• BMB Reports
• /
• v.35 no.2
• /
• pp.220-227
• /
• 2002

Two aspartate aminoransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT-2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8,0 and 9.0) and temperature ($60-65^{\circ}C$) were similar for both isoenzymes. In the temperature range of $45-65^{\circ}C$, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.3
• /
• pp.358-365
• /
• 1999

The effects of dry roasting whole lupin seeds (lupinus albus, WLS) at 110, 130 or $150{^{\circ}C}$ for 15, 30 or 45 minutes on the in sacco rumen degradation characteristics, optimal heating conditions of time and temperature and in vitro enzyme digestibility were determined. Ruminant degradation characteristics (RDC) of crude protein (CP) of WLS were determined by in sacco technique in dairy cows. Measure ROC were soluble (S), undegradable (U), potentially degradable (D) fractions, lag time (TO) and rate of degradation (Kd) of insoluble but degradable fraction. Based on measured ROC, percentage bypass CP (%BCP) and bypass CP (BCP in g/kg, DM) were calculated. Degradability of CP was significantly reduced by dry roasting (p<0.001). The interaction of dry roasting temperature and time had significant effects on D (p<0.05), Kd (p<0.01), U (p<0.01), %BCP (p<0.001) and BCP (p<0.001) but not on S (p=0.923>0.05). With increasing time and temperature, S, D, Kd and U varied from 31.8%, 67.4%, 10.3%/h and 0.8% in the raw WLS (RWLS) to 27.1 %, 35.8%, 3.6%/h, 38.4% in $150{^{\circ}C}/45\;min$, respectively. All these effects resulted in increasing %BCP from 25.9 in RWLS to 61.0% in the $150{^{\circ}C}/45\;min$. Therefore BCP increased form 111.2 to 261.2 g/kg DM, respectively. Both %BCP and BCP at $150{^{\circ}C}/45\;min$ increased nearly 2.5 times over the RWLS. The effects of dry roasting on %BCP and BCP seemed to be linear up to the highest value tested. Although ROC had been altered by dry roasting, the In vitro perpsin-cellulase digestibility was generally unchanged. It was concluded that dry roasting was effective in shifting CP degradation from rumen to the lower gastrointestinal tract to potential reduce unnecessary N loss in the rumen. It might be of great value in successfully synchronizing the rhythms of release of nitrogen and energy in the rumen, thus achieving a more efficient fermentation of diets with high proportions of lignocellulosic resources. To determine the optimal dry roasting conditions, the digestibility of each treatment in the cows will be measured in the next trial using mobile bags technique.

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.8
• /
• pp.1205-1214
• /
• 1999

Whole lupinus albus seeds were pressure toasted at temperatures of 100, 118 and $136^{\circ}C$ for 3, 7, 15 and 30 min to study rumen degradation and post-rumen digestion and to determine optimal heating conditions for the Dutch dairy feed industry. In sacco nylon bag and mobile bag techniques were employed for rumen and intestine incubations to determine ruminal degradation characteristics and intestinal digestion of crude protein (CP) in 4 lactation rumen cannulated and 4 lactating intestinal cannulated Dutch dairy cows fed 47% hay and 53% concentrate according to Dutch dairy requirements. Measured rumen degradation characteristics were soluble fraction (S), undegradable fraction (U), potentially degradable fraction (D), lag time (T0) and rate of degradation (Kd) of insoluble but degradable fraction. Percentage bypass feed protein (BCP), ruminal microbial protein synthesized based on available nitrogen (N_MP) and that based on available energy (E_MP), true protein supplied to the small intestine (TPSI), truly absorbed BCP (ABCP), absorbed microbial protein (AVP) in the small intestine, endogenous protein losses in the digestion (ENDP), true digested protein in the small intestine (TAP or DVE in Dutch) and degraded protein balance (PDB or OEB in Dutch) were totally evaluated using the new Dutch DVE/OEB System. Pressure toasting decreased (p<0.001) rumen degradability of CP. It reduced S (p<0.05) and Kd (p=0.06), increased D (p<0.05) and U (p<0.01) but did not alter T0 (p>0.05), thus resulting in dramatically increased BCP (p<0.001) with increasing time and temperature from 73.7 (raw) up to 182.5 g/kg DM ($136^{\circ}C/15min$). Although rumen microbial protein synthesized based on available energy (E_MP) was reduced, true protein (microbial and bypass feed protein) supplied to the small intestine (TPSI) was increased (p<0.001) from 153.1 (raw) to 247.6 g/kg DM ($136^{\circ}C/15min$). Due to digestibility of BCP in the intestine not changing (p>0.05) average 87.8%, the absorbed BCP increased (p<0.001) from 62.3 (raw) to 153.7 g/kg DM ($136^{\circ}C/15min$). Therefore DVE value of true digested protein in the small intestine was significantly increased (p<0.001) from 118.9 (raw) to 197.0 g/kg DM ($136^{\circ}C/15min$) and OEB value of degraded protein balance was significantly reduced (p<0.001) from 147.2 (raw) to 63.1 g/kg DM ($136^{\circ}C/15min$). It was concluded that pressure toasting was effective in shifting degradation of CP of lupinus albus from the rumen to small intestine without changing intestinal digestion. Further studies are required on the degradation and digestion of individual amino acids and on the damaging effects of processing on amino acids, especially the first limiting amino acids.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.7
• /
• pp.974-981
• /
• 2002

