1. Introduction
Full-fat soybean (FFSB) is obtained by the mechanical cracking of whole soybeans without cooking or oil extraction [
1] and can be a good source of energy and protein for poultry [
2]. However, the presence of several anti-nutritional factors (ANFs), most importantly trypsin inhibitors (TI) and lectins, is a major obstacle for the inclusion of raw FFSB in poultry diets [
3,
4,
5]. Trypsin inhibitors can bind to proteolytic enzymes, forming inactive complexes and impairing amino acid (AA) digestibility [
6]. Most FFSB proteinaceous ANFs are, however, heat-labile, and therefore proper hydrothermal processing, such as conditioning and expansion, will inactivate these factors and eliminate their adverse effects on AA utilization. Multiple research projects have indicated that, once properly processed, broilers can be fed up to 200–250 g FFSB/kg diet without any adverse effect on growth performance [
7,
8,
9,
10].
Among the broad spectrum of factors affecting the content and quality of nutrients in FFSB, processing conditions are of the greatest importance. The digestibility of AA in FFSB is influenced to a large extent by the adequacy of heat treatment to inactivate the ANFs. Both under- and over-processing are deleterious to AA digestibility and energy utilization. The feed industry has been monitoring the scope of thermal processing by using in vitro tests such as urease activity, protein solubility in potassium hydroxide (KOH), protein dispersibility index, trypsin inhibitor activity (TIA), and reactive lysine [
1]. Raw soybeans usually contain 20–35 mg/g TIA. To eliminate the detrimental effects of TI, TIA needs to be reduced to a level of below 4.0 mg/g [
9] or between 1.75 and 2.50 mg/g [
1,
11]. Excessive heat processing of FFSB has been reported to damage heat-labile AA such as Arg, Cys, and more specifically, Lys through the formation of Maillard reaction products [
12,
13].
Despite the increasing interest in the use of FFSB in poultry diets, only limited studies have investigated the effects of conditioning and expanding characteristics on the AA digestibility and apparent metabolizable energy (AME) of this feed ingredient [
14,
15]. It was hypothesized that temperature and time during the conditioning process and different specific energy inputs applied during the expanding process will influence the digestibility of AA and energy utilization by birds. The present study was initiated to investigate the impact of short-term and long-term conditioning and expansion on the nitrogen-corrected AME (AMEn) and standardized ileal digestibility (SID) of AA in FFSB for broiler chickens.
3. Results and Discussion
All birds remained healthy and readily consumed their assay diets throughout the study. No evidence of histopathological abnormalities was observed when the abdominal cavity was opened following euthanasia.
While in vitro protein quality indicators such as urease activity index, protein dispersibility index, KOH protein solubility test, and TIA have been used in the feed industry to evaluate the protein quality of soybean meal, their application to FFSB merits more investigation [
2]. Investigating the effect of dry extrusion temperature (115, 125, 135, 145, and 165 °C) on FFSB, Palic et al. [
27] concluded that urease activity has a limited application as an indicator of the degree of FFSB processing and should be used to identify only under-processed FFSB. According to Herkelman et al. [
28] and Perilla et al. [
29], TIA in FFSB is more reflective of its nutritional value and a better predictor of FFSB protein quality than urease activity for chickens. A KOH protein solubility of 78–85% has been suggested for properly heat-treated soybean meals [
1,
2,
22,
30]. Moreover, TIA values of below 4.0 mg/g for FFSB [
9] and of 1.75–2.50 mg/g for soybean meals are considered necessary to mitigate the negative impact of TI on AA digestibility [
1,
11]. The in vitro protein quality indicators of the FFSB with different heat treatments are shown in
