Effects of microwave irradiation on ruminal protein degradation and intestinal digestibility of cottonseed meal

Livestock Science 106 (2007) 176 – 181 www.elsevier.com/locate/livsci

Effects of microwave irradiation on ruminal protein degradation and intestinal digestibility of cottonseed meal A.A. Sadeghi a,⁎, P. Shawrang b a

b

Department of Animal Science, Faculty of Agriculture, Science and Research Branch, Islamic Azad University, P.O. Box 14515.4933, Tehran, Iran Department of Animal Science, Faculty of Agriculture, Tehran University, P.O. Box 4111, Karaj, Iran Received 4 February 2006; received in revised form 29 July 2006; accepted 1 August 2006

Abstract The objectives of this study were to determine the effects of microwave irradiation on ruminal degradability and intestinal digestibility of crude protein (CP) and to monitor the fate of true proteins of cottonseed meal (CSM) in the rumen. Duplicate nylon bags of untreated or irradiated CSM (microwave power of 800 W for 2, 4 and 6 min) were suspended in the rumen of four nonlactating Holstein cows for up to 48 h, and resulting data were fitted to non-linear degradation model to calculate degradation parameters of CP. Proteins of untreated and treated CSM bag residues were fractionated by gel electrophoresis. Intestinal digestibility of CP was measured using the mobile nylon bag technique. From in situ results, microwave irradiation decreased (P b 0.05) the water soluble fraction and degradation rate, but increased the potentially degradable fraction of CP. The effective degradability of CP decreased (P b 0.05) as processing period increased. Irradiation for 2 and 4 min increased (P b 0.05) intestinal digestibility of ruminally undegraded CP, but extending to 6 min lowered (P b 0.05) the beneficial effect that 4 min had on intestinal CP digestibility. Electrophoretic results indicated that globulin 9S in untreated CSM (whereas globulin 9S, globulin 5S and albumin 2S in microwave irradiated CSM) make the bulk of escaped protein. Based upon these results, microwave irradiation at a power of 800 W for 4 min had the greatest potential to increase digestible undegraded protein. © 2006 Published by Elsevier B.V. Keywords: Cottonseed meal; Microwave processing; Protein degradation; SDS-PAGE

1. Introduction Microwave processing has achieved a remarkable acceptance by the food industry, because heat is generated throughout the material. This effect leads to faster heating rates and shorter processing times compared to conventional heating, where heat is usually transferred from the surface to the interior. Another advantage includes energy ⁎ Corresponding author. Tel.: +98 912 6602719; fax: +98 261 2246752. E-mail addresses: [email protected], [email protected] (A.A. Sadeghi). 1871-1413/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.livsci.2006.08.006

efficiency, since most of the electromagnetic energy is converted into heat (Fakhouri and Ramaswamy, 1993). In our previous investigations (Sadeghi et al., 2005; Sadeghi and Shawrang, 2006b), effects of microwave irradiation period on protein degradation and starch fermentation in the rumen were identified. We found that 4 min irradiation period for decreasing protein degradation of soybean meal and, 5 min for increasing starch fermentation of corn grain were adequate and extending irradiation period over them had adverse effects. Proteins of cottonseed meal (CSM), which is used as a protein supplement for ruminants, are extensively

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degraded in the rumen. Conventional heat processing has been used to alter the extent of ruminal degradation of CSM protein (Broderick and Craig, 1980; Shirley et al., 1998), thus increasing escape of protein from rumen to intestine. As far as we know, information regarding effect of microwave irradiation on ruminal protein degradation of CSM and types of true protein of CSM (untreated or treated) escaping the rumen are lacking. The main objectives were to determine the effect of microwave irradiation at power of 800 W for 2, 4 and 6 min on crude protein (CP) degradation kinetics and intestinal digestibility of CSM and to monitor the fate of true proteins of untreated and microwave irradiated CSM in the rumen. 2. Materials and methods 2.1. Sample preparation and treatments Cottonseed meal samples were obtained from the Jahan oilseed manufactory located in Karaj (Iran). Cottonseed meal used in this study contained 298, 436, 86 and 67 g/kg CP, neutral detergent fiber, ether extract and ash, respectively. The DM of CSM 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 CSM to 250 g/kg. Three samples (500 g each) were subjected to microwave heating at a power of 800 W for 2, 4 and 6 min under agitation. 2.2. Animals and diets Four non-lactating Holstein cows with an average live weight of 622 ± 18 kg fitted with rumen fistulas and T-shaped duodenal cannulae were placed in individual 3.4 m × 4.9 m pens with concrete floors that were cleaned regularly. Cows were fed 15 kg dry matter; a total mixed ration containing 700 g/kg of DM of high quality alfalfa hay and 300 g/kg of DM concentrate. The concentrate consisted of ground barley, soybean meal, CSM, wheat bran, salt, dicalcium phosphate, and a vitamin + mineral premix (500, 110, 150, 210, 10, 10 and 10 g/kg DM, respectively). Diet was formulated to contain 171 g CP/kg of DM and fed twice daily at 08:00 and 16:00 h. 2.3. In situ degradability Nylon bags (9 cm × 21 cm) with a pore size of 46 μm were filled with approximately 5 g (sample size: bag surface area of 13 mg/cm2) of the samples ground to pass

