Chemical characterization and in vitro crude protein degradability of thin stillage derived from barley- and wheat-based ethanol production

Animal Feed Science and Technology 80 (1999) 247±256

Chemical characterization and in vitro crude protein degradability of thin stillage derived from barley- and wheat-based ethanol production A.F. Mustafa*, J.J. McKinnon, D.A. Christensen Department of Animal and Poultry Science, University of Saskatchewan, 72 Campus Drive, Saskatoon, SK, Canada S7N 5B5 Received 22 November 1998; received in revised form 12 April 1999; accepted 27 April 1999

Abstract A study was conducted to characterize the different carbohydrate and protein fractions of wheatand barley-based thin stillage samples. In vitro crude protein degradability of wheat- and barleybased thin stillage was estimated relative to soyabean (SBM) and canola (CM) meal using a protease enzyme assay. Results of the carbohydrate analysis showed that wheat thin stillage had similar neutral (NDF, average 328.5 g kgÿ1) and lower (P < 0.05) acid detergent fibre (ADF) than barley-based thin stillage. Relative to barley-based thin stillage, wheat thin stillage had higher (P < 0.05) crude protein (CP) and soluble CP content. However, the amount of CP associated with NDF and ADF was higher (P < 0.05) in barley-based thin stillage than in wheat thin stillage. Fractionation of true protein showed that most of the CP (average 707 g kgÿ1 of CP) was present in the slowly degradable true protein fraction and was similar in both byproducts. Glutamic acid was the main amino acid in thin stillage and was higher (P < 0.05) in wheat than in barley-based thin stillage. However, barley-based thin stillage had higher (P < 0.05) levels of lysine, methionine, arginine, threonine, leucine and isoleucine than wheat thin stillage. Results of the in vitro trial indicated that effective degradability of CP (g kgÿ1 of CP) followed the order (P < 0.05): SBM (665.0) > wheat thin stillage (614.0) > CM (531.0) > barley-based thin stillage (493.0). It was concluded that barley-based thin stillage had different chemical characteristics than wheat thin stillage. The reduced CP degradability of barley-based thin stillage relative to wheat thin stillage was attributed to a lower CP and a higher acid detergent in soluble CP level. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Thin stillage; Ethanol production; Barley; Wheat; Protein degradability *

Corresponding author. Tel.: +1-3069664156; fax: +1-3069664151 E-mail address: [email protected] (A.F. Mustafa) 0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 6 3 - 2

248

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

1. Introduction Whole stillage is the byproduct remaining from cereal grain-based ethanol production. The fermentation process utilizes starch while other nutrients such as protein and fibre are concentrated. Stillage can be fractionated into wet distillers grains and thin stillage which can be fed separately in wet form or dried and marketed as dried distillers grains or dried distillers grains plus solubles (Larson et al., 1993; Ham et al., 1994). Corn is by far the most common substrate used in the distilling industry (Lee et al., 1991). Fractionation and chemical characterization of corn thin stillage was previously reported (Wu et al., 1981). On dry matter basis, corn thin stillage contained 220 g kg ÿ1 starch, 160 g kgÿ1crude protein, 117 g kgÿ1neutral detergent fibre and 81 g kgÿ1 fat (Larson et al., 1993). In western Canada, wheat is the main grain used for ethanol production. Some of the chemical characteristics and the feeding value of wheat thin stillage were previously reported. Ojowi et al. (1996) showed that wheat-based thin stillage contained 84, 485, 96, 345 and 34 g kgÿ1 ether extract, crude protein, neutral and acid detergent fibre, respectively. The authors also found that feeding thin stillage as a fluid source improved daily gain and fat deposition of growing cattle grazing crested wheatgrass. Barley grain is another important crop grown in western Canada and can be used alone or in combination with wheat to reduce the cost of ethanol production. However, due to differences in hull and starch content, barley-based thin stillage and distillers grains are expected to have different chemical composition and feeding value than the corresponding wheat-based byproducts. Limited data are available on the chemical composition and ruminal protein degradability characteristics of barley-based thin stillage. The objectives of the present study were to determine the chemical composition of thin stillage derived from primarily a barley-based fermentation relative to that obtained from 100% wheat and to determine their crude protein degradability relative to canola and soyabean meal. 2. Materials and methods 2.1. Sample preparation and chemical analysis Samples of barley-based and wheat thin stillage (n = 5) were supplied by the PoundMaker Agventures, ethanol plant at Lanigan, Saskatchewan. The barley-based thin stillage samples were derived from fermentation runs where the fermentation substrate consisted of a 700 g kgÿ1 barley, 200 g kgÿ1 wheat and 100 g kgÿ1 rye/triticale mixture. The wheat thin stillage was derived from fermentation runs using only wheat as the substrate. Samples were freeze dried and analyzed for moisture, ash, ether extract (EE), crude protein (CP, Kjeldahl N x 6.25), acid detergent fibre (ADF) and acid detergent lignin (ADL) according to the methods of the Association of the Official Analytical Chemists (AOAC, 1990). Neutral detergent fibre was determined according to Van Soest et al. (1991). Neutral and acid detergent insoluble CP were determined on NDF and ADF

