal Sciences, Razi University, Kermanshah, Iran 2 Agricultural Research School, Nuclear Science and Technology Research Institute, Atomic Energy Organization, Karaj, Iran  [email protected] Received on 9/4/2015 and Accepted for Publication on 9/7/2015.

intake of up to 20% of the total feed intake does not possess deleterious or positive effects on fattening ration intake of feedlot calves. But, Oliveira et al. (2010) found that feeding a pomegranate extract to young calves for the first 70 d of life decreased intake of grains and whole tract digestibility of fat and CP, likely because of its high tannin content. Previous studies demonstrated higher

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© 2016 DAR Publishers/The University of Jordan. All Rights Reserved.

Ionizing Radiation of…

M. Zarei, F. Kafilzadeh, P. Shawrang

antioxidant capacity of the peels relative to other part

(EB) irradiation exposed to PP at doses of 5, 10, 15 and

pomegranate (Li et al., 2006; Tzulker et al., 2007),

20 kGy. Gamma-irradiation was completed by using a

mainly due to water-soluble polyphenols, anthocyanins

cobalt-60 irradiator at 20ºC. The dose rate determined by

and condensed tannins (Gil et al., 2000; Tzulker et al.,

Fricke dosimetry (Holm and Berry, 1970) was 0.36 Gy/s.

2007). General effects of tannins, for example, decrease

Three-paper packages of samples were irradiated to total

in vivo protein utilization, decrease growth, decrease

doses of 5, 10, 15 and 20 kGy in the presence of air. After

palatability and feed intake or decrease in various

irradiation and prior to sealing the plastic bags, samples

enzyme activities, and digestion kinetics (Makkar, 2003)

were allowed to air equilibrate for 2 h. Three poly-ethylene packages of samples were

and adversely effects rumen metabolism (Kumar and

exposed to 10 MeV EB of a Rhodotron accelerator

Singh, 1984).

model

Therefore, removal of these undesirable components

TT-200

(IBA

Co.,

Belgium),

Radiation

is essential to improve the nutritional quality of

Applications Research School (of Atomic Energy

pomegranate seeds and effectively utilize its potential as

Organization of Iran) to various doses (5, 10, 15 and 20

animal feed. There is scant information on its nutritive

kGy). All irradiations were performed at room

value for ruminants (Feizi et al., 2005; Shabtay et al.,

temperature in air, with 4 mA beam of 10 MeV

2008; Modarresi et al., 2010; Mirzaei-Aghsaghali et al.,

electrons. Regarding the low thickness of the samples

2011). The methods (Chen et al., 1995, Duodu et al.,

packages, single sided irradiation has been used. The

1999, Parker et al., 1999) used to deactivate the anti-

required doses were delivered to the samples by

nutrients not necessarily reduce or completely eliminate

adjusting the conveyer speed when each of the sample

these compounds; instead some methods reduce the

batches passed under the beam.

nutritive value. There is no information concerning

2.2. Chemical analysis Dry matter (DM) of PP was determined by drying at

effects of gamma radiation and electron beam on º

nutritive value of PP. Therefore, the major aim of the

60 C for 48 h. After drying, the samples were ground

present study was to evaluate and comparison the

through a 1 mm screen (Wiley mill, Arthur H. Thomas,

impacts of gamma radiation and electron beam on the

Philadelphia, PA), and DM, crud protein (CP) , ether

nutritional and antinutritional components, in vitro

extract (EE) and ash were analyzed according to

digestibility and rumen fermentation of PP.

procedures described by AOAC (1995). Non fiberus

2. MATERIALS AND METHODS

carbohydrate (NFC), neutral detergent fiber (NDF) and

2.1.

Samples

preparation

and

acid detergent fiber (ADF) were analyzed according to

irradiation

Van Soest et al. (1991). Condensed tannins (CT) were

treatments Pomegranate peel (PP) was obtained from the Neyriz

determined according to Galyean (1997) procedure and

Green Farm pomegranate Juice Factory in Fars, Iran.

results are expressed as catechin equivalents (mg of

Before the initiation of the study, PP was air dried.

CE/g of dry sample).

