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Effects of microwave irradiation on ruminal protein and starch degradation of corn grain
Animal Feed Science and Technology 127 (2006) 113–123
Effects of microwave irradiation on ruminal protein and starch degradation of corn grain A.A. Sadeghi a,∗ , P. Shawrang b a
Department of Animal Science, Faculty of Agriculture, Sciences and Research Campus, Islamic Azad University, Tehran, Iran b Department of Animal Science, Faculty of Agriculture, Tehran University, Karaj, Iran
Received 19 December 2004; received in revised form 15 June 2005; accepted 21 July 2005
Abstract This study was designed to evaluate effects of 800 W microwave irradiation for 3, 5 and 7 min on ruminal dry matter (DM), crude protein (CP) and starch degradation parameters of corn grain. Nylon bags of untreated or microwave treated corn were suspended in the rumen of three Holstein steers for 0–48 h, and resulting data were fitted to a non-linear degradation model to calculate effective rumen degradation (ERD). Microwave treatments decreased (P<0.05) the water soluble fraction and increased the potentially degradable fraction of CP, except for 7 min processing time that decreased (P<0.05) the potentially degradable fraction. Processing for 3 and 5 min had no effect, but for 7 min it decreased (P<0.05) ERD of CP. Microwave irradiation for 3 and 5 min increased (P<0.05) the water soluble fraction and decreased the potentially degradable fraction of starch. The degradation rate of the latter fraction increased (P<0.05) with 3 and 5 min processing times. Microwave irradiation for 7 min decreased (P<0.05) the water soluble fraction and the potentially degradable fraction of starch. Processing for 3 and 5 min increased (P<0.05), but for 7 min decreased (P<0.05) ERD of starch. From SDS-PAGE patterns, four major protein components in corn containing prolamin (i.e., zein), albumin, globulin and glutelin fractions were observed. Electrophoretic analysis of protein residues revealed that microwave processing decreased degradation of corn true protein. SDS-PAGE indicated that the bulk of the rumen undegradable protein for untreated corn was zein, while for 5 and 7 min microwave
Abbreviations: CP, crude protein; DM, dry matter; ERD, effective rumen degradation; NDFom, neutral detergent fiber; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis ∗ Corresponding author. Tel.: +98 261 224 8082; fax: +98 261 224 6752. E-mail addresses: [email protected], [email protected] (A.A. Sadeghi). 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.07.004
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treated corn it was zein and other proteins. In this study, the optimal processing time with 800 W microwave power was judged to be 5 min. © 2005 Elsevier B.V. All rights reserved. Keywords: Corn; Protein; Ruminal degradation; Starch; SDS-PAGE
1. Introduction In many parts of the world, corn grain is an important ingredient in diets for ruminants, serving mainly as an energy component, although starch is an excellent substrate for microbial growth in the rumen. The effect of heat processing on ruminal protein and starch degradation of corn grain has been evaluated (Mathison, 1996; Huntington, 1997), but there are no data on ruminal starch and crude protein degradation characteristics of microwave processed corn grain. Furthermore, effects of microwave processing on the amount of corn true protein escaping the rumen are lacking. Briefly, microwave energy penetrates a food or feed material and produces a volumetrically distributed heat source, due to molecular friction, resulting from dipolar rotation of polar solvents and from conductive migration of dissolved ions. The dipolar rotation is caused by variations of the electrical and magnetic fields in the organic components (Alton, 1998). Water, the major constituent of most food and feed products, is the main source for microwave interactions due to its dipolar nature. Heat is generated throughout the material, leading to faster heating rates and shorter processing times compared to conventional heating, where heat is usually transferred from the surface to the interior (Fakhouri and Ramaswamy, 1993). Other advantages include space savings and energy efficiency, since most of the electro-magnetic energy is converted into heat (Mermelstein, 1997). The purposes of this study were to evaluate effects of microwave processing on ruminal CP and starch degradation of corn grain, and to monitor the fate of corn true protein in the rumen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
2. Materials and methods 2.1. Samples and treatments Corn grain samples (n = 15) were obtained from commercial sources in Iran. The DM of corn was determined by freeze drying a 1 g sample in duplicate. Based upon this value, sufficient water was added to increase the moisture content of 2 kg of corn to 200 g/kg. Three samples (500 g each) were subjected to microwave heating at a power of 800 W for 3, 5 and 7 min. For chemical analysis, 10 g were ground to pass a 1 mm screen, packed in low-density polyethylene air locked pouches and stored at −18 ◦ C. The remainder of each sample was ground to pass a 2 mm screen for the ruminal in situ study and preserved as describe above.
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2.2. Chemical analysis The N level of corn was determined according to AOAC (Method 984.13; AOAC, 1995), and ash was determined by burning a 2 g sample in duplicate at 600 ◦ C for 2 h in a muffle furnace (Method 942.05; AOAC, 1995). Neutral detergent fiber (NDFom) was analyzed according to the method of Van Soest et al. (1991), using an automatic fiber analyzer (Fibertec System M, Tecator). NDFom was determined without using sodium sulfite, without washing with acetone, without ␣-amylase and expressed without residual ash. The method of McCleary et al. (1994) was used for determination of starch. There was no correction for sugar. Thus, the determined content of starch represents starch plus sugar. 2.3. Determination of rumen degradability The procedure of ruminal incubation followed the method of Mehrez and Ørskov (1977) in which 5 g of untreated or microwave treated corn were weighed in duplicate into nylon bags (9 cm × 21 cm, 45 m pore size). Each group of 48 samples (two replicates × eight incubation periods × three steers for each treatment) were prepared into individual nylon bags for assay. Bags were incubated in the ventral sac of the rumen of three Holstein steers (452 ± 14 kg) for 2, 4, 6, 8, 12, 16, 24 and 48 h. Steers were maintained on 873 g/kg alfalfa hay and 127 g/kg corn grain based supplement (938 g/kg of ground corn, 20 g/kg of molasses, 40 g/kg of trace mineralized salt, and 2 g/kg of Vitamin A, D, and E premix). Diet was offered at 20 g/kg of body weight daily in two equal portions (08:00 and 16:00 h). Immediately after removal from the rumen, bags were put in ice water to stop microbial fermentation, and washed under tap water until the rinsing water became colorless, then freeze-dried and weighed. Three bags were soaked in water bath for 30 min at 20 ◦ C to estimate the rapidly degradable and readily available DM, CP and starch fractions. After soaking, bags were washed by hand, rinsed and freeze dried. 2.4. Determination of protein sub-units Protein sub-units were fractionated by a SDS-PAGE discontinuous system (Laemmli, 1970). All ruminal undegradable fractions from each incubation period were freeze dried, ground (0.25 mm particle size) and replicate samples pooled. Twenty microgram of untreated or treated corn were placed into 750 l SDS-PAGE sample buffer. After 30 min of mixing (i.e., vortex and inverse), samples were immersed at 90 ◦ C for 3 min, and then centrifuged at 10 000 × g for 1 min. A 25 l aliquot of each sample was loaded into the sample well. Electrophoresis of proteins was on 12.5% resolving gel (1.0 mm × 110 mm × 140 mm) with 3.75% acrylamide stacking gel. The gels were kept at a constant current of 30 mA until the bromophenol blue marker dye reached the bottom of the gel. Protein fixation and staining were completed simultaneously using a solution of Coomassie brilliant blue. Gel destaining was accomplished by using a 300 ml/l methanol and 70 ml/l acetic acid solution. The subunits of the gel were monitored by densitometric scanning at 580 nm. One standard protein mixture included -galactosidase (116 kDa), bovine plasma albumin (66.0 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), Soybean trypsin inhibitor (21.5 kDa), -lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa) was used.