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

249

residues, respectively, using the Kjeldahl method (AOAC, 1990). Buffer soluble protein was determined according to the procedure of Roe et al. (1990). Sodium tungstate was used as a precipitating agent for measuring non-protein nitrogen (Licitra et al., 1996). Total starch was determined using the a-amylase amyloglucosidase method (Megazyme kit, NSW, Australia). The equations of Sinffen et al. (1992) were used to fractionate total carbohydrate and CP based on rate of rumen degradation. For carbohydrate the following fractions were estimated: fraction A (rapidly degradable), fraction B1 (intermediately degradable), fraction B2 (slowly degradable) and fraction C (unavailable cell wall). Total CP was fractionated into fraction A (non-protein nitrogen), fraction B (true protein) and fraction C (unavailable protein). True protein was further sub-fractionated into B1 (highly degradable), B2 (intermediately degradable) and B3 (slowly degradable) fractions. Samples of wheat- and barley-based thin stillage were analyzed for amino acids (AOAC, 1984) following oxidation in performic acid (16 h) and hydrolysis in 6 N HCl (24 h), respectively. The oxidation step was not used for phenylalanine, tyrosine and histidine. Tryptophan was measured after alkaline hydrolysis (Miller, 1967). All amino acids were determined using a Brinkmann (System 6300) High Performance Amino Acid Analyzer. 2.2. In vitro protein degradability The procedure of Roe et al. (1990) was used to estimate in vitro CP degradability of barely-based and wheat thin stillage. Equal portions (100 g) of the five barley-based and wheat thin stillage were composited to obtain a single batch of each byproduct. Samples of canola and soyabean meal used in a previous study (Mustafa et al., 1997b) were included for comparison purposes. Duplicate samples containing the equivalent of 0.2 g of air dry CP were weighed into 125 ml Erlenmeyer flasks and incubated in 40 ml of borate phosphate buffer (pH 6.7) at 398C for 1 h. Following incubation, 10 ml of fresh protease solution (0.33 units mlÿ1 protease enzyme from Streptomyces griseus, type XIV, Sigma, St. Louis, Mo) was added to each flask and the samples were incubated for 2, 4, 8, 12, 18, 24, 36 and 48 h at 398C. Zero hour disappearance was estimated by soaking samples in 40 ml borate phosphate buffer without the enzyme for 1 h at 398C. At the end of each incubation time, insoluble residues were filtered through Whatman No. 54 filter paper and residual nitrogen was determined using the Kjeldahl method (AOAC, 1990). The experiment was repeated three times for replication. Disappearance of CP at each incubation time was determined by subtracting residual CP from original CP. The data were then fitted to the equation of érskov and McDonald (1979): p ˆ a ‡ b…1ÿeÿct † where p (g kgÿ1) is CP disappearance at time t (h), a (g kgÿ1) is rapidly soluble CP, b (g kgÿ1) is insoluble but degradable CP and c (% hÿ1) is the rate constant at which b is degraded. The constants, a, b and c were estimated by an iterative least-square method using the nonlinear regression procedure of the Statistical Analysis System Institute (1989) with the constraints that a + b 4 100. In vitro effective CP degradability (ECPD)