Irradiations

of

samples

were

done

in

Radiation

2.3. In vitro gas production and digestibility

Applications Research School, Nuclear Science and Technology

Research

Institute,

Atomic

Three ruminally fistulated rams, used as rumen fluid

Energy

donors, were fed at 8:15 and 17:15 h daily a diet

Organization of Iran. Gamma ray (GR) and electron beam

containing lucerne hay and barley grain (70:30, DM

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basis) at the maintenance energy requirements. Rumen

general linear model procedure. All statistical analysis

fluid was collected before the morning feeding and

was carried out using SAS software (SAS v. 9.1;

strained into a pre-warmed thermos flask and then

Statistical Analysis System). Comparison of irradiation

filtered and flushed with CO2. About 125 mg of each

groups and control and between ionizing radiation

sample was weighed into tubes kept at approximately

(gamma and electron) was conducted by orthogonal

39ºC. Each sample was incubated in three replicates.

comparison. The least significant difference (LSD) was

Fifteen mL of buffered rumen fluid (in the proportion of

used to compare and estimate the differences between

20% rumen fluid + 80% buffer solution) was

irradiation treatments dose and untreated PP (control).

anaerobically dispensed in each tube. All tubes were

3. RESULTS

crimped and placed in a shaking incubator. According to

3.1. Effects on chemical composition

Theodorou et al. (1994) the pressure of gas produced in

Irradiation had no effect on DM, EE, CP, OM, ADF,

each tube was recorded by a pressure transducer

NDF and NFC (Table 1). Orthogonal comparisons

(Manometer Digital testo 512) at 2, 4, 6, 8, 12, 24, 48,

indicated that both GR and EB irradiation significantly

72 and 96 h after the start of the incubation. To estimate

decreased CT content of PP as compared to control.

the kinetics of gas production, data on cumulative gas

There was no difference between GR and EB irradiation.

volume produced were fitted using the model of Orskov

Gamma Ray and EB irradiation had no effect on NDF% with the exception of GR at the dose of 20 kGy

and McDonald (1979) as follows: −ct

Y = b(1 − e )

(32.27%), which significantly increased as compared to

Where b = the potential extent of gas production, c =

control (Table 2). Gamma ray and EB irradiation at the

the gas production rate constant for the insoluble fraction

doses of 5, 10, 15 and 20 kGy significantly decreased CT

(b), t = incubation time, y=gas produced at time “t”.

compared to control by 11, 38, 81 and 98% for GR and by

In a separate run of gas production, the method of

4, 46, 76 and 89% for EB respectively. In other words, the

Blummel et al. (1997) was adopted to determine the

decrease in CT was dose dependent. The decrease in the

partitioning factor (PF). The PF is defined as the ratio of

level of the CT was accompanied with an increase in

substrate truly degraded in vitro (mg) to the volume of

irradiation dose. Maximum and minimum level of CT

gas (ml) produced by it. Two-step digestion technique

content of irradiated PP observed at a doses of 5 kGy and

(Tilly and Terry, 1963) was used to determine in vitro

20 kGy EB (5.58 and 0.07 g/100 g DM), respectively.

digestibility of untreated and irradiated pomegranate peel. The ME contents of gas production (GP) was

3.2. In vitro study

calculated using equations of Menke et al. (1979) as

3.2.1 Gas production parameters

ME (MJ/kg DM) = 2.20 + 0.136 × GP + 0.057 × CP +

Cumulative gas production of radiation treatments

0.0029 × CP

2

was presented as gas production curves (Fig.1) and

where GP is the net gas production (ml) and CP is Crud

estimated variables are given in Table 3. Orthogonal

protein.

contrast showed that irradiation did not change gas production variables, but there were differences between

2.4. Statistical analyses

the effects of GR and EB irradiation at low doses of

Data were analyzed by analysis of variance using the

irradiation on gas production potential (b). Irradiation

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2008) and on the effect of gamma irradiation on

did not affect rate constant of gas production (c). As shown in Table 4 the increase of gas production

chemical composition of rapeseed and soybean seed

potential (b) was not significant. Irradiation did not

(Ebrahimi et al., 2009; Taghinejad et al., 2009).

affect gas production rate constants (c). There was no

According to Farag (1998), Ebrahimi et al. (2009),

different between GR and EB irradiation and fraction c

Taghinejad et al. (2009) and Shawrang et al. (2011),

ranged from 0.0894 to 0.1109 (ml/h).

irradiation at the small doses were not sufficient to change chemical composition.