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2.5. Statistics Digestion kinetics of DM, CP and starch were determined according to the equations of Ørskov and McDonald (1979) as: P = a + b(1 − e−ct ),
ERD = a + bc/(c + k)
where ‘P’ is DM, CP or starch disappearance (g/kg) at time t (h), ‘a’ the water soluble fraction (g/kg), ‘b’ the potentially degradable fraction (g/kg), ‘c’ the rate of degradation (h−1 ) of ‘b’ fraction, ERD the effective rumen degradation, ‘k’ the fractional ruminal outflow rate. Effective degradability was calculated with estimated solid outflow rates from the rumen of 0.02, 0.05 and 0.08 h−1 (AFRC, 1993). The degradability parameters for the nylon bags were analyzed as a randomized complete block design, using steers as blocks. Analysis was with the general linear means model of SAS (1996). When a significant difference occurred, means were separated using Duncan’s test (Steel and Torrie, 1980). Differences were considered to be significant if P<0.05.
3. Results 3.1. Ruminal DM, CP and starch degradability Ruminal DM, CP and starch degradation parameters of untreated and microwave treated corn are in Table 1. Coefficients a, b and c for DM were affected (P<0.05) by the 7 min microwave processing time. There were no differences (P>0.05) for degradation parameters of DM among untreated, 3 and 5 min microwave treated corn. Microwave processing for 7 min decreased (P<0.05) the a and b fractions of DM. Effective ruminal DM degradability for this processing time were lower (P<0.05) than that of untreated corn. There were differences (P<0.05) in CP and starch degradation parameters among untreated, 3, 5 and 7 min microwave treated corn. For CP, all coefficients were affected (P<0.05) by microwave irradiation, reducing values for a and c, but increasing values for b, except for 7 min processing time that decreased (P<0.05) the b fraction. No differences (P>0.05) were found in ERD of CP among untreated, 3 and 5 min microwave treated corn. The effective CP degradability of 7 min microwave treated corn was lower (P<0.05) than that for untreated corn. Microwave treatments for 3 and 5 min increased (P<0.05) the a fraction and decreased the b fraction of starch. The degradation rate of the b fraction increased with 3 and 5 min processing times. Microwave irradiation for 7 min decreased (P<0.05) the a and b fractions of starch. The ERD of starch for 3 and 5 min microwave treated corn were higher (P<0.05), but for 7 min were lower (P<0.05), than that for untreated corn. 3.2. Electrophoretic profile of corn protein sub-units The molecular weight of corn proteins is depicted in Fig. 1. The four major components were prolamins (two sub-units), albumins, globulins and glutelins. From 12.5% slab gel analysis, the globulins appeared to be composed mainly of sub-units with a molecular mass
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Table 1 Dry matter, crude protein and starch degradation parameters of untreated and microwave treated corn graina Degradation traitsb (g/kg)
c (h−1 )
ERD (g/kg) at outflow rate 0.02 h−1
0.05 h−1
0.08 h−1
0.051 a 0.052 a 0.052 a 0.043 b 0.003
746 a 740 a 738 a 683 b 16.9
572 a 570 a 569 a 514 b 16.4
478 a 477 a 477 a 427 b 18.0
972 a 968 a 958 a 846 b 14.7
0.043 a 0.036 b 0.039 ab 0.037 b 0.004
700 a 657 a 656 a 568 b 16.2
512 a 461 a 457 a 391 b 14.8
415 a 365 a 359 a 305 b 13.2
988 a 989 a 992 a 931 b 10.1
0.065 c 0.084 a 0.076 b 0.063 c 0.003
803 b 843 a 841 a 753 c 14.5
646 b 706 a 704 a 604 c 12.6
554 b 620 a 620 a 518 c 13.0
a
b
a+b
Dry matter Untreated 3 min microwave 5 min microwave 7 min microwave SEM
164 a 162 a 165 a 157 b 8.3
812 a 799 a 795 a 769 b 15.6
976 a 961 a 960 a 926 b 15.3
Crude protein Untreated 3 min microwave 5 min microwave 7 min microwave SEM
115 a 92 b 68 c 59 c 8.9
857 b 876 a 890 a 787 c 12.4
Starch Untreated 3 min microwave 5 min microwave 7 min microwave SEM
201 c 234 b 267 a 194 c 6.5
787 a 755 b 725 c 737 bc 15.8
Means in the same column with different letters differ (P<0.05). a The CP, NDFom, ash and starch content of corn were 89, 78, 14 and 710 g/kg DM, respectively. b a is the soluble fraction as measured by washing loss from nylon bags; b is the potentially degradable fraction; c is the rate of degradation of fraction b (h−1 ); ERD is the effective rumen degradability of DM, CP or starch (g/kg DM, CP or starch) measured at outflow rates of 0.02, 0.05 and 0.08 h−1 .
Fig. 1. SDS-PAGE patterns of corn proteins (A, lines 1 and 2) and protein standard (A, line 3), and electrophoretic profile of untreated corn proteins incubated in the rumen (B).