250

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

of each protein supplement was calculated according to the equation of érskov and McDonald (1979):   c ECPD ˆ a ‡ b c‡k where k is the rumen flow rate (5% hÿ1). 2.3. Statistical analysis All data were analyzed as a completely randomized design using the General Linear Model procedure of Statistical Analysis System Institute (1989). For chemical composition data, number of replicates was five while for the in vitro study, the number of replicates was three. When a significant difference was found, means were separated using the Student Newman's Keul procedure (Steel and Torrie, 1980). 3. Results and discussion 3.1. Chemical Composition of Thin Stillage Dry matter content of barley-based and wheat thin stillage was 59.7  11.9 and 84.0  3.5 g kgÿ1, respectively. Relative to wheat thin stillage, barley-based thin stillage had higher (P < 0.05) ash but similar ether extract levels (Table 1). Ojowi et al. (1996) reported a lower EE (96 g kgÿ1) level for wheat thin stillage than the value reported in this study. Similarly, Ham et al. (1994) and Larson et al. (1993) reported a lower EE value (92 g kgÿ1) for corn-based thin stillage. Carbohydrate analysis (Table 1) indicated that barley-based thin stillage had higher (P < 0.05) total and non-structural carbohydrate and ADF levels than wheat thin stillage. However, both byproducts had similar NDF, ADL and starch contents. The NDF and ADF values reported for wheat thin stillage are in good agreement with those reported by Ojowi et al. (1996). However, barley-based and wheat thin stillage used in this study contained more NDF and less starch than corn thin stillage (Larson et al., 1993; Ham et al., 1994). The low residual starch levels in barley-based and wheat thin stillage indicate that the fermentation process was efficient in removing starch from barley and wheat grain. The main carbohydrate fraction in barley-based and wheat thin stillage was the A fraction (rapidly degradable) and was higher (P < 0.05) in barley-based than wheat thin stillage (Table 1). According to Sniffen et al. (1992) the A carbohydrate fraction consists of soluble sugars and pectins. These results imply that most of the carbohydrate in both byproducts should be highly degradable in the rumen. No difference was observed in the B1 (intermediately degradable), B3 (slowly degradable) and C (unavailable carbohydrate) fractions between barley-based and wheat thin stillage. Total CP analysis and fractionation is presented in Table 2. Relative to barley-based thin stillage, wheat thin stillage had higher (P < 0.05) CP, soluble CP and non-protein nitrogen content. However, the amount of CP associated with NDF and ADF were higher (P < 0.05) in barley-based than in wheat thin stillage. A Similar CP value was reported

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

251

Table 1 Ash, ether extract and carbohydrate fractions of barley- and wheat-based thin stillage (DM basis, n = 5) SEMa

Thin stillage Barley-based ÿ1

Wheat-based

Ash (g kg ) Ether extract (g kgÿ1)

b

97.0 128.0

83.0c 136.0

4.1 13.5

Carbohydrate analysis (g kgÿ1) Neutral detergent fibre (NDF) Acid detergent fibre Acid detergent lignin (g kgÿ1 of NDF) Starch Nonstructural carbohydrate (NSC) Starch (g kgÿ1 of NSC) Total carbohydrate

317.0 77.0b 43.0 7.0 376.0b 18.0 404.0b

340.0 40.0c 35.0 22.0 281.0c 74.0 317.8c

13.7 4.5 5.7 7.3 16.7 20.1 12.2

Carbohydrate fractions (g kgÿ1 of DM) A (Rapidly degradable) B1 (Intermediately degradable) B2 (Slowly degradable) C (Unavailable)

