Effect of Dry Heat Temperature and Moist Heat Pressure on Canola Meal for Ruminant Utilisation . Part I : Nutritional , Protein Solubility and Degradability Characteristics

The effects of a range of barrel dry heat temperatures (20 to 180 C), and moist heat pressure (MHP) (120 C 15 min 192 kPa) on general nutritional, protein solubility, and in vitro protein degradability characteristics of canola meal were investigated. Increasing dry heat temperature was negatively correlated with meal crude protein (CP), soluble CP, neutral detergent fibre, acid detergent insoluble CP, and rapidly degradable (B1 Fraction) protein; and positively with NPN, intermediately degradable (B2 Fraction) protein, dry matter, lipid, carbohydrate, and in vitro rumen-undegradable protein. Relative to control meal, MHP increased in vitro rumen-undegradable protein, and in vitro CP digestibility; and decreased soluble protein, and 0.5% KOH solubility. Positive increases of Fraction A and B2, as B1 decreased, suggest barrel temperature induces protein hydrolysis and conversion of rapidly to intermediately degraded protein, respectively. The changes observed may have a great effect on ruminal degradation and supply of protein and AA for ruminant utilisation.


Introduction
Due to economic reasons, there is interest in increasing milk production per cow, through dietary modifications such as rumen-undegradable protein (RUP) supplementation (Paz et al., 2014;Thanh & Suksombat, 2015).Dietary RUP passes through the rumen to form a potential direct source of protein to satisfy post-ruminal AA requirements during lactation (NRC, 2001).Canola (rapeseed, Brassica spp.napus, rapa, and juncea) meal is a derivative of seed oil production utilised as a protein supplement in dairy cattle feed (Sánchez & Claypool, 1983), due to its desirable AA profile and digestibility (Santos, 2011).Current literature reports RUP in canola meal ranges from 10.1 to 75.0%CP (NRC, 2001;Purser & Woodroofe, 2004).Factors contributing to variation of RUP include species (Theodoridou & Yu, 2013), processing conditions (Newkirk et al., 2003), and physical and chemical treatments (McKinnon et al., 1991).To extract seed oil and generate meal, solvent-based and mechanical (e.g., cold-press, expeller, and extrusion) processing technologies exist.Expeller extraction utilises dry heat (95 to 135 o C) (Newkirk, 2009), and, cold-press extraction mechanically presses seeds by frictional force ( 65 o C) (Leming & Lember, 2005).Deacon et al. (1988) proposed heat of expeller extraction establishes cross-linkages among and within peptides chains, and to carbohydrates to increase RUP.Toghyani et al. (2014) detailed the effects of expeller barrel dry heat temperature (90, 95, 100 o C) on ileal AA digestibility of canola meal in broiler chickens.
To reduce ruminal degradation, and accordingly, increase the post-ruminal supply of canola protein, other heat treatments have been evaluated.Dry heating (125 o C 10 min) of canola meal increased digestible RUP without compromising intestinal digestibility (McKinnon et al., 1995).Dry heating (125 o C 20 min) of expeller canola meal reduced rumen degradability, and when fed to primiparous cows increased milk production (Jones et al., 2001).Moist heat pressure (MHP, autoclaving, 117 kPa 127 o C 15 or 30 min) treatment of canola meal induced partial protein denaturation to decrease the ruminal protein degradability and increase ruminal bypass-AA for digestion and absorption in the small intestine (Moshtaghi Nia & Ingalls, 1992, 1995).Wright et al. (2005) reported dietary inclusion of MHP solvent-extraction canola meal (2% H 2 O 100 o C 120 min) increased milk production in dairy cows by 0.5 kg per d.Although, Khalili et al. (1999) found the dietary inclusion of control and MHP rapeseed cold-press cake elicited similar milk responses in mid-lactation dairy cattle.
Limited knowledge exists of the effects of low, moderate and high expeller barrel dry heat, and the effects of MHP, on the general nutritional and protein degradability characteristics of canola meal for ruminant utilisation.The hypothesis tested in the current study was that the general nutritional, protein solubility and degradability of expeller-extracted canola meal would differ depending on the processing and treatment conditions.The objectives of this study were to investigate the effects of barrel dry heat temperature range and MHP on general nutritional, protein solubility and degradability characteristics of canola meal.