3.2.2 In vitro digestibility In vitro DM and OM digestibility of irradiated and

Gamma ray and EB irradiation had no significantly

untreated PP are presented in Table 3 and 4. Generally,

effect on NDF content with the exception of 20 kGy GR

orthogonal contrast showed that gamma ray and EB

(32.27%), which significantly increased as compared

irradiation had no effects on DM and OM digestibility of

control. Khosravi et al. (2012) reported that EB

PP. But electron irradiation at a dose of 5 kGy decreased

irradiation increased ADF content of pomegranate seed

DM digestibility of PP as compared to control. There

without effects on NDF content of it. Electron beam

were no differences between the DM and OM

irradiation in high doses (50, 100 and 150 kGy) reduced

digestibility of GR and EB irradiated PP (Table 3).

NDF and ADF content of soybean and cotton seed meal (Tahan et al., 2012). However, Ebrahimi-Mahmoudabad

3.2.3 Partitioning factor and metabolizable energy

(2011) reported that EB-irradiation at doses of 15, 30

Irradiation did not change PF, gas volume at 24 hour

and 45 kGy had no significant effect on NDF content of

and ME (Table 5). The effects of irradiation on

whole cottonseed, soybean and canola seeds. Tahan et

partitioning factor and gas production volume at 24 hour

al. (2012) revealed that the differences could be due to

incubation (GV24) were significantly differed between low

different doses and kind of irradiation and laboratory

and upper doses of gamma radiation and between GR and

circumstance in cell wall analysis. However it seems that

EB irradiation in low doses. Table 6 showed that the

besides of this, free radicals formation, depolymerization

increase of ME at a dose of 20 kGy GR as same as all

(chain-scission) or cross-linking of cellulose and glucose

doses of EB were not significant as compared to control.

chain (Polvi and Nordlund, 2014, Khan et al., 2006,

Means of PF was not different between irradiated

Pekel et al., 2004), had important influences. The

and untreated PP (mean average 9.17 mg DM truly

radiation-induced reactions in the macromolecules of the

degraded/ml gas produced in 24 h). The Correlation

cellulose materials are known to be initiated through

coefficient of chemical composition and experimental

rapid localization of the absorbed energy within the

parameters

molecules to produce long- and short-lived radicals.

of

gamma

and

electron

irradiated

Dela Rosa et al. (1983) found that radiation treatment

pomegranate peel are presented in Table 4 and 5. 4. DISCUSSION

has an effect on the structure of cellulose present in

4.1. Effects on chemical composition

agricultural cellulosic raw materials. The cellulose

Irradiation treatments had no effect on DM, EE, CP,

contains carbon, oxygen, and hydrogen atoms, and each

OM, ADF and NFC of PP. Result was agreed with

atom of this unit has practically equal probability of

previous work on the effect of EB irradiation on

being ionized to take part in chemical reactions such as

proximate composition of lotus seed (Bhat and Sridhar,

chain scission, cross-linking, and so forth. The efficiency

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of these two types of reactions depends mainly on the

effect of ionizing radiation on phenolic compounds may

polymer structure and radiation dose (Charlesby, 1981).

be partly due to the higher extractability of these

Although GR and EB irradiation did not significantly

compounds in irradiated samples as a result of

affect NDF%, there were some differences between low

alternations in cellular compounds and release of bound

(5 and 10 kGy GR) and upper doses (15 and 20 kGy) of

or insoluble phenolic especially at high doses of

GR irradiation and between GR and EB in low and

irradiation. Mechanism of gamma action on tannin has

upper doses. Ebrahimi-Mahmoudabad and Taghinejad-

been related to generation of the hydroxyl and

Roudbaneh (2011) found that GR irradiation had no

superoxide anion radicals (Riley, 1994), but mode of EB

effect on chemical composition and fiber content of

action on tannins has not been demonstrated. Further

feeds. So our result showed that irradiation at low doses

research studies are needed to clear its mode of

could not change chemical composition of PP. Further

reduction in tannin contents.

studies are needed to evaluate the definite effect of

4.2. In vitro study

radiation on cell wall content.