359.0b 6.0 9.0 32.0

259.0c 22.0 19.0 29.0

13.1 7.3 6.8 5.0

a

SEM = Pooled standard error of the mean. Means in the same row with different superscripts are different (P < 0.05). c Means in the same row with different superscripts are different (P < 0.05). b

for wheat thin stillage by Ojowi et al. (1996). However, Wu (1986) reported a lower CP level for barley thin stillage than the average value reported in this study. The CP levels of barley-based and wheat thin stillage in our study are higher than those reported for corn thin stillage (Larson et al., 1993; Ham et al., 1994). Total true protein content was similar Table 2 Crude protein analysis and fractionation of barley- and wheat-based thin stillage (DM basis, n = 5) SEMa

Thin stillage Barley-based

Wheat-based

Crude protein analysis Crude protein (CP, g kgÿ1) Soluble protein (g kgÿ1 of CP) Non-protein nitrogen (g kgÿ1 of CP) Neutral detergent insoluble protein (g kgÿ1 of CP) Acid detergent insoluble protein (g kgÿ1 of CP)

371.0b 190.0b 170.7b 749.0c 115.0c

457.0c 295.0c 286.1c 652.0b 7.8b

12.8 13.6 12.5 14.3 5.6

True protein fractions (g kgÿ1 of CP) Total B1 (Rapidly degradable) B2 (Intermediately degradable) B3 (Slowly degradable)

714.6 18.9 52.0 643.7

699.3 13.0 65.3 631.0

16.7 8.3 10.7 9.8

a

SEM = Pooled standard error of the mean. Means in the same row with different superscripts are different (P < 0.05).

b,c

252

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

Table 3 Amino acid (AA) composition of barley- and wheat-based thin stillage (DM basis, n = 5) SEMa

Thin stillage Barley-based

Wheat-based

ÿ1

Essential (g kg of AA) Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Non-essential (g kgÿ1 of AA) Alanine Aspartic acid Cystine Glutamic acid Glycine Proline Serine Total AA (g kgÿ1 of CP) a

47.5b 3.9c 43.4b 79.8b 38.5b 19.6b 21.4c 39.6b 3.6 55.9b

38.5c 19.7b 40.6c 74.9c 22.2c 17.2c 45.3b 33.1c 4.6 49.7c

1.85 0.65 0.52 0.82 2.54 0.53 0.48 0.25 0.32 0.64

46.7b 63.4b 23.8b 298.2c 44.8 122.0 48.5b 865.1b

39.2c 53.2c 21.4c 339.6b 43.3 113.7 47.7c 832.5c

0.52 1.62 0.36 5.82 0.84 2.67 0.19 6.61

SEM = Pooled standard error of the mean. Means in the same row with different superscripts are different (P < 0.05).

b,c

in barley-based and wheat thin stillage (average 707.0 g kgÿ1 of CP). The slowly degradable true protein (B3) fraction constituted most of the true protein and was similar in both byproducts (average 637.4 g kgÿ1 of CP). The amino acid composition showed high levels of glutamic acid and proline for both barley-based and wheat thin stillage (Table 3). Similar results were previously reported for barley (Wu, 1986), wheat (Wu et al., 1981) and sorghum (Wu and Sexson, 1984) thin stillage. Relative to wheat thin stillage, barley-based thin stillage contained more (P < 0.05) arginine, isoleucine, leucine, lysine, methionine, threonine, valine, aspartic acid, cystine and serine. However, wheat thin stillage had higher (P < 0.05) levels of histidine, alanine and glutamic acid than barley-based thin stillage. These results suggest that in terms of amino acid composition, protein quality of barley-based thin stillage is superior to wheat thin stillage. In agreement with our findings, Wu (1986) concluded that thin stillage derived from barley had a superior amino acid composition than the corresponding product derived from corn, wheat or sorghum. Data on ruminal degradability of thin stillage amino acids are not available. However, several studies with other protein sources such as canola meal (Mustafa et al., 1997a), meat and bone meal, and poultry byproducts (Klemesrud et al., 1997) have shown that non-essential amino acids are more degradable than non-essential amino acids. If one