Canola Seed
Commercial bulk-handling canola seed was provided by MSM Milling (Manildra, NSW, Australia).The heterogeneous seed lot was stored at room temperature (RT, ~21 o C), in an air-tight hessian polypropylene bag within a dark and dry cupboard.

Barrel Dry Heat and Moist Heat Pressure of Canola
To prepare canola meals, seed (~200 g) was passed separately through a primed bench-top screw-press expeller (Model.DSZYJ-200A/B, 220V, 50 Hz, 50 rpm) at a barrel dry heat temperature of either 20 o C (RT, cold-press) or, a pre-heated temperature of 60, 80, 100, 120, 140, 160 or 180 o C (expeller), then repeated two more times (n = 3 × 8, 24).The meals were individually ground in an electric mill (Breville Grinder, CG2B) and passed through a 1 mm sieve.The MHP treatment was completed by placing each meal (30 g) in a separate flat rectangle polypropylene container and autoclaving using a steriliser (Atherton Centenary Series, Melbourne, Australia) set on the Hard Goods Dry Cycle No. 1.1, for 15 min (192 kPa 120 o C).For each triplicate meal, an independent sterilising cycle was performed.The meals were stored in the dark at RT.

Preparation of Canola Meal Suspensions
The meals were ground (< 5 µM) by placing 5 g of meal in a stainless-steel screw-top grinding jar (50 mL) with a ø 25 mm grinding ball.The jar was positioned in a mill (MM301, Retsch, GmbH, Hann, Germany) and shaken for 30 s (frequency 20 per s) followed by a 15 s rest, thrice.To generate suspensions, ground meal (200 mg) was added to deionised H 2 O (10 mL) and shaken for 30 min using a Heidolph Multi Reax set at 10.The suspensions were stored in the dark at 4 o C.

General Nutritional Characteristics
The meals were analysed for dry matter (DM, AOAC 930.15), lipid (%DM, AOAC 992.06),CP (6.25 × N) by Leco Dumas N combustion (AOAC 992.23), and carbohydrate (%DM (Masuko et al., 2005)).To determine the quantity of carbohydrate (%DM), each meal suspension (40 µL) was added separately to deionised H 2 O (10 µL), concentrated sulphuric acid (150 µL) and 5% phenol in deionised H 2 O (30 µL) in a clear flat bottom non-absorbent 96 F Microwell microplate (Nunc, #269620).The plate was incubated for 5 min at 90 o C in a shallow water bath, rested for 5 min at RT, wiped dry, placed in a CLARIOstar 5.20 R5 microplate reader, shaken at 500 rpm for 10 s and measured for Abs 490nm .Values were corrected by deducting an average of blank measurements.A standard curve (0 to 10 nmol) was established with a 1 M stock solution of D-mannose (Sigma) prepared in deionised H 2 O.

Protein Solubility and Fractionation
The meals were analysed for soluble protein (Licitra et al., 1996) and solubility in 0.5% KOH (Pastuszewska et al., 1998).The latter was performed by stirring samples of the meal (5 g) in 0.5% KOH (33.3 mL) for 20 min, centrifuging at 1250 ×g for 10 min, and quantifying the protein in the supernatant by Leco Dumas N combustion.The meals were analysed in duplicate (n = 2 × 8, 16) for NPN (tungstic acid (Licitra et al., 1996)), acid detergent fibre (ADF), neutral detergent fibre (NDF), ADIN and neutral detergent insoluble N (NDIN) by the Australian Oil Reference Laboratory (Department of Primary Industries, NSW, Australia).Results were utilised to calculate true protein: %, CP -NPN.The meals were partitioned into protein fractions based on characteristics of degradability according to the Cornell Net Carbohydrate and Protein System (CNCPS) as described (Sniffen et al., 1992).Using CNCPS, Fraction A is NPN, Fraction B is degradable protein containing B1 (soluble protein, rapidly soluble in the rumen), B2 (intermediate degradation, Total CP -(A + B1 + B3 + C)), B3 (slowly degraded in the rumen, NDIN -ADIN), and Fraction C is undegradable protein (ADICP).