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a 2 mm screen according to Nocek (1988). Duplicate bags filled with untreated or irradiated CSM were incubated in the rumen for periods of 0, 2, 4, 6, 8, 12, 16, 24 and 48 h. All bags were simultaneously placed in the rumen, just before the cows were offered their first meal in the morning (i.e., 08:00 h). After retrieved from the rumen, bags were washed with tap water and stored at − 20 °C. After thawing, bags were washed three times for 5 min in a turbine washing machine. The same procedure was applied to two bags to obtain the 0 h value. All residues were freeze-dried and analyzed for DM and CP to establish degradation kinetics of CSM. 2.4. Mobile bag technique Post ruminal disappearance of protein was determined using mobile bag technique described by De Boer et al. (1987). Approximately, 1 g of sample DM were placed in each nylon bag (3.5 cm × 6 cm, 46 μm pore size), then inserted (16 bags included of four replicates × four cows per treatment) into plastic mesh cylinders (26 cm × 8 cm, 0.57 mm pore size) and incubated in the rumen for 16 h. After removal from the rumen, the bags were washed with tap water and incubated in a pepsinhydrochloric acid solution (pH 2) at 37.5 °C for 3 h with constant stirring as described by Kirkpatrick and Kennelly (1984). After rumen and pepsin digestion the bags were inserted into the small intestine via the duodenal cannulae at the rate of one bag every 15 min, then removed from the voided faeces, rinsed in cold running water and then machine washed. Finally, all residues were freeze-dried and weighed to determine DM and CP disappearance. 2.5. Monitoring of protein subunits The protein subunits were fractionated by an SDSPAGE discontinuous system according to the method of Laemmli (1970). All ruminally undegraded fractions from each incubation period were freeze-dried, ground and replicates pooled. Then 20 mg of dried untreated and treated CSM were placed into 750 μl SDS-PAGE sample buffer. After 30 min of mixing, samples were immersed at 90 °C for 3 min, and then centrifuged at 10,000×g for 1 min. A 30 μl aliquot of each protein sample was then loaded into the sample well. Electrophoresis of proteins was performed on 12.5% resolving gel (1.0 × 110 × 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.

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The gel destaining was accomplished by using a 300 ml/ l methanol and 70 ml/l acetic acid solution. The subunits of the gel were then monitored by densitometric scanning at 580 nm. One standard protein mixture containing β-galactosidase (116 kDa), bovine plasma albumin (66.0 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), restriction endonuclease Bsp981 (25.0 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa) was used. 2.6. Chemical analyses Cottonseed meal samples were analyzed for DM by freeze-drying a 1 g sample in duplicate. The nitrogen in feeds and residues after rumen and intestinal incubation was determined according to AOAC (Method 984.13; AOAC, 1995). Table 1 Ruminal degradation characteristics of dry matter and crude protein of untreated and microwave irradiated cottonseed meal Parameters

Untreated cottonseed meal

Microwave irradiated cottonseed meal at power of 800 W for:

S.E.M.

2 min

4 min

6 min

0.226b 0.687b 0.913b 0.061b

0.198c 0.713a 0.911b 0.047c

0.184d 0.721a 0.905c 0.043c

0.0091 0.0124 0.0150 0.0054

Effective rumen degradation 0.02/h 0.796a 0.743b a 0.05/h 0.678 0.603b 0.08/h 0.605a 0.523b

0.698c 0.543c 0.461c

0.676d 0.517d 0.436d

0.0130 0.0127 0.0111

Crude protein a b a+b c (/h)

0.143c 0.796a 0.939b 0.038c

0.119d 0.814b 0.933b 0.035d

0.0065 0.0104 0.0132 0.0056

0.664c 0.486c 0.399c

0.637d 0.454d 0.366d

0.0123 0.0116 0.0108

Dry matter a b a+b c (/h)

0.301a 0.625c 0.926a 0.076a

0.251a 0.706d 0.957a 0.052a

0.188b 0.758c 0.946ab 0.044b

Effective rumen degradation 0.02/h 0.760a 0.709b 0.05/h 0.610a 0.543b a 0.08/h 0.529 0.457b

Intestinal crude protein digestibility at 12 h ruminal incubation period 0.522d 0.578c 0.651a 0.619b 0.0134

Fig. 1. Molecular weights of standard protein (line 1) and subunits of cottonseed meal protein (line 2).