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Fig. 2. Electrophoretic profile of 5 min (A) and 7 min (B) microwave treated corn proteins incubated in the rumen.
ranging from 25 to 50 kDa. Corn prolamins (i.e., zein) were easily identified after SDSPAGE and consisted of two major sub-units of 22 and 24 kDa. The SDS-PAGE slab gel analysis of untreated, 5 and 7 min microwave treated corn proteins are in Figs. 1 and 2, respectively. Each line was for the hours of ruminal incubation (i.e., 0, 2, 4, 6, 8, 12, 24 and 48 h). From the SDS-PAGE patterns, and densitometric scans (Fig. 3), albumin, globulin and glutelin sub-units of untreated and microwave treated corn were degraded at the shortest and middle incubation periods, respectively, whereas the prolamin sub-units (i.e., zein) were not degraded completely until 48 h.
4. Discussion 4.1. Ruminal DM and CP degradation In this study, the DM a fraction (164 g/kg) of untreated corn was similar to previous reports of Nocek (1987, 166 g/kg) and Herrera-Saldana et al. (1990, 186 g/kg). The DM degradation rate (0.051 h−1 ) was slower than reported by Nocek (1987, 0.091 h−1 ) but close to values reported by Herrera-Saldana et al. (1990, 0.047 h−1 ). Microwave irradiation for 7 min decreased effective DM degradability of corn, and most of this reduction was due to reduction in ERD of CP and starch. Crude protein in untreated corn had a degradability curve characterized by an a fraction of 115 g/kg, b fraction of 857 g/kg, and degradation rate (c) of 0.043 h−1 . Tamminga et al. (1990) reported a higher CP a fraction value of 150 g/kg. Variation in this fraction among studies could be due to differences in feed particle size and/or differences in analytical techniques. Grings et al. (1992) reported a similar degradation rate (0.041 h−1 ), but Nocek
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Fig. 3. Densitometrical scanning of corn (A), untreated (B) and 5 min microwave treated corn (C).
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(1987) reported a slightly faster degradation rate (0.068 h−1 ). The predicted effective CP degradability values of untreated corn at rumen outflow rates of 0.02, 0.05, and 0.08 h−1 were 700, 512 and 415 g/kg respectively, consistent with Batajoo and Shaver (1998, 400 g/kg) at an outflow rate of 0.08 h−1 . Effective CP degradability of 3, 5 and 7 min microwave treated corn at outflow rate of 0.05 h−1 decreased by 9, 10 and 23% units compared with untreated corn, respectively. These effects were mainly attributed to microwave heating, but recent reports have shown, or suggested, that there are non-thermal microwave effects in terms of energy required to produce various types of molecular transformations and alterations (Banik et al., 2003). The literature does not contain information on effects of microwave irradiation on ruminal protein degradation of feedstuffs. 4.2. Ruminal starch degradation The starch a fraction of untreated corn was 201 g/kg, supporting Herrera-Saldana et al. (1990, 210 g/kg). Tamminga et al. (1990, 276 g/kg) and Cerneau and Michalet-Doreau (1991, 265 g/kg) reported higher values. This fraction contains soluble sugars (i.e., glucose, fructose, sucrose and fructans), as well as soluble non-starch polysaccharides (i.e., arabinose, xylose, mannose, galactose and uronic acids; Aman and Hesselman, 1984). Discrepancies in reported in situ disappearance values can be attributed to varietal differences in the grain incubated, in situ technique, and/or basal diet (Nocek, 1988). The starch b fraction was 787 g/kg, higher than reported by Cerneau and MichaletDoreau (1991, 735 g/kg). As expected (Ørskov, 1986), starch in untreated corn was slowly degraded. This could be related to association of the protein matrix with starch granules, as well as type and proportion of proteins in the endosperm (Rooney and Pflugfelder, 1986; McAllister et al., 1993). Effective starch degradability of untreated corn at rumen outflow rates of 0.02, 0.05, and 0.08 h−1 were 803, 646 and 554 g/kg, respectively. Similar ERD of starch in corn have been reported by Herrera-Saldana et al. (1990, 619 g/kg) at rumen outflow rates of 0.06 h−1 , and Cerneau and Michalet-Doreau (1991, 578 g/kg) at a rumen outflow rates of 0.08 h−1 . The degradability of starch depends, in part, on the surface area of the starch granule which is exposed and able to contact starch degrading enzymes. Microwave processing for 3 and 5 min increased ERD of starch, due to an increase in starch a fraction. Starch b fractions decreased and the degradation rate of the b fraction increased with these treatments, probably because linkages between the protein matrix and the starch granule were disrupted. It seems likely that changes in the amorphous amylose part of the starch granule occurs during microwave treatment (Lewandowicz et al., 2000). Most technological treatments increased starch degradability, mainly by particle size reduction, gelatinization and protein matrix disruption. Gelatinization led to chemical and physical changes in the starch granule. Disruption of hydrogen bonds and water absorption facilitated microbial or enzyme degradation of the starch granule. Microwave irradiation for 7 min decreased the ERD of starch, due to a decrease in the starch a fraction. Lewandowicz et al. (2000) reported that microwave irradiation causes a drop in solubility and crystallinity of starch. They concluded that a higher gelatinization temperature of microwave irradiated starches may indicate an association, and a
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more stable configuration, in a granular structure. These observations suggest a great importance of the amylose content relative to susceptibility of starches to microwave treatment. Very little research has been completed on effects of microwave irradiation on chemical modification of starches (Muzimbaranda and Tomasik, 1994; Sikora et al., 1997). 4.3. Molecular weight of corn protein sub-units Several albumin and globulin polypeptides over a large range of molecular mass were separated by SDS-PAGE (Fig. 1). Corn prolamins (i.e., Zein) were easily identified after SDS-PAGE, and consisted of two major sub-units of 22 and 24 kDa. Evidence of the bifractional pattern of zein when separated by SDS-PAGE in a denatured state has been reported (Hoseney, 1986). Because corn glutelins consist of several polypeptides linked through disulfide bonds, use of reducing agents in SDS-PAGE sample buffer creates several additional protein components that can be characterized by SDS-PAGE (Landry and Moureaux, 1981). In this study, three main polypeptides, of 31, 50 and 55 kDa, were identified by SDS-PAGE analysis of glutelins residues in the presence of the reducing agent -mercaptoethanol. A similar profile of corn glutelins in 1% SDS sample buffer has been reported (Wilson et al., 1981). 4.4. Individual protein degradation in the rumen Zein, the prolamin fraction, was relatively resistant to ruminal proteolysis. Wadhwa et al. (1993) reported that, irrespective of protein source and dietary regimen, prolamin degradation was minimal in the rumen. Conversely, albumin, globulin and glutelin subunits contributed very little to the protein residues following incubation in the rumen. The hydrophobic nature of zein may be responsible for its low degradation by microorganisms (Wilson et al., 1981). The marked variability in ruminal extents of degradation also may be associated with different tertiary structures of protein sources (Nugent and Mangan, 1978). Corn prolamin sub-units are rich in non-polar amino acids and have a rod-like molecular structure compared with globular water-soluble proteins (Hoseney, 1986). Furthermore, the presence of three-dimensional disulfide linkages in corn glutelin sub-units, which increases the complexity of the tertiary structure, may limit the action of microbial proteases and reduce ruminal degradation of zein. Electrophoretic and densitometric analyses of protein residues revealed that the prolamin, albumin, globulin and glutelin sub-units of microwave treated corn were more resistant to ruminal degradation than untreated corn. Analysis of densitometric scans revealed that, by 48 h, zein represented the bulk of the residual protein for 5 and 7 min microwave treated corn. The diverse composition of corn proteins makes it difficult to set a single temperature where all protein sub-units denature or aggregate simultaneously. Heat treatment of protein will result in its denaturation, and probably transform the proteins to a more enzyme resistant structure. In addition, heat processing can result in formation of chemical cross-linkages between amino acids and reducing sugars (i.e., Maillard polymerization), or between proteins (i.e., iso-peptide bonds), reactions that will probably make feed proteins more resistant to degradation in the rumen.