Faba beans (vicia faba) (FB) and lupin seeds (Lupinus Albus) (LS) were dry roasted at three temperatures (110, 130, $150^{\circ}C$) for 15, 30 or 45 min to determine the effects of dry roasting on rumen degradation of crude protein and starch free organic matter ($^{PSF}OM$). Rumen degradation characteristics of $^{PSF}OM$ were determined by the nylon bag incubation technique in dairy cows fed 60% hay and 40% concentrate. Measured characteristics of $^{PSF}OM$ were undegradable fraction (U), degradable fraction (D), soluble fraction (S), lag time (T0), and the rate of degradation (Kd). Based on the measured characteristics, rumen availability ($RA^{PSF}OM$) and bypass $^{PSF}OM$ ($B^{PSF}OM$) were calculated. Dry roasting did not have a greater impact on rumen degradation characteristics of $^{PSF}OM$ (p>0.05). S varied from 32.1 (raw) to 30.0, 27.8, 30.8% (LS) and 15.4 (raw) to 14.4, 20.8, 20.9% (FB); D varied from 65.4 (raw) to 66.3, 66.9, 55.9% (LS) and 54.9 (raw) to 55.0, 51.0, 64.7% (FB); U varied from 2.6 (raw) to 7.3, 7.0, 7.7% (LS) and 29.7 (raw) to 30.6, 28.2, 14.4% (FB); Kd varied from 6.0 (raw) to 7.3, 7.0, 7.7% (LS) and 22.4 (raw) to 24.4, 21.1, 7.9% (FB); $B^{PSF}OM$ varied from 35.5 (raw) to 33.8, 36.6, 38.2% (LS) and 41.3 (raw) to 41.5, 39.7, 47.6% (FB) at 110, 130 and $150^{\circ}C$, respectively. Therefore dry roasting did not significantly affect $RA^{PSF}OM$, which were 353.7, 367.9, 349.6, 336.9 (g/kg DM) (LS) and 12.82, 127.0, 133.7, 117.1 (g/kg DM) (FB) at 110, 130 and $150^{\circ}C$, respectively. These results alone with our previously published reports indicate dry roasting had the differently affected pattern of rumen degradation characteristics of various components in LS and FB. It strongly increased bypass crude protein (BCP) and moderately increased starch (BST) with increasing temperature and time but least affected $^{PSF}OM$. Such desirable degradation patterns in dry roasted LS and FB might be beneficial to the high yielding cows which could use more dry roasted $^{PSF}OM$ as an energy source for microbial protein synthesized in the rumen and absorb more amino acids and glucose in the small intestine.