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

253

Table 4 Mineral composition of barley- and wheat-based thin stillage (DM basis, n = 5) SEMa

Thin stillage Barley-based ÿ1

Calcium (g kg ) Phosphorous (g kgÿ1) Magnesium (g kgÿ1) Copper (mg kgÿ1) Iron (mg kgÿ1) Manganese (mg kgÿ1) Sodium (mg kgÿ1) Potassium (mg kgÿ1) Zinc (mg kgÿ1) a

b

5.3 11.3 5.4c 5.4 493.2b 52.2c 0.6b 1.6 84.5b

Wheat-based 4.2c 12.1 5.9b 5.7 419.1c 110.1b 0.2c 1.6 63.8c

0.07 0.45 0.13 0.61 19.00 1.92 0.02 0.01 0.86

SEM = Pooled standard error of the mean. Means in the same row with different superscripts are different (P < 0.05).

b,c

were to extrapolate these results to the present study, one could conclude that most of the glutamic acid and proline in thin stillage will be degraded in the rumen. Barley-based thin stillage contained more (P < 0.05) calcium, iron, sodium and zinc than wheat thin stillage (Table 4). However, magnesium and manganese levels were higher (P < 0.05) in wheat than in barley-based thin stillage. Data on the mineral composition of thin stillage are limited. However, the mineral concentrations of barleybased and the wheat thin stillage in this study were higher than those of wheat and barley grains as reported by Boila et al. (1993) suggesting again that the fermentation process concentrates nutrients. Lee et al. (1991) showed that thin stillage contained higher ash levels than distillers grains or original cereal grains. 3.2. In vitro protein degradability of thin stillage The excessive fineness of barley-based and wheat thin stillage dry matter makes it difficult to estimate ruminal protein degradability using the nylon bag technique. Other researchers have used in vitro techniques based on protease enzymes to measure protein degradability of different protein supplements (Assoumani et al., 1992; Susmel et al., 1993). In this study canola and soyabean meal were included for comparison purposes. In vitro CP disappearance at different incubation times showed significant differences between treatments (Fig. 1). For the majority of the incubation times, CP disappearance followed the order (P < 0.05): soyabean meal > wheat thin stillage > canola meal > barley-based thin stillage. However, at 0 h incubation the order was (P < 0.05) canola meal > wheat thin stillage > soyabean meal > barley-based thin stillage. The rapidly soluble CP fraction was highest (P < 0.05) for wheat thin stillage and canola meal, intermediate for soyabean meal and lowest (P < 0.05) for barley-based thin stillage (Table 4). This is consistent with the chemical analysis data (Table 1) which showed a higher buffer soluble protein value for wheat than barley-based thin stillage. The results also agree with other studies which reported higher rapidly

254

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

Fig. 1. In vitro crude protein disappearance (CPD) of barley- and wheat-based thin stillage relative to canola and soyabean meal.