2.7. Statistical analyses Disappearances data of DM or CP (including 0 h values) were fitted for each cow to the exponential model of Ørskov and McDonald (1979) as: P = a + b(1 − e−ct ). In this model, the constants “a” and “b” represent, respectively, the water soluble fraction and the non-soluble but degradable component, which disappears at a constant fractional rate “c” per unit time. The effective rumen degradability (ERD) was calculated using ERD = a + bc / (c + k), assuming outflow rates (k) of 0.02, 0.05 and 0.08/h. Data were analyzed as a randomized complete block design, using animals as blocks. Analyses were carried out using the general linear models procedure of Statistical Analysis System (SAS, 1996). When a significant difference was found, means were separated using Duncan test (Steel and Torrie, 1980). Differences were considered to be significant if P b 0.05. 3. Results 3.1. Effects on ruminal degradability and intestinal digestibility

a, b, c, d

Means in the same row with different superscripts differ (P b 0.05). a, soluble fraction of feed DM or CP as measured by washing loss from nylon bags; b, potentially degradable fraction of feed DM or CP; c, rate of degradation of fraction b (/h); ERD, effective rumen degradability of DM or CP measured at rumen outflow rate k = 0 02, 0.05, and 0.08/h.

The in situ DM and CP degradability parameters of untreated and microwave irradiated CSM are shown in Table 1. There were differences (P b 0.05) for DM and CP degradation parameters among untreated, 2, 4 and 6 min microwave irradiated CSM. Increasing irradiation

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Fig. 2. Densitometrical scanning of cottonseed meal proteins (line 2 in Fig. 1).

time decreased (P b 0.05) the water soluble fraction “a” and increased (P b 0.05) the potentially degradable fraction “b” of DM and CP. The degradation rate of the b fraction decreased (P b 0.05) with increases in irradiation time. Effective degradability of DM and CP decreased (P b 0.05) as irradiation time increased. The effective CP degradability of 2, 4 and 6 min microwave irradiated CSM at outflow rate of 0.05/h decreased (P b 0.05) by 11%, 20% and 25%, respectively. Compared to untreated CSM, microwave irradiation for 2, 4 and 6 min increased (P b 0.05) intestinal CP

digestibility. Extending the irradiation period of CSM to 6 min lowered (P b 0.05) the beneficial effect that 4 min had on intestinal CP digestibility. 3.2. Effects on subunits profile of cottonseed meal true proteins The molecular weights of CSM proteins and its densitometrical scanning are depicted in Figs. 1 and 2, respectively. Three major protein components were observed: globulin 9S, globulin 5S and albumin 2S,

Fig. 3. Electrophoretic patterns of untreated (a) and 4 min microwave irradiated cottonseed meal (b) incubated in the rumen for various times up to 48 h.

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accounting for approximately 30%, 35% and 35% of buffer soluble CSM proteins, respectively. The approximate molecular weights of globulin 9S subunits were 55.2 and 48.8 kDa; 24.1, 23.4 and 21.5 kDa for globulin 5S subunits, and 19.8, 15.2 and 13.9 kDa for albumin 2S subunits. Electrophoretic patterns of untreated and 4 min microwave irradiated CSM proteins are depicted in Fig. 3. Electrophoretic analysis of untreated CSM proteins (Fig. 3a) revealed that the subunits of albumin 2S were degraded completely within shortest incubation period, but the subunits of globulin 9S as well as globulin 5S were more resistant to degradation. In microwave irradiated CSM for 2, 4 and 6 min, three subunits of albumin 2S were degraded in the middle of incubation period, whereas the subunits of globulin 5S were degraded in the longest incubation period. The subunits of globulin 9S were not degraded completely even after 48 h of incubation time. 4. Discussion 4.1. Ruminal degradation and intestinal digestibility of cottonseed meal Degradation parameters and effective degradability of CP are in the normal range for untreated CSM reported previously (Broderick and Craig, 1980; Shirley et al., 1998). A decrease in protein solubility resulting from microwave irradiation, observed in the present work, gives evidence of the occurring cross-linking of chains and proteins aggregation through heating. Solubility of proteins relates to surface hydrophobic (protein–protein) and hydrophilic (protein–solvent) interaction. When secondary and tertiary structures of a protein through heating are unfolded, the hydrophobic groups interact and reduce water binding (Taha and Mohamed, 2004). Microwave effects were mainly attributed to heating, but recent reports (Banik et al., 2003) have shown that there are non-thermal microwave effects in terms of energy required to produce various types of protein transformations and alterations. In the literature, we could not find references dealing with the effects of microwave irradiation on protein degradation of CSM in the rumen. Previous experiments have shown that rate and extent of ruminal degradation of CSM protein are reduced due to conventional heat treatment (Broderick and Craig, 1980; Shirley et al., 1998). In our previous investigations (Sadeghi et al., 2005; Sadeghi and Shawrang, 2006a), microwave irradiation of soybean meal and Canola meal for 2 and 4 min decreased protein degradation and increased in vitro CP digestibility, but