4.2.1 Gas production profiles

The results indicated that GR and EB irradiation

Orthogonal contrasts demonstrated that irradiation

significantly decreased CT of PP as compared to control.

did not affect gas production parameters, but there were

There was no difference between GR and EB radiation

differences between GR and EB radiation effects in low

in CT reduction. With increasing dose of GR and EB

doses on potential extent of gas production (b). The gas

irradiation, CT was significantly decreased compared to

production potential (b), is associated with degradability

control. So decrease in CT was dose dependent.

of feed (Khazaal et al., 1995). The lower values obtained

Maximum and minimum level of CT content of PP

for gas production parameters in 5 kGy GR as compared

observed at a doses of 5 kGy and 20 kGy EB (5.58 and

to 20 kGy GR and all doses of EB radiation, might

0.07 g/100 g dry matter) respectively. There is a

indicate a better nutrient availability for rumen

difference in the effect of ionizing radiation on phenolic

microorganisms in these doses in relation to 5 kGy GR.

compounds and tannins. Some reports (Bhat et al., 2007;

In this study, irradiation did not affect rate constant of

Siddhuraju et al., 2002) indicated that gamma irradiation

gas production (c). Fraction c ranged from 0.0894 to

increased phenolic and others (Shawrang et al., 2011;

0.1109, which is agreed with Behgar et al. (2011) who

Abu-Tarboush, 1998; behgar et al., 2011; Villavicencio

reported that irradiation did not change the rate of gas

et al., 2000) reported that it decreased tannin in foods or

production of pistachio hull. No data were found by

feeds. Mali et al. (2011) showed that total phenolic

authors

content of gamma irradiated PP at a dose of 10.0 kGy

detannification and in vitro gas production of PP.

significantly increased as compared to control (16.80 ±

Generally, the negative correlation of CT and cell wall

0.15 g GE/100 g DW). Stajner et al. (2007) reported that

contents with gas parameters was reported by several

tannin content of soybean seeds increased (21.6%) by

studies (Ndlovy and Nherera, 1997, Nsahlai et al., 1994).

gamma irradiation at the dose of 1 kGy. Generally

However, this result is not consistent with the effects of

irradiation resulted in the degradation of tannin (Variyar

cell wall content, the significantly negative correlation of

et al., 1998) and a change in its molecular conformation

CT and gas production parameters was observed in this

(Topuz and Ozdemir, 2004). The differences in the

study (Table 7 and 8). Recently Behgar et al. (2011)

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regarding

the

effect

of

irradiation

on



indicated that gamma radiation at the doses of 30, 40 and

nutrients digestibility vary with the concentration of

60 kGy decreased gas production potential (b) of

these metabolites, chemical structure and with the source

pistachio hull compared to the control. They reported

of the plant used (Abarghuei et al., 2010).

that decrease in fraction (b) might be related to increase of total phenolic content, which are toxic and suppressed

4.2.3 Partitioning factor and metabolizable energy

the growth of microorganism in the rumen (Chesson et

Irradiation did not change PF, gas volume at 24 hour

al., 1982). In contrast, Larbi et al. (1998) reported a

and ME. Similarly, ME did not increase at a dose of 20

weak relationship between CT and gas production of tree

KGy GR and in all doses of EB and the means of

leaves during wet and dry season in West Africa. A

metabolizable energy was 7.20 (MJ/kg DM). Mirzaei-

possible reason for these anomalies could be differences

Aghsaghali et al. (2011) and Taher-Maddah et al. (2012)

in the nature of tannins (Jackson et al., 1996) and

reported that amounts of ME of dried PP were 8.85 and

chemical compositions. Results indicated that effects of

8.61 MJ/kg DM, respectively. The effect of irradiation on PF and gas production

tannin on gas production are complex and vary among

volume at 24 hour incubation (GV24) were significantly

browse species.

different between lower and upper doses of GR and 4.2.2 In vitro digestibility

between GR and EB radiation in low doses. Means of PF

Generally irradiation had no effects on DM and OM

was not different between irradiated and untreated PP

digestibility of PP. But, the DM digestibility of electron

(mean average 9.17 mg DM truly degraded/ml gas

irradiated PP at a dose of 5 kGy decreased as compared

produced in 24 h). Values of PF are above theoretical

to control. There were no differences between GR and

levels (2.75–4.41) for conventional feeds (Blummel et

EB irradiation. The digestibility value of untreated PP

al., 1997). This is likely due to material (i.e., tannins)

was higher than that reported by Taher-Maddah (2012)

that appears to be degraded but does not contribute to

and Mirzaei-Aghsaghali et al. (2011). Discrepancies in

production of gas through fermentation. Partitioning

digestibility values may be due to differences in methods

factor increased with irradiation but it was not

of estimating digestibility and the variety of peel.