Table 4. Except for the samples LT conditioned at 90 °C (T1, T2, and T3), the KOH protein solubility reduced with LT conditioning at 100 °C for 6 and 9 min, regardless of expanding specific energy input. Expansion with specific energy input of 28 kWh/t resulted in lower KOH protein solubility than those expanded with 18 kWh/t at 3 min (T7 = 92.2 vs. T4 = 96.4), 6 min (T8 = 87.8 vs. T5 = 90.5), and 9 min (T9 = 85.7 vs. T6 = 89.3), with the lowest KOH protein solubility determined in T9. The TIA decreased in all heat-treated samples compared to raw FFSB (T0), with the lowest values recorded for T9 and T6, followed by T5, T8, and T7. The highest TIA recorded for raw FFSB in this study agrees with previous studies demonstrating the negative impact of TI on the digestibility of AA and highlights the fact that raw FFSB must not be used in poultry diets [
4,
31]. It is also recognized that TI are heat sensitive components and can be effectively reduced or eliminated through the proper heat treatment of raw FFSB. Similar to the current findings, Leeson and Atteh [
8] reported a reduction in TIA from 58.7 mg/g in raw soybeans to 16.1, 14.8, 9.3, and 8.4 mg/g following the extrusion of soybeans at 80, 100, 120, and 140 °C, respectively. In the current study, the lowest KOH protein solubility recorded in T9 and the remarkable reduction in TIA in T9 and T6 might suggest that LT conditioning at 100 °C for 9 min is required to properly deactivate the TI and is more efficient when combined with a specific energy input of 28 kWh/t during the expansion. The T9 sample with 3.9 mg/g TIA and 85.7% KOH protein solubility was the only heat-treated sample in the current study to meet the suggested maximum for TIA (4.0 mg/g) [
9] and KOH protein solubility (78–85%) [
2] in adequately heat-treated soybean products.
The AA composition of raw FFSB used in the present study was within the range previously reported [
2,
15,
32]. The ST conditioning and LT conditioning at different temperatures and times, and expansion with different specific energy inputs, had no notable effects on the contents of Lys and rLys (
Table 4), CP, and individual and total AA (
Table 5) in FFSB samples. This finding suggests that the heat treatments applied had minimal impact on the gross content of AA and mainly influence the nutritional quality through their impact on digestibility of nutrients [
33]. Severe thermo-mechanical treatments can favor the formation of Maillard reaction products [
20,
34,
35]. Free amino groups from AA, the epsilon-amino group, and free aldehyde groups from reducing sugars can interact and result in the destruction of some AA, Lys in particular, that can be characterized by the reduction in rLys [
20]. The lack of ST and LT conditioning effect on the concentration of Lys, rLys, and rLys:Lys ratio in this study suggests that the heat processing conditions applied in this study were not destructive to any AA.
Despite increasing interest in the use of FFSB in poultry diets, there are only sporadic data on the influence of heat processing conditions on the AMEn and SID AA of FFSB for poultry [
14,
15]. The influence of heat treatment on the AME, AMEn, N retention, and SID AA of FFSB in broilers is summarized in
Table 6. All heat treatment processes investigated in this study significantly (
p < 0.001) increased the AME, AMEn, and N retention in FFSB samples compared to raw FFSB (T0). The raw FFSB showed the lowest (
p < 0.05) AME, AMEn, and N retention. Among the heat-treated samples, T3, T5, T7, T8, and T9 showed similar (
p > 0.05) AME and AMEn values but were higher (
p < 0.05) than other samples. The highest (
p < 0.05) N retention was recorded in T5, followed by T3 and T8. Among all heat-treated samples, the smallest improvements in AME, AMEn, and N retention were achieved in T2 and T4. Compared to the raw sample, T5 supported 4.49 and 3.88 MJ/kg higher AME (15.54 versus 11.05 MJ/kg) and AMEn (14.29 versus 10.41 MJ/kg), respectively. No further improvements in AME or AMEn were observed with increases in LT conditioning time and/or expansion of specific energy input above T5 (LT conditioned at 100 °C for 6 min and expanded with 18 kWh/t specific energy input). The high concentration of fat and its digestion extent make major contributions to metabolizable energy of FFSB. As indicated by Kan et al. [
14], the increase in AMEn because of heat treatment could be attributed to an increase in digestibility of fat, and perhaps protein, in FFSB.