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5. Conclusion The 3 and 5 min microwave irradiation had no effect on ruminal protein degradation, but increased starch degradability. Microwave heating for 7 min decreased ruminal CP and starch degradability. SDS-PAGE indicated that the bulk of the rumen undegradable protein in untreated corn was zein, while for microwave treated corn it was zein and other proteins. Further study is needed to determine effects of different microwave powers and times on corn starch degradation and protein sub-unit profiles after incubation in the rumen. Acknowledgements The authors gratefully thank the Tehran Sciences and Research Campus, Islamic Azad University, for financial support, and they would also especially like to thank Prof. A. Nikkhah for his comments, and Dr. M.M. Shahrebabak for statistical guidance and Mrs. Saeedeh Mousavy for technical assistance. References Agricultural Food Research Council, 1993. Energy and Protein Requirements of Ruminants. AFRC Technical Committee on Responses to Nutrients. CAB International, Wallingford, UK. Alton, W.J., 1998. Microwave pasteurization of liquids. Eng. Paper. 2, 98–211. Aman, P., Hesselman, K., 1984. Analysis of starch and other main constituents of cereal grains. Swed. J. Agric. Res. 14, 135–142. AOAC, 1995. Official Methods of Analysis, 16th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Banik, S., Bandyopadhyay, S., Ganguly, S., 2003. Bio-effects of microwave. Bio-resource Technol. 87, 155–159. Batajoo, K.K., Shaver, R.D., 1998. In situ dry matter, crude protein, and starch degradabilities of selected grains and by-product feeds. Anim. Feed Sci. Technol. 71, 165–176. Cerneau, P., Michalet-Doreau, B., 1991. In situ starch degradation of different feeds in the rumen. Reprod. Nutr. Dev. 31, 65–73. Fakhouri, M.O., Ramaswamy, H.S., 1993. Temperature uniformity of microwave heated foods as influenced byproduct type and composition. Food Res. Inter. 26, 89–95. Grings, E.E., Roffler, R.E., Deitelhoff, D.P., 1992. Responses of dairy cows to additions of distillers dried grains with solubles in alfalfa-based diets. J. Dairy Sci. 75, 1946–1955. Herrera-Saldana, R.E., Huber, J.T., Poore, H.M., 1990. Dry matter, crude protein, and starch degradability of five cereal grains. J. Dairy Sci. 73, 2386–2393. Hoseney, R.C., 1986. Cereal Proteins. Page 69 in Principle of Cereal Science and Technology. Am. Assoc. Cereal Chem., St. Paul, MN, USA. Huntington, G.B., 1997. Starch utilisation by ruminants: from basic to the bulk. J. Anim. Sci. 75, 852–867. Laemmli, U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 680–689. Landry, J., Moureaux, T., 1981. Physicochemical properties of maize glutelins as influenced by their isolation conditions. J. Agric. Food. Chem. 29, 1205–1211. Lewandowicz, G., Jankowsk, T., Fornal, J., 2000. Effect of microwave radiation on physico-chemical properties and structure of cereal starches. Carbohyd. Polym. 42, 193–199. Mathison, G.W., 1996. Effects of processing on the utilisation of grain by cattle. Anim. Feed Sci. Technol. 58, 113–125. McAllister, T.A., Phillippe, R.C., Rode, L.M., Cheng, K.J., 1993. Effect of the protein matrix on the digestion of cereal grains by ruminal microorganisms. J. Anim. Sci. 71, 205–212.