soluble CP for canola meal than soyabean meal (Khorasani et al., 1994; Mustafa et al., 1997b). The slowly degradable CP fraction was higher (P < 0.05) for barley-based than wheat thin stillage and canola meal and was higher (P < 0.05) for soyabean meal than barleybased thin stillage (Table 4). No difference in potentially degradable CP was observed between wheat thin stillage and canola meal. In accordance with these results, Mustafa et al. (1997b) and Khorasani et al. (1994) found that soyabean meal contained higher potentially degradable CP than canola meal. Rate of degradation of potentially degradable CP had the order (P < 0.05) soyabean meal > wheat thin stillage > barleybased thin stillage > canola meal. The lower rate of CP degradation of barley-based thin stillage relative to wheat thin stillage can be attributed at least in part, to the higher level of unavailable CP in barley-based thin stillage. This would agree with the work of Boila and Ingalls (1994) who reported a reduced ruminal rate of CP degradation of dried distillers grains as a result of high levels of acid detergent insoluble CP. The higher rate of CP degradation of soyabean meal relative to canola meal in the present study is supported by the findings of Mustafa et al. (1997b). In vitro effective CP degradability was highest (P < 0.05) for soyabean meal and lowest (P < 0.05) for barley-based thin stillage (Table 5). Canola meal had lower (P < 0.05) effective CP degradability than wheat thin stillage

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

255

Table 5 In vitro crude protein (CP) kinetic parameters and effective degradability of barley- and wheat-based thin stillage relative to canola and soyabean meal (n = 3) Canola meal Soyabean meal SEMa

Thin stillage Barley-based Wheat-based ÿ1

Soluble (g kg of CP) Slowly degradable (g kgÿ1 of CP) Degradation rate (% hÿ1) Effective degradability (g kg-1)f

186.0b 500.0d 7.9b 493.0e

294.0c 457.0b 11.8d 614.0d

301.0c 442.0b 5.5e 531.0b

229.0d 561.0c 17.4c 665.0c

12.4 10.9 0.5 5.2

a

SEM = Pooled standard error of the mean. Means in the same row with different superscripts are different (P < 0.05). f Calculated assuming rumen flow rate of 5% hÿ1. b,c,d,e

but higher (P < 0.05) than barley-based thin stillage (Table 3). Decreased effective degradability for barley-based thin stillage relative to wheat thin stillage may have resulted from a combination of lower soluble CP and rate of degradation of potentially degradable CP. The results of the present study showed differences in effective CP degradability between barley-based and wheat thin stillage despite the fact that no differences in the true protein fractions were found between the two types of thin stillage (Table 2). This might indicate that CP degradability of thin stillage was more affected by the level of acid detergent insoluble CP than by the other protein fractions. The results of this experiment also suggest that barley-based thin stillage is a better source of rumen undegraded protein than wheat thin stillage. 4. Conclusions The results reported in the present study indicate that thin stillage derived primarily from barley, is different from wheat. In terms of chemical composition, the barley-based thin stillage had more fibre and less protein than the wheat thin stillage. The concentration of most amino acids was also higher in the barley-based than in the wheat thin stillage. Differences in CP content as well as distribution of CP between different cell wall components resulted in wheat thin stillage having a higher in vitro CP degradability than barley-based thin stillage. Data from this study indicate that thin stillage derived from fermentation of a mixture of cereal grains consisting of barley, wheat, rye, and triticale exhibit lower ruminal protein degradability and higher amino acid concentrations than wheat thin stillage. Acknowledgements The authors would like to thank the Applied Technology Group at the Degussa Corporation (NJ, USA) for the amino acid analysis and the Pound-Maker Agventures, ethanol plant at Lanigan, Saskatchewan for providing the thin stillage samples.

