MOJ ISSN: 2374-6920 MOJPB

Proteomics & Bioinformatics
Review Article
Volume 5 Issue 1 - 2017
Evaluation of Pomegranate Pomace Supplemented with Different Levels of Polyethylene Glycol using In Vitro Gas Production Technique
Maghsoud Besharati* and Einollah Abdi
Department of Animal Science, University of Tabriz, Iran
Received: October 08, 2016 | Published: February 06, 2017
*Corresponding author: Maghsoud Besharati, Department of Animal Science, Ahar Faculty of Agriculture and Natural Resources, University of Tabriz, Ahar, Iran, Email:
Citation: Besharati M, Abdi E (2017) Evaluation of Pomegranate Pomace Supplemented with Different Levels of Polyethylene Glycol using In Vitro Gas Production Technique. MOJ Proteomics Bioinform 5(1): 00150. DOI: 10.15406/mojpb.2017.05.00150


The object of this study was to examine the chemical composition, including the tannin content of pomegranate pomace supplemented withdifferent levels of tannin-binding Agent (Polyethylene Glycol) and gas production amount using in vitro gas production technique. The treatment contained 0, 75, 150, 300 and 450mg PEG per each serum bottle, respectively. Approximately 300 mg of dried and ground (2mm) Pomegranate pomace were weighed and placed into serum bottles. CP, ADF, NDF, EE, ASH, TP and TT contents in pomegranate pomace were 8.7%, 27.6%, 32.3%, 1.7%, 9.2%, 5.4% and 4.6%, respectively. At the 2 h incubation times, the gas production amount of PP, PP + 75 mg PEG, PP + 150 mg PEG, PP + 300 mg PEG and PP + 450mg PEG were 29.83, 32.89, 23.26, 25.83 and 19.37 ml/g DM. At the first incubation times (2 and 4h), the PP + 75mg PEG treatments had the highest in vitro gas production amount within treatments (P<0.05). At all incubation times PP+75mg PEG treatment had the highest in vitro gas production amount within treatments (P<0.05). The addition of PEG at levels 75 and 450mg increased the in vitro gas production amount.

Keywords: By-product; In vitro gas production; Polyethylene glycol; Pomegranate pomace; Tannin


BPF: By-Product Feedstuffs; PVP: Poly Vinyl Pyrrolidone; PEG: Polyethylene Glycol; PP: Pomegranate Pomace; PG: Polyethylene Glycol; DM: Dry Matter; CP: Crude Protein; EE: Ether Extract; ADF: Acid Detergent Fiber; NDF: Neutral Detergent Fibre; TP: Total Phenol; TT: Total Tannins


By-product feedstuffs (BPF) obtained from the processing of commercial crops and the food processing industry [1]. Increased disposal costs in many parts of the world lead to increase interest in BPF as alternative feeds for ruminants [2]. Increasing agricultural industrial factories for producing pomegranate juice leads to production of pomegranate peel and the annual production of this by-product approximately 120,000 metric tons in Iran [3]. The pomegranate fruit consists of seeds, the juice and the peels [4].

A major restriction to increasing livestock productivity in some developing countries is the shortage and fluctuating quantity and quality of the year-round supply of conventional feedstuffs. These countries experience serious shortages in animal feeds of the conventional type. In order to meet high demand for livestock products and to fulfill the future hopes of feeding the millions and safeguarding their food security, the better utilization of non-conventional feed resources which do not compete with human food is imperative. There is also a need to identify and introduce new and lesser known food and feed crops. An important class of non-conventional feeds is BPF which are obtained during harvesting or processing of a commodity in which human food or fibre is derived. The amount of BPF generally increases as the human population increases and economies grow [1,5,6].