A significant (
p < 0.001) effect of heat treatment on the SID of protein, all individual indispensable (IAA), dispensable (DAA), and average of all AA was observed (
Table 6). The raw FFSB (T0) had the poorest (
p < 0.05) digestibility of protein and all individual AA, highlighting the documented fact that raw FFSB should be avoided in poultry diets [
4,
31]. Among the heat-treated samples, the lowest SID protein and average of all AA was recorded for T2, followed by T4, T1, and T3. When heat-treated, the highest (
p < 0.05) SID for all indispensable AA was recorded for T5, the lowest (
p < 0.05) SID for T2 and T4, with the other heat-treated samples being intermediate. A similar pattern was observed for all dispensable AA, with T5 and T2 generating the greatest (
p < 0.05) and poorest (
p < 0.05) digestibility among heat-treated samples, respectively, with other samples being intermediate.
Similar to patterns observed for the AME, AMEn, and N retention, the highest SIDs of CP and AA were recorded in T5 and confirm that, under the conditions of the present study, the LT conditioning at 100 °C for 6 min prior to expansion with 18 kWh/t specific energy input is sufficient to improve the SID of CP and AA in FFSB. It is pertinent to note that despite a striking increase of 45% in average SID of AA in T5 (78.3%) compared to T0 (54.0%), the KOH protein solubility only declined by 2.8 percentage points (93.3 vs. 90.5%) and to a level beyond the industry recommended KOH protein solubility of 78–85% for properly heat-treated soybean meals [
2,
22]. These findings suggest that (i) guidelines for KOH protein solubility suggested for soybean meal might not be applicable to FFSB, and (ii) KOH protein solubility is not a good indicator to assess whether the FFSB has been properly processed, and therefore should not be used as the sole measure of optimal processing and AA digestibility in FFSB. Interestingly, increases in LT conditioning time from 6 to 9 min or expansion of specific energy input from 18 to 28 kWh/t, although reducing the KOH protein solubility and TIA further, did not lead to any extra benefits to AA digestibility. It is also noteworthy that the highest average SID of all AA and individual AA observed in T5 did not correspond to the lowest values for KOH protein solubility and TIA, suggesting that broiler chickens might be tolerant to higher levels of TI (TIA of 10.6 mg/g in T5) in FFSB than the previously reported optimum value of <4.0 mg/g [
9]. Further deactivation of TI in T9 (TIA of 3.90 mg/g) failed to benefit AA digestibility. In contrast, Clarke and Wiseman [
9] measured the digestibility of AA in FFSB samples with different TIA contents of 14.8, 9.6, 4.5, and 1.9 mg/g, and recorded the highest AA digestibility in the FFSB sample with TIA of 1.9 mg/g.
Despite numerical reductions in the SID AA, intensifying the level of heat treatment beyond T5 (LT conditioned at 100 °C for 6 min and expanded with 18 kWh/t energy input) did not significantly (
p > 0.05) deteriorate the AA digestibility. However, it should be recognized that heat-induced structural changes in an AA might not be accurately captured by the digestibility measurements as the damaged AA can be digested and absorbed without the ability to participate in metabolic reactions in the animal body [
36]. Nevertheless, Oliveira et al. [
37] reported no effect of different FFSB extrusion temperatures (125, 130, 135, and 140 °C) on the performance and carcass composition of broilers (22–45 d) and suggested that FFSB extruded at temperatures of 125 to 140 °C can be used in broiler diets after 22 d of age. Similarly, in the study by Herkelman et al. [
28], maximum broiler performance was achieved when FFSB was heated at 121 °C for 40 min compared to heating times of 10, 20, 30, 60, and 90 min. Perilla et al. [
29] concluded that the optimum heating temperature during the wet extrusion of FFSB for broilers lies between 122 °C and 126 °C.