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McCleary, B.V., Solah, V., Gibson, T.S., 1994. Quantitative measurement of total starch in cereal flours and products. J. Cereal Sci. 20, 51–58. Mehrez, A.Z., Ørskov, E.R., 1977. A study of the artificial bag technique determining the digestibility of feeds in the rumen. J. Agri. Sci. (Camb.) 88, 645–650. Mermelstein, N.H., 1997. How food technology covered microwaves over the years. Food Technol. 51, 82–84. Muzimbaranda, C., Tomasik, P., 1994. Microwaves in physical and chemical modification of starch. Starch 46, 469–476. Nocek, J.E., 1987. Characterization of in situ dry matter and nitrogen digestion of various corn grain forms. J. Dairy Sci. 70, 2291–2301. Nocek, J.E., 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci. 71, 2051–2069. Nugent, J.H.A., Mangan, J.L., 1978. Rumen proteolysis of fraction I leaf protein, casein and bovine serum albumin. Hoc. Nutr. Soc. 37, 48 (Abstract). Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. Camb. 92, 499–503. Ørskov, E.R., 1986. Starch digestion and utilization in ruminants. J. Anim. Sci. 63, 1624–1634. Rooney, L.W., Pflugfelder, R.L., 1986. Factor affecting starch digestibility with special emphasis on sorghum and corn. J. Anim. Sci. 63, 1607–1623. SAS Institute Inc., 1996. Statistical Analysis System (SAS) User’s Guide. SAS Institute, Cary, NC, USA. Sikora, M., Tomasik, P., Pielichowski, K., 1997. Chemical starch modification in the field of microwaves. II. Reactions with a-amino and ahydroxy acids. Polish J. Food Nut. Sci. 47, 23–30. Steel, R.G.D., Torrie, J.H., 1980. Principles and Procedures of Statistics: A Biometrical Approach, 2nd ed. McGraw-Hill, NY, USA. Tamminga, S., van Vuuren, A.M., van der Koelen, C.J., Ketelaar, R.S., van der Togt, P.L., 1990. Ruminal behavior of structural carbohydrates, non-structural carbohydrates and crude protein from concentrate ingredients in dairy cows. Neth. J. Agric. Sci. 38, 513–521. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wadhwa, M., Makkar, G.S., Ichhponani, J.S., 1993. Disappearance of protein supplements and their fractions in sacco. Anim. Food Sci. Technol. 40, 285–293. Wilson, C.M., Shewry, P.R., Miflin, B.J., 1981. Maize endosperm proteins compared by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Cereal Chem. 58, 275–282.
Effects of microwave irradiation on ruminal protein and starch degradation of corn grain A.A. Sadeghi a,∗ , P. Shawrang b a
Department of Animal Science, Faculty of Agriculture, Sciences and Research Campus, Islamic Azad University, Tehran, Iran b Department of Animal Science, Faculty of Agriculture, Tehran University, Karaj, Iran
Received 19 December 2004; received in revised form 15 June 2005; accepted 21 July 2005
Abstract This study was designed to evaluate effects of 800 W microwave irradiation for 3, 5 and 7 min on ruminal dry matter (DM), crude protein (CP) and starch degradation parameters of corn grain. Nylon bags of untreated or microwave treated corn were suspended in the rumen of three Holstein steers for 0–48 h, and resulting data were fitted to a non-linear degradation model to calculate effective rumen degradation (ERD). Microwave treatments decreased (P<0.05) the water soluble fraction and increased the potentially degradable fraction of CP, except for 7 min processing time that decreased (P<0.05) the potentially degradable fraction. Processing for 3 and 5 min had no effect, but for 7 min it decreased (P<0.05) ERD of CP. Microwave irradiation for 3 and 5 min increased (P<0.05) the water soluble fraction and decreased the potentially degradable fraction of starch. The degradation rate of the latter fraction increased (P<0.05) with 3 and 5 min processing times. Microwave irradiation for 7 min decreased (P<0.05) the water soluble fraction and the potentially degradable fraction of starch. Processing for 3 and 5 min increased (P<0.05), but for 7 min decreased (P<0.05) ERD of starch. From SDS-PAGE patterns, four major protein components in corn containing prolamin (i.e., zein), albumin, globulin and glutelin fractions were observed. Electrophoretic analysis of protein residues revealed that microwave processing decreased degradation of corn true protein. SDS-PAGE indicated that the bulk of the rumen undegradable protein for untreated corn was zein, while for 5 and 7 min microwave
Abbreviations: CP, crude protein; DM, dry matter; ERD, effective rumen degradation; NDFom, neutral detergent fiber; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis ∗ Corresponding author. Tel.: +98 261 224 8082; fax: +98 261 224 6752. E-mail addresses: [email protected], [email protected] (A.A. Sadeghi). 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.07.004
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treated corn it was zein and other proteins. In this study, the optimal processing time with 800 W microwave power was judged to be 5 min. © 2005 Elsevier B.V. All rights reserved. Keywords: Corn; Protein; Ruminal degradation; Starch; SDS-PAGE
1. Introduction In many parts of the world, corn grain is an important ingredient in diets for ruminants, serving mainly as an energy component, although starch is an excellent substrate for microbial growth in the rumen. The effect of heat processing on ruminal protein and starch degradation of corn grain has been evaluated (Mathison, 1996; Huntington, 1997), but there are no data on ruminal starch and crude protein degradation characteristics of microwave processed corn grain. Furthermore, effects of microwave processing on the amount of corn true protein escaping the rumen are lacking. Briefly, microwave energy penetrates a food or feed material and produces a volumetrically distributed heat source, due to molecular friction, resulting from dipolar rotation of polar solvents and from conductive migration of dissolved ions. The dipolar rotation is caused by variations of the electrical and magnetic fields in the organic components (Alton, 1998). Water, the major constituent of most food and feed products, is the main source for microwave interactions due to its dipolar nature. Heat is generated throughout the material, leading to faster heating rates and shorter processing times compared to conventional heating, where heat is usually transferred from the surface to the interior (Fakhouri and Ramaswamy, 1993). Other advantages include space savings and energy efficiency, since most of the electro-magnetic energy is converted into heat (Mermelstein, 1997). The purposes of this study were to evaluate effects of microwave processing on ruminal CP and starch degradation of corn grain, and to monitor the fate of corn true protein in the rumen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
2. Materials and methods 2.1. Samples and treatments Corn grain samples (n = 15) were obtained from commercial sources in Iran. The DM of corn was determined by freeze drying a 1 g sample in duplicate. Based upon this value, sufficient water was added to increase the moisture content of 2 kg of corn to 200 g/kg. Three samples (500 g each) were subjected to microwave heating at a power of 800 W for 3, 5 and 7 min. For chemical analysis, 10 g were ground to pass a 1 mm screen, packed in low-density polyethylene air locked pouches and stored at −18 ◦ C. The remainder of each sample was ground to pass a 2 mm screen for the ruminal in situ study and preserved as describe above.