The addition of pomegranate yield by-products in ruminant diets can improve the utilization of low-quality roughages mainly through the supply of protein to rumen microbes, but the presence of tannins in these byproducts prevents not only their optimal utilization but also that of the roughages and byproducts. The addition of a tannin-complexing agent, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG) to tannin-rich diets is another attractive option to enhance the feeding value of such diets.
For about 3 decades, it has been known that tannins bind to PVP and PEG. PVP and PEG are also considered to break already formed tannin-protein complexes, as their affinity for tannins is higher than for proteins. This property of these tannin-complexing agents, in particular of PEG, has been exploited by various workers to alleviate the effects of tannins [7]. The addition of PEG results in the formation of PEG-tannin complexes which inactivates tannins [8]. The PEG may be preferred for inactivation of tannins in feed stuffs as its binding to tannins was highest at near neutral pH values [7]. Addition of PEG to tannin-containing feeds increased in vitro gas and SCFA production and in vitro degradation of nitrogen. Therefore, there appears to be a potential for improving the utilization of tannin-containing feeds by the use of tannin binding agent such as PEG without altering the genetic pool of tannin-containing plants. Inclusion of energy sources with the aim of synchronizing nitrogen degradability and availability of energy increased the efficiency of microbial protein synthesis in the presence of PEG [9]. This approach can be used both by farmers and by the industry. Farmers can give PEG directly to animals through water, by mixing it with a small amount of concentrate, by spraying it on tannin-rich feedstuffs or better still as a part of nutrient blocks. Industry can incorporate PEG in a pelleted diet composed of ingredients including tannin-rich byproduct(s) [8].

There is little information available regarding the nutritive value of pomegranate pomace (PP) produced in Iran. The aim of this study was to determine the chemical composition, including tannin content of pomegranate pomace supplemented withdifferent levels of tannin-binding Agent (Polyethylene Glycol) and gas production characteristics using in vitro gas production technique.

Materials and Methods

Pomegranate by-product

Pomegranate pomace (PP) was obtained from fruit juice manufacturing factory of Tabriz, Iran.

Chemical composition

Pomegranate pomace dry matter (DM, method ID 934.01), ash (method ID 942.05), ether extract (EE, method ID 920.30) and crude protein (CP, method ID 984.13) were determined by procedures of AOAC [10]. The NDF and ADF concentrations were determined using the methods of Van Soest et al. [11] without sodium sulphite. NDF was analysed without amylase with ash included. Total phenolics (TP) were measured using the Folin Ciocalteau method [12]. Total tannin (TT) was determined after adding insoluble polyvinylpyrrolidone and reacting with Folin Ciocalteau reagent [12].

In vitro gas production trial

The dry matter degradability of each by-product was determined by in vitro fermentation with ruminal fluid. Ruminal fluid was collected approximately 2h after morning feeding from two cannulated sheep receiving alfalfa hay, barley and soybean meal. Ruminal fluid was immediately squeezed through four layers of cheesecloth and was transported to the laboratory in a sealed thermos. The resulting ruminal fluid was purged with deoxygenated CO2 before use as the inoculum. Gas production was measured by Fedorak & Hurdey [13] method. The treatment contained 0, 75, 150, 300 and 450 mg PEG per each serum bottle, respectively. Approximately 300 mg of dried and ground (2mm) Pomegranate pomace were weighed and placed into serum bottles. There were 3 replicates per treatment. Buffered rumen fluid with McDougal buffer (20ml) was pipetted into each serum bottle [14]. The in vitro gas production volume was measured after 2, 4, 6, 8, 12, 16, 24, 36, and 48 h of incubation. Total gas values were corrected for the blank incubation, and reported gas values are expressed in ml per 1 g of DM.

Statistical analysis

Data obtained from in vitro gas production study was subjected to analysis of variance as a completely randomized design by the GLM procedure of SAS Institute Inc [15] and treatment means were compared by the Duncan test.

Results and Discussion

The chemical compositions of pomegranate pomace are shown in Table 1. CP, ADF, NDF, EE, ASH, TP and TT contents in pomegranate pomace were 8.7%, 27.6%, 32.3%, 1.7%, 9.2%, 5.4% and 4.6%, respectively. Chemical compositions of pomegranate pomace in the current study were inconsistent with findings of Taher-Maddah et al. [16]. Feizi et al. [17] reported that DM, OM, CP, crude fiber, and EE values of pomegranate seeds were 94.8, 96.8, 11.4, 38.9, and 1.0%, respectively. These differences in chemical composition of by-products may be due to a difference in cultivar, growing conditions, varieties, and different de-hulling process methods [16].











Pomegranate Pomace









Table 1: Chemical composition of Pomegranate pomace (% of DM).