Rumen Undegradable Protein
The meals were analysed for RUP utilising an in vitro simulated rumen proteolysis procedure by Krishnamoorthy et al. (1983) validated in vivo (R 2 = 0.61).The meal (0.5 g) was weighed into a 125 mL Erlenmeyer flask and incubated at 39 °C for 1 h in 40 mL borate-phosphate (BP) buffer (pH 8.0).Streptomyces griseus protease (Type XIV 5.4 U/mg protein, Sigma P-5147, St Louis, MO, USA) solution (0.33 U/mL, 10 mL BP-buffer) was added, and the meal was incubated at 39 °C for 18 h.All flasks were placed on ice to suspend proteolytic activity before filtering.The residue was collected on quantitative filter paper (22 µm pore, No. 541, Whatman), rinsed with distilled H 2 O and air-dried overnight.Residual CP was determined by combusting the whole filter paper by Leco Dumas N combustion.

Intestinal Digestion of Protein in Ruminants
The meals were analysed in duplicate (n = 2 × 8, 16) for in vitro CP digestibility utilising the HCl-pepsin pre-digestion procedure of Calsamiglia & Stern, (1995) validated in vivo (r = 0.91).In a 50 mL Falcon tube sample (15 mg CP) was suspended in 10 mL pH 1.9, 0.1 N HCl solution of 1 g/L pepsin (Sigma P-7012), vortexed, and incubated at 38 o C for 1 h in a shaking H 2 O bath.Pancreatin solution (13.5 mL: 0.5 M KH 2 PO 4 pH 7.8 containing 3 g/L pancreatin, Sigma P-7545) and 1 N NaOH (0.5 mL) was added, and the tube was vortexed, incubated at 38 o C for 24 h in a shaking H 2 O bath, vortexing every ~ 8 h.To cease the reaction TCA (3 mL) was added, the tube was vortexed, rested (15 min), and then centrifuged (10,000 ×g, 15 min).The supernatant was analysed for soluble N, as described.Results were utilised to calculate % pepsin-pancreatin digestion of protein:

Statistical Analysis
Statistical analyses of data were performed using the statistical software OriginLab v 95E (Origin, Northampton, MA, USA).To establish differences, the one-way ANOVA mathematical model used for analysis was: where, Y ij is an observation on the dependent variable ij; µ is the population mean for the variable, T j is the effect of treatment (i = MHP and/or barrel dry heat temperature), as a fixed effect.The independent barrel runs at each temperature were experimental replications and e ij value is the random error associated with the observation ij.A post-hoc Fisher Least Significant Difference test was performed to determine the statistical significance of differences between individual means, declared at P < 0.05.Normal distribution was established by performing an Anderson-Darling test, P > 0.05.The Spearman correlation coefficient (r s ) with a two-tailed test of significance (P < 0.05) was used to define strength and association of relationships between dry heat temperature and dependent variables.Polynomial regression was performed to determine the Coefficient of Determination (R 2 ), using the equation:

Results
The effects of dry heat (20 to 180 o C) with MHP on general nutritional characteristics of canola meal are presented in Table 1 (Table A1 and Figure A1).The DM content of MHP meals (r s = 0.78) and control meals (R 2 = 0.95) increased with temperature and the average DM content was higher (P < 0.05) in MHP meals (94.1%) than in the control (93.3%) meals (without MHP).The CP content of the all meals decreased with temperature (R 2 = 0.90, 0.89); however, the average CP content did not differ (P > 0.05) between the control (33.2%) and MHP meals (34.1%).Greatest CP for control meal was at 60 and 80 o C, and from 60 to 100 o C for MHP meals.
Lipid content also increased with dry heat temperature (R 2 = 0.98, 0.95).The average lipid content was similar (P > 0.05) in MHP (19.7%) and control meals (17.3%).Lipid extraction was most efficient at 80 and 100 o C for control, and from 60 to 100 o C for MHP meals.Carbohydrate content increased with temperature (r s = 0.95, 0.50, P < 0.05), with the average carbohydrate content similar in the control (11.5%) and MHP meals (11.5%).