significant compared to control. This is probably due to

Apparent total-tract digestibility of nutrients in four

both depolymerization of tannins from feed and

Holstein cows were not affected by PP extract (PPE)

inhibition of cell solubles which contributing to DM

supplementation (0, 400, 800 and 1200 ml PPE/cow per

loss, but not to gas production, because gas production is

day)

(2013).

inhibited by tannins (Makkar, 2004). Similarly, Baba et

Accordingly, Oliveira et al. (2010) found that feeding a

al. (2002) observed above theoretically possible PF

pomegranate extract containing 16.9% gallic acid

values when evaluating tanniniferous forages.

as

reported

by

Abarghuei

et

al.

equivalent (in 0, 5 and 10 gr per day) to young calves

5. CONCLUSION

had no influence DM, OM, or starch digestibility during

Irradiation had no effect on chemical composition,

their first 70 d of life. In contrast, Jami et al. (2012)

but they could change NDF content of PP. The present

showed that using 1–4% PP extract improved DM, CP,

study reveals that gamma-ray and electron beam

and NDFom digestibility in dairy cows. These results

irradiation has the potential to reduce anti-nutritional

indicated that the effects of secondary metabolites on

factors without any adverse effects on nutritional value

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of pomegranate peel. Decrease in condensed tannin

maintaining the nutritive value of pomegranate peel with

content of pomegranate peel was dose dependent. The

reducing anti nutritional factors.

increase of gas production potential (b) was not

ACKNOWLEDGMENTS

significant as compared to control. The increase of dry

Authors are grateful to the Nuclear Agriculture

matter digestibility at doses of 10 and 15 KGy electron

Research School, Nuclear

Science and Technology

beam was not significant too. The digestibility value of

Research Institute, Atomic Energy Organization of Iran

non-irradiated pomegranate peel was higher than other

for the irradiation operations. The authors are grateful to

reports. Irradiation did not change the in vitro OMD and

Neyriz Green Farm pomegranate juice factory, Mr.

partitioning factor. In conclusion, irradiation as a

Haem for his assistance to preparing pomegranate peel

physical method of preservation proved its efficacy in

samples.

Table 1. Orthogonal compare means of pomegranate peel before and after irradiation (Means Square) Treatments

df

OM

CP

EE

NDF

ADF

NFC

CT

Irradiation vs. control

1

4.87

0.001

0.02

0.006

1.86

6.31

19.14**

GR vs. control

1

4.78

0.00

0.02

0.03

0.07

7.98

18.17**

5 and 10 GR vs. control

1

2.76

0.006

0.02

18.67

1.11

37.08

2.83**

15 and 20 GR vs. control

1

4.96

0.003

0.01

15.66

3.04

1.42

37.2**

5 and 10 vs. 15 and 20 GR

1

0.45

0.02

0.001

102.81**

8.77

53.07

29.26**

EB vs. control

1

3.81

0.005

0.01

0.002

8.50

3.13

16.30**

5 and 10 EB vs. control

1

2.0

0.19

0.01

7.34

20.35

0.01

3.04**

15 and 20 EB vs. control

1

4.69

0.09

0.01

6.84

0.65

9.64

31.68**

5 and 10 vs. 15 and 20 EB

1

0.52

0.83

0.00

43.55*

20.54

11.75

22.64**

GR vs. EB

1

0.18

0.01

0.001

0.15

5 and 10 GR vs. 5 and 10 EB 15 and 20 GR vs. 15 and 20 EB

1 1

0.07 0.11

0.40 0.20

0.002 0.00

74.17

20.52

1.91

0.12

**

10.84

43.82

0.005

*

9.79

18.49

0.33*

64.80

GR: Gamma Ray, EB: Elecreon Beam, df: degree of freedom, OM: Organic Matter, CP: Crud Protein, EE: Ether Extract, NDF: Neutral Detergent Fiber, ADF: Acid detergent Fiber, NFC: Non Fibrus Carbohydrat, CT: Condense Tannin (mg of CE/g of dry sample); *

P