• Asian-Australasian Journal of Animal Sciences
• /
• v.13 no.10
• /
• pp.1377-1387
• /
• 2000

Two laboratory techniques: (1) an in vitro method with two procedures for measuring protein degradabilities and (2) an in vitro method with three procedures for measuring protein solubility, were investigated to determine which laboratory techniques could most accurately predict the quantity of rumen protein degradation kinetics of legume seeds after dry roasting under various conditions, in terms of (1) rumen protein disappearance ($D_j$, where j=0, 2, 4, 8, 12, 24 and 48 h incubation), (2) rumen protein effective degradability (EDCP), (3) the parameters describing rumen degradation characteristics (the soluble fraction: S, the potentially degradable fraction: D, undegradable fraction: U, lag time: T0 and the degradation rate: Kd) and (4) rumen bypass protein (BCP), which were determined by the method accepted internationally at present, in sacco nylon bag technique using the standardized Dutch method. Feeds evaluated were the raw and dry roasted whole faba (Vicia faba) beans (WFB) and whole lupin (Lupinus albus) seeds (WLS), each was dry roasted under various conditions (at 110, 130 or $150^{\circ}C$ for 15, 30 or 45 min). In vitro protein degradability ($D_1$_Auf and $D_{24}$_Auf) were determined using the modified Aufr re method by enzymatic hydrolysis for 1 h and 24 h using a protease extracted from Streptomyces griseus in a borate-phosphate buffer. In vitro protein solubility ($bf_1$_S, $bf_2$_S, $bf_3$_S) was measured in a borate-phosphate buffer with three different procedures. Results from laboratory techniques (in vitro) were correlated and linearly regressed with in sacco results. Of the three procedures of in vitro protein solubility evaluated, none of them could predict in sacco results with good precision. The highest Pearson correlation coefficient ($R^2$) was less than 0.50. Of two procedures of in vitro protein degradability studied, the $D_1$_Auf values were closely correlated with in sacco parameters: Kd, EDCP and %BCP with high R' values: 0.82, 0.85 and 0.85, respectively, and closely correlated with in sacco $D_j$ at 2, 4, 8 and 12 h rumen incubation with high $R^2$ values: 0.83, 0.91, 0.93 and 0.83, respectively. The $D_{24}$_Auf values could not predict in sacco results. The highest $R^2$ value was less then 0.40. These results indicated that in vitro protein solubility measured in borate-phosphate failed to identify differences in the rate and extent of protein degradation of legume seeds after dry roasting under various conditions and thus should not be used to predict rumen degradation, particularly for heat processed feedstuffs. But in vitro protein degradability using the modified Aufr re method by enzymatic hydrolysis for 1 h or possibly an intermediate time (>1 h and <24 h) is a promising laboratory procedure to detect effectiveness of dry roasting legume seeds on rumen protein degradation characteristics and could be used as a simple laboratory method to predict the rate and extent of protein degradation in the rumen in sacco with high accuracy. The equations to predict EDCP, Kd and BCP of dry roasted legume seeds (WLS and WFB) under various conditions are as follow: For both: EDCP (%)=-1.37+1.06*$D_1$_Auf ($R^2=0.85$, p<0.01). For both: Kd (%/h)=-21.81+0.49*$D_1$_Auf ($R^2=0.82$, p<0.01). For both: %BCP=103.37-1.07*$D_1$_Auf ($R^2=0.85$, p<0.01).

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.7
• /
• pp.1054-1062
• /
• 1999