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2.2. Chemical analysis The N level of corn was determined according to AOAC (Method 984.13; AOAC, 1995), and ash was determined by burning a 2 g sample in duplicate at 600 ◦ C for 2 h in a muffle furnace (Method 942.05; AOAC, 1995). Neutral detergent fiber (NDFom) was analyzed according to the method of Van Soest et al. (1991), using an automatic fiber analyzer (Fibertec System M, Tecator). NDFom was determined without using sodium sulfite, without washing with acetone, without ␣-amylase and expressed without residual ash. The method of McCleary et al. (1994) was used for determination of starch. There was no correction for sugar. Thus, the determined content of starch represents starch plus sugar. 2.3. Determination of rumen degradability The procedure of ruminal incubation followed the method of Mehrez and Ørskov (1977) in which 5 g of untreated or microwave treated corn were weighed in duplicate into nylon bags (9 cm × 21 cm, 45 m pore size). Each group of 48 samples (two replicates × eight incubation periods × three steers for each treatment) were prepared into individual nylon bags for assay. Bags were incubated in the ventral sac of the rumen of three Holstein steers (452 ± 14 kg) for 2, 4, 6, 8, 12, 16, 24 and 48 h. Steers were maintained on 873 g/kg alfalfa hay and 127 g/kg corn grain based supplement (938 g/kg of ground corn, 20 g/kg of molasses, 40 g/kg of trace mineralized salt, and 2 g/kg of Vitamin A, D, and E premix). Diet was offered at 20 g/kg of body weight daily in two equal portions (08:00 and 16:00 h). Immediately after removal from the rumen, bags were put in ice water to stop microbial fermentation, and washed under tap water until the rinsing water became colorless, then freeze-dried and weighed. Three bags were soaked in water bath for 30 min at 20 ◦ C to estimate the rapidly degradable and readily available DM, CP and starch fractions. After soaking, bags were washed by hand, rinsed and freeze dried. 2.4. Determination of protein sub-units Protein sub-units were fractionated by a SDS-PAGE discontinuous system (Laemmli, 1970). All ruminal undegradable fractions from each incubation period were freeze dried, ground (0.25 mm particle size) and replicate samples pooled. Twenty microgram of untreated or treated corn were placed into 750 l SDS-PAGE sample buffer. After 30 min of mixing (i.e., vortex and inverse), samples were immersed at 90 ◦ C for 3 min, and then centrifuged at 10 000 × g for 1 min. A 25 l aliquot of each sample was loaded into the sample well. Electrophoresis of proteins was on 12.5% resolving gel (1.0 mm × 110 mm × 140 mm) with 3.75% acrylamide stacking gel. The gels were kept at a constant current of 30 mA until the bromophenol blue marker dye reached the bottom of the gel. Protein fixation and staining were completed simultaneously using a solution of Coomassie brilliant blue. Gel destaining was accomplished by using a 300 ml/l methanol and 70 ml/l acetic acid solution. The subunits of the gel were monitored by densitometric scanning at 580 nm. One standard protein mixture included -galactosidase (116 kDa), bovine plasma albumin (66.0 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), Soybean trypsin inhibitor (21.5 kDa), -lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa) was used.
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2.5. Statistics Digestion kinetics of DM, CP and starch were determined according to the equations of Ørskov and McDonald (1979) as: P = a + b(1 − e−ct ),
ERD = a + bc/(c + k)
where ‘P’ is DM, CP or starch disappearance (g/kg) at time t (h), ‘a’ the water soluble fraction (g/kg), ‘b’ the potentially degradable fraction (g/kg), ‘c’ the rate of degradation (h−1 ) of ‘b’ fraction, ERD the effective rumen degradation, ‘k’ the fractional ruminal outflow rate. Effective degradability was calculated with estimated solid outflow rates from the rumen of 0.02, 0.05 and 0.08 h−1 (AFRC, 1993). The degradability parameters for the nylon bags were analyzed as a randomized complete block design, using steers as blocks. Analysis was with the general linear means model of SAS (1996). When a significant difference occurred, means were separated using Duncan’s test (Steel and Torrie, 1980). Differences were considered to be significant if P<0.05.
3. Results 3.1. Ruminal DM, CP and starch degradability Ruminal DM, CP and starch degradation parameters of untreated and microwave treated corn are in Table 1. Coefficients a, b and c for DM were affected (P<0.05) by the 7 min microwave processing time. There were no differences (P>0.05) for degradation parameters of DM among untreated, 3 and 5 min microwave treated corn. Microwave processing for 7 min decreased (P<0.05) the a and b fractions of DM. Effective ruminal DM degradability for this processing time were lower (P<0.05) than that of untreated corn. There were differences (P<0.05) in CP and starch degradation parameters among untreated, 3, 5 and 7 min microwave treated corn. For CP, all coefficients were affected (P<0.05) by microwave irradiation, reducing values for a and c, but increasing values for b, except for 7 min processing time that decreased (P<0.05) the b fraction. No differences (P>0.05) were found in ERD of CP among untreated, 3 and 5 min microwave treated corn. The effective CP degradability of 7 min microwave treated corn was lower (P<0.05) than that for untreated corn. Microwave treatments for 3 and 5 min increased (P<0.05) the a fraction and decreased the b fraction of starch. The degradation rate of the b fraction increased with 3 and 5 min processing times. Microwave irradiation for 7 min decreased (P<0.05) the a and b fractions of starch. The ERD of starch for 3 and 5 min microwave treated corn were higher (P<0.05), but for 7 min were lower (P<0.05), than that for untreated corn. 3.2. Electrophoretic profile of corn protein sub-units The molecular weight of corn proteins is depicted in Fig. 1. The four major components were prolamins (two sub-units), albumins, globulins and glutelins. From 12.5% slab gel analysis, the globulins appeared to be composed mainly of sub-units with a molecular mass
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Table 1 Dry matter, crude protein and starch degradation parameters of untreated and microwave treated corn graina Degradation traitsb (g/kg)
c (h−1 )
ERD (g/kg) at outflow rate 0.02 h−1
0.05 h−1
0.08 h−1
0.051 a 0.052 a 0.052 a 0.043 b 0.003
746 a 740 a 738 a 683 b 16.9
572 a 570 a 569 a 514 b 16.4
478 a 477 a 477 a 427 b 18.0
972 a 968 a 958 a 846 b 14.7
0.043 a 0.036 b 0.039 ab 0.037 b 0.004
700 a 657 a 656 a 568 b 16.2
512 a 461 a 457 a 391 b 14.8
415 a 365 a 359 a 305 b 13.2
988 a 989 a 992 a 931 b 10.1
0.065 c 0.084 a 0.076 b 0.063 c 0.003
803 b 843 a 841 a 753 c 14.5
646 b 706 a 704 a 604 c 12.6
554 b 620 a 620 a 518 c 13.0
a
b
a+b
Dry matter Untreated 3 min microwave 5 min microwave 7 min microwave SEM
164 a 162 a 165 a 157 b 8.3
812 a 799 a 795 a 769 b 15.6
976 a 961 a 960 a 926 b 15.3
Crude protein Untreated 3 min microwave 5 min microwave 7 min microwave SEM
115 a 92 b 68 c 59 c 8.9
857 b 876 a 890 a 787 c 12.4
Starch Untreated 3 min microwave 5 min microwave 7 min microwave SEM
201 c 234 b 267 a 194 c 6.5
787 a 755 b 725 c 737 bc 15.8
Means in the same column with different letters differ (P<0.05). a The CP, NDFom, ash and starch content of corn were 89, 78, 14 and 710 g/kg DM, respectively. b a is the soluble fraction as measured by washing loss from nylon bags; b is the potentially degradable fraction; c is the rate of degradation of fraction b (h−1 ); ERD is the effective rumen degradability of DM, CP or starch (g/kg DM, CP or starch) measured at outflow rates of 0.02, 0.05 and 0.08 h−1 .