Discussion
In this study, the effects of expeller barrel dry heat and MHP on general, protein solubility, and in vitro protein degradability characteristics of canola meals were investigated.Initial analysis of the general nutritional characteristics of cold-press (20 o C) and expeller (100 o C) meals, respectively, were similar values to those published for CP (33.1 vs. 36.4to 45.0% (Leming & Lember, 2005;Seneviratne et al., 2011) 2014).A decline in CP at higher temperatures (≥ 160 o C, P < 0.01) is indicative of increased retention of CP in extracted oil (thereby decreasing the CP content of the meal), or the degradation of thermolabile proteins at higher temperatures (Jung et al., 2012).A reduction of carbohydrate (11.5%) levels compared to a prior report (15% (Newkirk, 2009)), may be explained by a variance of carbohydrate levels among canola and rapeseed types (Naczk & Shahidi, 1990).
These results differed from other studies reporting little association between barrel dry heat and general nutritional characteristics (Toghyani et al., 2014) and decreases in meal lipid at higher processing temperatures (Clandinin et al., 1956).While MHP had no effect on CP, lipid and carbohydrate contents, DM increased (control 93.3% vs. MHP 94.1%, P < 0.05).In comparison, Samadi et al. (2013) reported MHP of canola seed had no effect on DM and CP, but reduced carbohydrate and increased lipid (control 94.9%, 25.2%, 29.0%, 41.8% vs. autoclave (120 o C 1 h) treated 94.9%, 25.0%, 26.4%, 44.6%, respectively).Increased lipid in meal processed at higher barrel temperatures may have further catalysed Maillard reactions since lipid is a known proactive contributor to these reactions in other systems (Farmer, 1996).
Although protein solubility of expeller and cold-press meals in 0.5% KOH or borate buffer were similar, Smulikowska et al. (2006) reported a reduction of protein solubility of expeller meal compared to cold-press rapeseed meal.A negative association of BP-buffer protein solubility with dry heat temperature suggests the formation of insoluble complexes at higher temperatures.Based on the soluble protein classifications outlined by Pastuszewska et al. (2003), the expelled meals (55.3%) were very well processed and of high nutritional value (55 to 60% solubility); however, the MHP meals (33.2%) were over processed and declined in nutritional value (< 45% solubility).The in vitro RUP values of the cold-press (26.4%) and expeller (average 32.2%) meals were higher than previous reports by Kaldmäe et al. (2010) (10.8%) and Shannak et al. (2000) (17.8%), respectively, particularly at higher dry heat temperatures.Heat during the expelling process can induce the formation of insoluble peptide chain and carbohydrate complexes, which contribute to greater RUP in these meals (Deacon et al., 1988).Consequently, the positive association of RUP with dry heat temperature (P < 0.01) suggests the formation of insoluble complexes under higher temperature conditions.The application of MHP (15 min 192 kPa 120 o C) considerably increased the formation of RUP (32.3 vs. 64.4%,P < 0.01).Moshtaghi Nia and Ingalls (1992) similarly reported the application of MHP (15 min 117 kPa 127 o C) increased RUP (69.9 vs. 25.6%).Heating of meal was theorised to promote protein denaturation and reduce solubility to favour more rumen escape of un-denatured protein to the lower gastrointestinal tract (Van Soest, 1994).Levels of IVCPD were similar to those reported by (Mustafa et al., 2000) (9.78% vs. 16.4% CP).Notwithstanding dry heat temperature associated variances in ruminal protein availability, changes were not reflected in IVCPD values, which remained similar among dry heat temperatures.Increases in IVCPD after MHP were less than those reported for toasting (11.7 vs. 49.1%),but still suggest protein denaturation and formation of insoluble protein complexes with irreversible bonds by MHP, in turn, reducing available protein (Toghyani et al., 2014).Levels of ADF and NDF were similar to previous reports (CCC, 2015;Newkirk, 2009), while ADICP content was similar, and NDICP was reduced relative to feed library values (DairyOne, 2016;NRC, 2001).These results imply lower levels of digestible fibre associated protein, known to contribute to RUP (Chrenkova et al., 2014).Compared to a previous report of Shannak et al. (2000) for expeller rapeseed meal, protein fraction levels of Fraction A (NPN) and B2 were lower, B1 was higher, and B3 and C were similar.These results imply lower levels of digestible fibre associated protein.Positive increases of Fraction A and B2 as B1 decreased imply increased dry heat temperature induces protein hydrolysis and conversion of rapidly to intermediately degraded protein, respectively.Future studies may aim to test different expeller barrel types and monitor the barrel retention time and exit temperature of meal.