DM: Dry Matter; CP: Crude Protein; EE: Ether Extract; ADF: Acid Detergent Fiber;
NDF: Neutral Detergent Fibre; TP: Total Phenol; TT: Total Tannins

Gas production volumes (ml/g DM) from in vitro incubation of PP supplemented with different levels of Polyethylene Glycol at different incubation times are shown in Table 2 and Fig. 1. The volume of in vitro gas production increased with increasing time of incubation. Although there are other models available to describe the kinetics of gas production, the Ørskov & McDonald [18] was chosen due to the relationship of its parameters with intake, digestibility and degradation characteristic of forages and concentrate feedstuffs had been documented. Sommart et al. [16] show that in vitro gas volume is a good parameter from which to predict digestibility, fermentation end product and microbial protein synthesis of the substrate by rumen microbes in the in vitro system. Gas volumes also have shown a close relationship with feed intake [20] and growth rate in cattle [21].


Incubation Times (H)




















PP + 75mg PEG










PP + 150mg PEG










PP + 300mg PEG










PP + 450mg PEG




















Table 2: Total gas production volume (ml/g DM) in incubation times.

The means within a column without common letter differ (p<0.05).

Figure 1: The gas production volume of PP with polyethylene glycol.

At the 2h incubation times, the in vitro gas production amount of PP, PP + 75mg PEG, PP + 150 mg PEG, PP + 300mg PEG and PP + 450mg PEG were 29.83, 32.89, 23.26, 25.83 and 19.37 ml/g DM. At the first incubation times (2 and 4 h), the PP + 75 mg PEG treatments had the highest in vitro gas production amount within treatment (P<0.05). At the all incubation times PP + 75mg PEG treatment had the highest in vitro gas production volume within treatment (P<0.05). After 48 incubation, the treatments PP + 75 mg PEG and PP + 300 mg PEG respectively had highest and lowest in vitro gas production among treatments (P<0.05).

Kamalak et al. [22] reported that total and soluble condensed tannins, NDF and ADF were negatively correlated with estimated parameters of gas production. The results in our study are consistent with those of Feizi et al. [17] who obtained that tannins of pomegranate peel have negative effect on in vitro rumen fermentation. Tannins are considered to have both adverse and beneficial effects in ruminant animals. High concentrations of tannins may reduce intake, digestibility of protein and carbohydrates, and animal performance through their negative effect on palatability and digestion [4]. In the last few years there is an increasing interest of nutritionists in bioactive plant factors-phytofactors as natural feed additives, tannins and etc. that can modify the rumen fermentation processes (e.g., defaunation), improve the protein metabolism and, at the same time, reduce ammonia production and emission, and curb methane production and emission to the atmosphere. High diversity of bioactive phytofactors contained in many plant species has been identified as a potential factor affecting the above-mentioned processes [23].

The PEG supplementation had significant effect on in vitro gas production of PP (Table 2). These results are in agreement with the findings of Getachew et al. [24], Getachew et al. [25], Seresinhe and Iben [25] and Singh et al. [27]. Tannins lead to form a less digestible complex with crude proteins and may bind and inhibit the endogenous protein, such as digestive enzymes [29]. Tannin can adversely affect the microbial and enzyme activities [29-32]. Hagerman et al. [33] showed that tannins reduced crude protein digestibility. In another study, McNeill et al. [14] reported that by increasing condensed tannin in diet, nitrogen digestibility decreased from 0.805 to 0.378 and excretory nitrogen in sheep feces increased from 4.3 to 9.7 g/d. Besharati & s Taghizadeh [5] reported that addition of dried grape by-product to basal diets had effect on digestibility of crude protein (P<0.05), also increasing of dried grape by-product supplementation level had linear effect on crude protein digestibility of diets (P<0.05). The substantial reduction in nitrogen digestibility as a result of the presence of tannins was similar to that reported in sheep fed Lotus pedunculatus as a sole diet [34] and when Lotus pedunculatus was fed with ryegrass (Lolium perenne) [35], with and without polyethylene glycol. Polyethylene glycol has a high affinity to tannins and makes tannins inert by forming tannin polyethylene glycol complexes [8]. Polyethylene glycol also can also liberate protein from the preformed tannin-protein complexes [36]. The increase in the gas production in the presence of polyethylene glycol is possibly due to an increase in the available nutrients to rumen micro-organisms, especially the available nitrogen. McSweeney et al. [37] showed that the addition of polyethylene glycol caused a significant and marked increase in the rate and extent of ammonia production in the rumen [38-39].


Addition of polyethylene glycol (at levels 75 and 450 mg) could overcome adverse effects of tannins on nutrient availability as indicated by gas production parameters. Addition of polyethylene glycol aids inactivated effects of tannins and increased gas production. However there is little information about possibility of using polyethylene glycol in tannin-rich feedstuffs for ruminants. Polyethylene glycol supplementation to improve the nutritive value of pomegranate pomace should be further analyzed in detail: whether or not it is economic due to high cost of polyethylene glycol, before large scale implementation.