The effects of dry roasting whole faba beans (WFB) and whole lupin seeds (WLS) at 110, 130 or $150{^{\circ}C}$ for 15, 30 or 45 min on rumen (RDCP%), estimated intestinal (IDCP%) and total tract disappearance of CP (TDCP%) and intestinal availability (IARUCP%) of rumen undegraded CP (RUCP%) were determined. The RDCP values were estimated by in sacco technique by incubating nylon bags for 8, 12 and 24 h in the rumen of dairy cows. The IDCP and IARUCP values were estimated using a sequence of ruminal incubation, in vitro incubation in acid-pepsin for 1 h and then in pancreatin for 24 h of three-step in vitro procedure technique. Dry roasting at 130 and $150^{\circ}C$ decreased RDCP with correspondingly increasing IDCP. The IDCP value generally increased from 12.3(raw) to 8.6, 14.8 and 39.6% (WFB) and from 28.3 (raw) to 33.7, 36.2 and 56.2% (WLS) at 8 h rumen incubation; from 2.9 (raw) to 2.9, 4.6 and 23.3% (WFB) and from 19.6 (raw) to 19.0, 24.0 and 46.6% (WLS) at 12 h rumen incubation; from 1.3 (raw) to 1.9, 1.7 and 11.0% (WFB) and from 4.4 (raw) to 4.2, 10.7 and 36.7% (WLS) at 12 h rumen incubation as the temperatures rose to 110, 130 and $150{^{\circ}C}$ respectively. The TDCP values were always high and increased by time in the rumen, the average values of which were 97.9, 96.6; 99.2, 96.9 and 99.6, 98.7% for WFB and WLS, respectively, at 8, 12 and 24 h rumen incubation. But within the same retention time, TDCP was generally unchanged. The average IARUCP increased from 87.3 (raw) to 87.4, 88.7 and 92.0% (WFB); from 87.6 (raw) to 88.9, 91.5 and 93.0% (WLS) at roasting temperatures of 110, 130 and $150{^{\circ}C}$, respectively. It was concluded that dry roasting can shift the digestion of CP from rumen to the lower gastrointestinal tract without depressing the digestion of RUCP. The best processing condition in this study was dry roasting at $150{^{\circ}C}$ for 45 min in terms of effects on the disappearances and availability of CP. Research data on intestinal availability of individual amino acids need to be further investigated.

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.6
• /
• pp.871-880
• /
• 1999

The effects of dry roasting (110, 130, $150^{\circ}C$ for 15, 30, 45 min) on potential ruminant protein nutritional values in terms of: a), rumen bypass protein (BCP); b), rumen bypass starch (BST); c), fermented organic matter (FOM); d), true absorbed bypass protein (ABCP); e) microbial protein synthesized in the rumen based on available energy (E_MP); f), microbial protein synthesized in the rumen based on available nitrogen (N_MP); g), true protein supplied to the small intestine (TPSI); h), true absorbed rumen synthesized microbial protein (AMP); i), endogenous protein losses (ENDP); j), true digested protein in the small intestine (DVE); k), degraded protein balance (OEB) of whole lupin seeds (WLS) and faba beans (WFB) were evaluated by the new Dutch DV/OEB protein evaluation system. Dry roasting significantly increased BCP, BST, TPSI, ABCP, DVE (p<0.001) and decreased FOM, E_MP, AMP, N_MP and OEB (p<0.001) with increasing temperatures and times except that when temperature was at $110^{\circ}C$. The values of BCP, BST, TPSI, ABCP and DVE at $150^{\circ}C/45min$ for WLS and WFB were increased 2.2, 3.7; -, 2.0; 1.7, 1.7; 2.3, 3.7 and 1.7, 1.7 times and the values of FOM, E_MP, AMP, N_MP and OEB at $150^{\circ}C/45min$ for WLS and WFB were decreased by 15.3, 25.8; 18.1, 25.8; 18.7, 25.8; 54.6, 41.6 and 82.3% 54.7%, respectively, over the raw WLS and WFB. The results indicated that though dry roasting reduced microbial protein synthesis due to reducing FOM, TPSI didn't decrease but highly increased due to increasing BCP more than enough for compensation of the microbial protein decreasing. Therefore the net absorbable DVE in the small intestine was highly increased. The OEB values were significantly reduced for both WLS and WFB but not to the level of negative. It indicated that microbial protein synthesis might not be impaired due to the sufficient N supplied in the rumen, but the high positive OEB values in the most treatments except of $150^{\circ}C$ for 30 and 45 min of WLS (The OEB values: 54.8 and 26.0 g/kg DM) indicated that there were the large amounts of N loss in the rumen. It was concluded that dry roasting at high temperature was effective in shifting protein degradation from rumen to intestines and it increased the DVE values without reaching the negative OEB values. No optimal treatment was found in WLS due to the too high OEB values in all treatments. But dry roasting at $150^{\circ}C$ for 30 and 45 min might be optimal treatments for WLS due to the very lower OEB values.