irradiation period over 4 min decreased in vitro digestibility of CP. Irradiation of CSM could modify the molecular properties of the proteins, which result in alteration of CSM proteins by covalent cross-linkages formed in proteins after heating. Proteins can be converted to higher molecular weight aggregates, due to the generation of inter-protein cross-linking reactions, hydrophobic and electrostatic interactions. The cross-linking process results in formation of chemical bonds between two adjacent protein molecules. This reaction increases the molecular weight of the protein until the material is eventually bound into an insoluble three-dimensional network (Englard and Seifter, 1990). In this condition, accessibility of protein molecule will probably reduce for microbial attack. Microwave irradiation for 4 min increased intestinal digestibility of CP, because of denaturation. Irradiation may induce the unfolding of the protein and denaturation, exposing hydrophobic amino acids (especially aromatics) that are position groups for active site of pepsin and trypsin enzymes (Murray et al., 2003). Irradiation for 6 min decreased CP digestibility. Several of the reactions responsible for the reduction in ruminal degradation (i.e., the Maillard reaction) may also reduce intestinal digestibility of protein. 4.2. SDS-PAGE analysis of untreated and treated CSM proteins Cottonseed contains three major classes of proteins, 2S, 5S and 9S, in approximately equal amounts. The 5S and 9S proteins are typical globulin storage proteins while the 2S proteins are albumins but are also classified as storage proteins based upon their amino acid composition, developmental properties and high levels in the seed as indicated by Youle and Huang (1979). Because of their abundance, these proteins are mainly responsible for the nutritional quality of CSM. From the slab gel analysis of cottonseed protein, molecular weights of 55.2, 48.8, 24.1, 23.4, 21.5, 19.8, 15.2 and 13.9 kDa for subunits of CSM were estimated in this study. These findings agreed with the report of Youle and Huang (1979). In microwave irradiated CSM, there were crosslinked products of the degraded protein molecules that could not penetrate the running gels (Fig. 3b). Heat treatment of protein will result in denaturation of protein and probably transform the proteins to a more resistant structure. In addition, heat processing can result in formation of cross-linkages between amino acids and reducing sugars (i.e., the Maillard reaction), or between

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proteins (i.e., iso-peptide bonds). In inherent folded structure of globular proteins, many hydrophobic amino acids are buried inside the protein. Heating induces the unfolding of the protein and denaturation, thus exposing non-polar groups that were previously blocked. The hydrophobic interactions lead to aggregation, followed by coagulation and precipitation (Englard and Seifter, 1990). These reactions will probably make the feed protein more resistant against degradation in the rumen. 5. Conclusion The results of this study indicated that microwave irradiation of CSM at power of 800 W for 4 min appeared to be an effective means of increasing digestible rumen undegradable protein content. Electrophoretic analysis revealed that globulin 9S in untreated CSM (whereas globulin 9S, globulin 5S and albumin 2S in microwave irradiated CSM) make the bulk of escaped protein. Further study is needed to examine effects of different microwave intensities and time periods applied to CSM on protein subunits profile after incubation in the rumen and other nutritional properties of CSM, particularly on gossypol content. Acknowledgment The authors are grateful to the Tehran Science and Research Branch, Islamic Azad University for financial support and Tehran University for allowing use of cows. They would also like to thank Prof. A. Nikkhah for his comments, Dr. M. Moradi for statistical guidance, Mr. Davoodi for preparing cottonseed meal samples in Jahan oilseed manufactory and Mr. Noori and Mrs. S.S. Mosavi for laboratory assistance. References 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. Bioresour. Technol. 87, 155–159.

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