  1. Besharati M, Taghizadeh A, Janmohammadi H, Moghadam GA (2008) Evaluation of some by-products using in situ and in vitro gas production techniques. American J Anim Vet Sci 3(1): 7-12.
  2. Bampidis VA, Robinson PH (2006) Citrus by-products as ruminant feeds: A review. J Anim Feed Sci Technol 128(3-4): 175-217.
  3. Mirzaei-Aghsaghali A, Maheri-Sis N, Mansouri H, Razeghi ME, Mirza-Aghazadeh A, et al. (2011) Evaluating nutritional value of apple pomace for ruminants using in vitro gas production technique. ARPN J Agr Biol Sci 1(1): 100-106.
  4. Shabtay A, Eitam H, Tadmor Y, Orlov A, Meir A, et al. (2008) Nutritive and antioxidative potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed. J Agric Food Chem 56(21): 10063-10070.
  5. Besharati M, Taghizadeh A (2009) Evaluation of dried grape by-product as a tanniniferous tropical feedstuff. J Anim Feed Sci Technol 152(3): 198-203.
  6. Besharati M, Taghizadeh A (2011) Effect of Tannin-Binding Agents (Polyethylene Glycol and Polyvinylpyrrolidone) Supplementation on In Vitro Gas Production Kinetics of Some Grape Yield Byproducts. ISRN Vet Sci 2011: 780540.
  7. Makkar HPS (2003) Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Rumin Res 49(3): 241-256.
  8. Makkar HPS, Blümmel M, Becker K (1995) Formation of complexes between polyvinyl pyrrolidone and polyethylene glycol with tannins and their implications in gas production and true digestibility in in vitro techniques. Br J Nutr 73(6): 897-913.
  9. Getachew G, Makkar HPS, Becker K (2000) Effect of polyethylene glycol on in vitro degradability of nitrogen and microbial protein synthesis from tannin rich browse and herbaceous legumes. Br J Nutr 84(1): 73-83.
  10. AOAC (1999) Official Methods of Analysis of AOAC international. AOAC international. Maryland, USA.
  11. Van Soest PJ, Robertson JB, Lewis BA (1991) Methods of dietary fiber, and neutral detergent fiber and non-starch polysaccharides in relation on animal nutrition. J Dairy Sci 74(10): 3583-3597.
  12. Makkar HPS (2000) Quantification of Tannins in Tree Foliage. A Laboratory Manual for the FAO/IAEA Co-ordinated Research Project on Use of Nuclear and Related techniques to Develop Simple Tannin Assays for Predicting and Improving the safety and Efficiency of Feeding Ruminants on Tanniniferous Tree Foliage. Joint FAO/IAEA, FAO/IAEA of Nuclear Techniques in Food and Agriculture. Animal Production and Health Sub-programme, FAO/IAEA Working Document. IAEA, Vienna, Austria.
  13. Fedorak PM, Hurdy DE (1983) A simple apparatus for measuring gas production by methanogenic cultures in serum bottles. Environ Technol Leu 4(10): 425-432.
  14. McNeill DM, Komolong M, Gobiun N, Barber D (2000) Influence of dietary condensed tannins on microbial CP supply in sheep. In: Brooker JD (Ed.), Tannins in Livestock and Human Nutrition. ACIAR Proceedings 92: 57-61.
  15. SAS Inc (2002) Sas user’s Guide: statistics. Statistical Analysis Systems Institute Inc. Cary NC, USA.
  16. Taher-Maddah M, Maheri-Sis N, Salamatdousnobar R, Ahmadzadeh A (2012) Estimating fermentation characteristics and nutritive value of ensiled and dried pomegranate seeds for ruminants using in vitro gas production technique. Open Veterinary J 2(1): 40-45.
  17. Feizi R, Ghodratnama A, Zahedifar M, Danesh-Mesgaran M, Raisianzadeh M (2005) Apparent digestibility of pomegranate seed fed to sheep. Proceedings of British Society of Animal Science 222.
  18. Ørskov ER, Mcdonald I (1979) The Estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J Agric Sci Camb 92: 499-503.
  19. Sommart K, Parker DS, Rowlinson P, Wanapat M (2000) Fermentation characteristics and microbial protein synthesis in an in vitrosystem using cassava, rice straw and dried ruzi grass as substrates. Asian-Aust J Anim Sci 13(8): 1084-1093.