256

A.F. Mustafa et al. / Animal Feed Science and Technology 80 (1999) 247±256

References Association of Official Analytical Chemists, 1984. Official methods of analysis, 14th ed., AOAC, Arlignton, VA. Association of Official Analytical Chemists, 1990. Official methods of Analysis, 15th ed., Association of Official analytical chemists, Washington, DC. Assoumani, M.B., Vedeau, F., Jacquot, L., Sinffen, C.J., 1992. Refinement of an enzymatic method for estimating the theoretical degradability of proteins in feedstuffs for ruminants. Anim. Feed Sci. Technol. 39, 357±368. Boila, R.J., Ingalls, R.J., 1994. The ruminal degradation of dry matter, nitrogen and amino acids in wheat-based distillers' grains in sacco. Anim. Feed Sci. Technol. 48, 57±72. Boila, R.J., Campbell, L.D., Stothers, G.H., Crow, G.H., Ibrahim, E.A., 1993. Variation in the mineral content of cereal grains grown at selected locations throughout Manitoba. Can. J. Anim. Sci. 73, 421±429. Ham, G.A., Stock, R.A., Klopfenstein, T.J., Larson, E.M., Shain, D.H., Hanke, H.E., 1994. Wet corn distillers byproducts compared with dried corn distillers grains with solubles as a source of protein and energy for ruminants. J. Anim. Sci. 72, 3246±3257. Khorasani, G.R., Robinson, P.H., Kennelly, J.J., 1994. Evaluation of solvent and expeller linseed meals as protein sources for dairy cattle. Can. J. Anim. Sci. 74, 479±485. Larson, E.M.R.A., Stock, R.A., Klopfenstein, T.J., Sind, M.H., Huffman, R.P., 1993. Feeding value of wet distillers byproducts for finishing ruminants. J. Anim. Sci. 71, 2228±2236. Lee, W.J., Sosulski, W.F., Sokhansanj, S., 1991. Yield and composition of soluble and insoluble fractions from corn and wheat stillages. Cereal Chem. 68, 559±562. Licitra, G., Mernandez, T.M., Van Soest, P.J., 1996. Standardization of procedures for nitrogen fractionation of ruminant feeds. Anim. Feed Sci. Technol. 57, 347±358. Miller, E.L., 1967. Determination of the tryptophan content of feedstuffs with particular reference to cereals. J. Sci. Food and Agric. 18, 381±386. Mustafa, A.F., McKinnon, J.J., Christensen, D.A., 1997a. In situ amino acid disappearance from regular, low and high fiber canola meal. Can. J. Anim. Sci. 77, 533±535. Mustafa, A.F., McKinnon, J.J., Thacker, P.A., Christensen, D.A., 1997b. Effect of borage meal on nutrient digestibility and performance of ruminants and pigs. Anim. Feed Sci. Technol. 64, 273±285. Ojowi, M.O., Christensen, D.A., Mckinnon, J.J., Mustafa, A.F., 1996. Thin stillage from wheat based ethanol production as a nutrient supplement for cattle grazing crested wheatgrass pastures. Can. J. Anim. Sci. 76, 547±553. érskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J. Agric. Sci. 92, 499±503. Roe, M.B., Sniffen, C.J., Chase, L.E., 1990. Techniques for measuring protein fractions in feedstuffs. In: Proc. Cornell Nutr. Conf., Ithaca, NY, p. 81. Sniffen, C.J., O,Connor, J.D., Van Soest, P.J., Fox, D.J., Russell, J.B., 1992. A net carbohydrate and protein system for evaluating cattle diets II. Carbohydrate and protein availability. J. Anim. Sci. 70, 3562±3577. Statistical Analysis System (SAS) Institute, 1989. SAS user's guide: Statistics. SAS Institute, Cary, NC. Steel, R.G., Torrie, J.H., 1980. Principles and Procedures of Statistics, 2nd ed., McGraw-Hill, New York, NY. Susmel, P., Mill, C.R., Colotti, M., Stefanon, B., 1993. In vitro solubility and degradability of nitrogen in concentrate ruminant feeds. Anim. Feed Sci. Technol. 42, 1±13. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fibre, neutral detergent fibre, neutral detergent fibre and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583±3597. Wu, Y.V., 1986. Fractionation and characterization of protein-rich material from barley after alcohol distillation. Cereal Chem. 63, 142±145. Wu, Y.V., Sexson, K.R., 1984. Fractionation and characterization of protein-rich material from sorghum alcohol distillation. Cereal Chem. 61, 388±391. Wu, Y.V., Sexson, K.R., Wall, L.S., 1981. Protein rich residue from wheat alcohol distillation: fractionation and characterization. Cereal Chem. 58, 343±347.