Fig. 1. SDS-PAGE patterns of corn proteins (A, lines 1 and 2) and protein standard (A, line 3), and electrophoretic profile of untreated corn proteins incubated in the rumen (B).
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Fig. 2. Electrophoretic profile of 5 min (A) and 7 min (B) microwave treated corn proteins incubated in the rumen.
ranging from 25 to 50 kDa. Corn prolamins (i.e., zein) were easily identified after SDSPAGE and consisted of two major sub-units of 22 and 24 kDa. The SDS-PAGE slab gel analysis of untreated, 5 and 7 min microwave treated corn proteins are in Figs. 1 and 2, respectively. Each line was for the hours of ruminal incubation (i.e., 0, 2, 4, 6, 8, 12, 24 and 48 h). From the SDS-PAGE patterns, and densitometric scans (Fig. 3), albumin, globulin and glutelin sub-units of untreated and microwave treated corn were degraded at the shortest and middle incubation periods, respectively, whereas the prolamin sub-units (i.e., zein) were not degraded completely until 48 h.
4. Discussion 4.1. Ruminal DM and CP degradation In this study, the DM a fraction (164 g/kg) of untreated corn was similar to previous reports of Nocek (1987, 166 g/kg) and Herrera-Saldana et al. (1990, 186 g/kg). The DM degradation rate (0.051 h−1 ) was slower than reported by Nocek (1987, 0.091 h−1 ) but close to values reported by Herrera-Saldana et al. (1990, 0.047 h−1 ). Microwave irradiation for 7 min decreased effective DM degradability of corn, and most of this reduction was due to reduction in ERD of CP and starch. Crude protein in untreated corn had a degradability curve characterized by an a fraction of 115 g/kg, b fraction of 857 g/kg, and degradation rate (c) of 0.043 h−1 . Tamminga et al. (1990) reported a higher CP a fraction value of 150 g/kg. Variation in this fraction among studies could be due to differences in feed particle size and/or differences in analytical techniques. Grings et al. (1992) reported a similar degradation rate (0.041 h−1 ), but Nocek
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Fig. 3. Densitometrical scanning of corn (A), untreated (B) and 5 min microwave treated corn (C).
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(1987) reported a slightly faster degradation rate (0.068 h−1 ). The predicted effective CP degradability values of untreated corn at rumen outflow rates of 0.02, 0.05, and 0.08 h−1 were 700, 512 and 415 g/kg respectively, consistent with Batajoo and Shaver (1998, 400 g/kg) at an outflow rate of 0.08 h−1 . Effective CP degradability of 3, 5 and 7 min microwave treated corn at outflow rate of 0.05 h−1 decreased by 9, 10 and 23% units compared with untreated corn, respectively. These effects were mainly attributed to microwave heating, but recent reports have shown, or suggested, that there are non-thermal microwave effects in terms of energy required to produce various types of molecular transformations and alterations (Banik et al., 2003). The literature does not contain information on effects of microwave irradiation on ruminal protein degradation of feedstuffs. 4.2. Ruminal starch degradation The starch a fraction of untreated corn was 201 g/kg, supporting Herrera-Saldana et al. (1990, 210 g/kg). Tamminga et al. (1990, 276 g/kg) and Cerneau and Michalet-Doreau (1991, 265 g/kg) reported higher values. This fraction contains soluble sugars (i.e., glucose, fructose, sucrose and fructans), as well as soluble non-starch polysaccharides (i.e., arabinose, xylose, mannose, galactose and uronic acids; Aman and Hesselman, 1984). Discrepancies in reported in situ disappearance values can be attributed to varietal differences in the grain incubated, in situ technique, and/or basal diet (Nocek, 1988). The starch b fraction was 787 g/kg, higher than reported by Cerneau and MichaletDoreau (1991, 735 g/kg). As expected (Ørskov, 1986), starch in untreated corn was slowly degraded. This could be related to association of the protein matrix with starch granules, as well as type and proportion of proteins in the endosperm (Rooney and Pflugfelder, 1986; McAllister et al., 1993). Effective starch degradability of untreated corn at rumen outflow rates of 0.02, 0.05, and 0.08 h−1 were 803, 646 and 554 g/kg, respectively. Similar ERD of starch in corn have been reported by Herrera-Saldana et al. (1990, 619 g/kg) at rumen outflow rates of 0.06 h−1 , and Cerneau and Michalet-Doreau (1991, 578 g/kg) at a rumen outflow rates of 0.08 h−1 . The degradability of starch depends, in part, on the surface area of the starch granule which is exposed and able to contact starch degrading enzymes. Microwave processing for 3 and 5 min increased ERD of starch, due to an increase in starch a fraction. Starch b fractions decreased and the degradation rate of the b fraction increased with these treatments, probably because linkages between the protein matrix and the starch granule were disrupted. It seems likely that changes in the amorphous amylose part of the starch granule occurs during microwave treatment (Lewandowicz et al., 2000). Most technological treatments increased starch degradability, mainly by particle size reduction, gelatinization and protein matrix disruption. Gelatinization led to chemical and physical changes in the starch granule. Disruption of hydrogen bonds and water absorption facilitated microbial or enzyme degradation of the starch granule. Microwave irradiation for 7 min decreased the ERD of starch, due to a decrease in the starch a fraction. Lewandowicz et al. (2000) reported that microwave irradiation causes a drop in solubility and crystallinity of starch. They concluded that a higher gelatinization temperature of microwave irradiated starches may indicate an association, and a