  20. Blummel M, Becker K (1997) The Degradability characteristics of fifty-four roughages and roughage neutral detergent fibers as described by in vitrogas production and their relationship to voluntary feed intake. Br J Nutr 77(5): 757-768.
  21. Blummel M, Ørskov ER (1993) Comparison of in vitrogas production and nylon bag degradability of roughages in predicting feed intake in cattle. J Anim Feed Sci Technol 40(2-3): 109-119.
  22. Kamalak A, Canbolat O, Gurbuz Y, Ozay O, Ozkan CO, et al. (2007) Chemical composition and in vitro gas production characteristics of several tannin containing tree leaves. Res J Agric Biol Sci 3(6): 983-986.
  23. Szumacher-Strabel M, Cieslak A (2010) Potential of phytofactors to mitigate rumen ammonia and methane production. J Anim Feed Sci Technol 19: 319-337.
  24. Getachew G, Makkar HPS, Becker K (2001) Method of polyethylene glycol application to tannin-containing browses to improve microbial fermentation and efficiency of microbial protein synthesis from tannin-containing browses. J Anim Feed Sci Technol 92(1-2): 51-57.
  25. Getachew G, Crovetto GM, Fondevila M, Krishnamoorthy U, Singh B, et al. (2002) Laboratory variation of 24 h in vitro gas production and estimated metabolizable energy values of ruminant feeds. J Anim Feed Sci Technol 102(1-4): 169-180.
  26. Seresinhe T, Iben C (2003) In vitro quality assessment of two tropical shrub legumes in relation to their extractable tannins content. J Anim Physiol Anim Nutr 87(3-4): 109-115.
  27. Singh B, Sahoo A, Sharma R, Bhat TK (2005) Effect of polyethylene glycol on gas production parameters and nitrogen disappearance of some tree forages. J Anim Feed Sci Tech 123-124: 351-364.
  28. Kumar R, Singh M (1984) Tannins: their adverse role in ruminant nutrition. J Agric Food Chem 32(3): 447-453.
  29. Singleton VL (1981) Naturally occurring food toxicants: Phenolic substances of plant origin common in foods. Advan Food Res 27: 149-242.
  30. Lohan OP, Lall D, Vaid J, Negi SS (1983) Utilization of oak tree fodder in cattle ration and fate of oak leaf tannins in the ruminant system. Indian J Anim Sci 53: 1057-1063.
  31. Barry TN, Duncan SJ (1984) The role of condensed tannins in the nutritional-value of Lotus-pedunculatus for sheep. 1. Voluntary intake. Br J Nutr 51(3): 485-491.
  32. Makkar HPS, Singh B, Negi SS (1989) Relationship of rumen degradability with microbial colonization, cell wall constituents and tannin levels in some tree leaves. Anim Prod 49: 299-303.
  33. Hagerman AE, Robbins CT, Weerasuriya Y, Wilson TC, McArthur C (1992) Tannin chemistry in relation to digestion. J Range Manage 45(57-62): 57-62.
  34. Waghorn GC, Shelton ID, McNabb WC, McCutcheon SN (1994) Effects of condensed tannins in Lotus pedunculatus on its nutritive value for sheep. 2. Nitrogenous aspects. J Agric Sci Cambridge 123: 109-119.
  35. Waghorn GC, Shelton ID (1995) Effect of condensed tannins in Lotus pedunculatus on the nutritive value of ryegrass (Lolium perenne) fed to sheep. J Agric Sci Cambridge 125: 291
  36. Barry TN, Manley TR, Duncan SJ (1986) The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 4. Site of carbohydrate and protein digestion as influenced by dietary reactive tannin concentration. Br J Nutr 55(1): 123-137.
  37. McSweeney CS, Palmer B, Bunch R, Krause DO (199) In vitro quality assessment of tannin-containing tropical shrub legumes: protein and fibre digestion. J Anim Feed Sci Technol 82(3-4): 227.
  38. Schofield P, Mbugua DM, Pell AN (2001) Analysis of condensed tannins: a review. J Anim Feed Sci Tech 91(1-2): 21-40.
  39. McDougall EEI (1948) The composition and output of sheep in salvia. Biochem J 43(1): 99-109.
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