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more stable configuration, in a granular structure. These observations suggest a great importance of the amylose content relative to susceptibility of starches to microwave treatment. Very little research has been completed on effects of microwave irradiation on chemical modification of starches (Muzimbaranda and Tomasik, 1994; Sikora et al., 1997). 4.3. Molecular weight of corn protein sub-units Several albumin and globulin polypeptides over a large range of molecular mass were separated by SDS-PAGE (Fig. 1). Corn prolamins (i.e., Zein) were easily identified after SDS-PAGE, and consisted of two major sub-units of 22 and 24 kDa. Evidence of the bifractional pattern of zein when separated by SDS-PAGE in a denatured state has been reported (Hoseney, 1986). Because corn glutelins consist of several polypeptides linked through disulfide bonds, use of reducing agents in SDS-PAGE sample buffer creates several additional protein components that can be characterized by SDS-PAGE (Landry and Moureaux, 1981). In this study, three main polypeptides, of 31, 50 and 55 kDa, were identified by SDS-PAGE analysis of glutelins residues in the presence of the reducing agent -mercaptoethanol. A similar profile of corn glutelins in 1% SDS sample buffer has been reported (Wilson et al., 1981). 4.4. Individual protein degradation in the rumen Zein, the prolamin fraction, was relatively resistant to ruminal proteolysis. Wadhwa et al. (1993) reported that, irrespective of protein source and dietary regimen, prolamin degradation was minimal in the rumen. Conversely, albumin, globulin and glutelin subunits contributed very little to the protein residues following incubation in the rumen. The hydrophobic nature of zein may be responsible for its low degradation by microorganisms (Wilson et al., 1981). The marked variability in ruminal extents of degradation also may be associated with different tertiary structures of protein sources (Nugent and Mangan, 1978). Corn prolamin sub-units are rich in non-polar amino acids and have a rod-like molecular structure compared with globular water-soluble proteins (Hoseney, 1986). Furthermore, the presence of three-dimensional disulfide linkages in corn glutelin sub-units, which increases the complexity of the tertiary structure, may limit the action of microbial proteases and reduce ruminal degradation of zein. Electrophoretic and densitometric analyses of protein residues revealed that the prolamin, albumin, globulin and glutelin sub-units of microwave treated corn were more resistant to ruminal degradation than untreated corn. Analysis of densitometric scans revealed that, by 48 h, zein represented the bulk of the residual protein for 5 and 7 min microwave treated corn. The diverse composition of corn proteins makes it difficult to set a single temperature where all protein sub-units denature or aggregate simultaneously. Heat treatment of protein will result in its denaturation, and probably transform the proteins to a more enzyme resistant structure. In addition, heat processing can result in formation of chemical cross-linkages between amino acids and reducing sugars (i.e., Maillard polymerization), or between proteins (i.e., iso-peptide bonds), reactions that will probably make feed proteins more resistant to degradation in the rumen.
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5. Conclusion The 3 and 5 min microwave irradiation had no effect on ruminal protein degradation, but increased starch degradability. Microwave heating for 7 min decreased ruminal CP and starch degradability. SDS-PAGE indicated that the bulk of the rumen undegradable protein in untreated corn was zein, while for microwave treated corn it was zein and other proteins. Further study is needed to determine effects of different microwave powers and times on corn starch degradation and protein sub-unit profiles after incubation in the rumen. Acknowledgements The authors gratefully thank the Tehran Sciences and Research Campus, Islamic Azad University, for financial support, and they would also especially like to thank Prof. A. Nikkhah for his comments, and Dr. M.M. Shahrebabak for statistical guidance and Mrs. Saeedeh Mousavy for technical assistance. References Agricultural Food Research Council, 1993. Energy and Protein Requirements of Ruminants. AFRC Technical Committee on Responses to Nutrients. CAB International, Wallingford, UK. Alton, W.J., 1998. Microwave pasteurization of liquids. Eng. Paper. 2, 98–211. Aman, P., Hesselman, K., 1984. Analysis of starch and other main constituents of cereal grains. Swed. J. Agric. Res. 14, 135–142. AOAC, 1995. Official Methods of Analysis, 16th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Banik, S., Bandyopadhyay, S., Ganguly, S., 2003. Bio-effects of microwave. Bio-resource Technol. 87, 155–159. Batajoo, K.K., Shaver, R.D., 1998. In situ dry matter, crude protein, and starch degradabilities of selected grains and by-product feeds. Anim. Feed Sci. Technol. 71, 165–176. Cerneau, P., Michalet-Doreau, B., 1991. In situ starch degradation of different feeds in the rumen. Reprod. Nutr. Dev. 31, 65–73. Fakhouri, M.O., Ramaswamy, H.S., 1993. Temperature uniformity of microwave heated foods as influenced byproduct type and composition. Food Res. Inter. 26, 89–95. Grings, E.E., Roffler, R.E., Deitelhoff, D.P., 1992. Responses of dairy cows to additions of distillers dried grains with solubles in alfalfa-based diets. J. Dairy Sci. 75, 1946–1955. Herrera-Saldana, R.E., Huber, J.T., Poore, H.M., 1990. Dry matter, crude protein, and starch degradability of five cereal grains. J. Dairy Sci. 73, 2386–2393. Hoseney, R.C., 1986. Cereal Proteins. Page 69 in Principle of Cereal Science and Technology. Am. Assoc. Cereal Chem., St. Paul, MN, USA. Huntington, G.B., 1997. Starch utilisation by ruminants: from basic to the bulk. J. Anim. Sci. 75, 852–867. Laemmli, U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 680–689. Landry, J., Moureaux, T., 1981. Physicochemical properties of maize glutelins as influenced by their isolation conditions. J. Agric. Food. Chem. 29, 1205–1211. Lewandowicz, G., Jankowsk, T., Fornal, J., 2000. Effect of microwave radiation on physico-chemical properties and structure of cereal starches. Carbohyd. Polym. 42, 193–199. Mathison, G.W., 1996. Effects of processing on the utilisation of grain by cattle. Anim. Feed Sci. Technol. 58, 113–125. McAllister, T.A., Phillippe, R.C., Rode, L.M., Cheng, K.J., 1993. Effect of the protein matrix on the digestion of cereal grains by ruminal microorganisms. J. Anim. Sci. 71, 205–212.
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