1. Introduction
Cassava (
Euphorbiaceae family), sweet potato, and potato are known as the world’s three major potato crops, of which cassava is highly adaptable, resistant to drought and acidic soil, and can be grown on poor land. At the same time, cassava is a potentially high biomass-producing crop [
1]. Widely planted in tropical and some subtropical countries and regions [
2,
3]. In recent years, with the continuous development of the cassava industry and the gradual diversification of uses [
2,
4], the demand for dried cassava in China has increased significantly, reaching 7.1827 million tons in 2021, a year-on-year increase of 43.9% [
5]. However, the cassava planting area continues to shrink, which leads to a continuous reduction in domestic raw material supply and a significant reduction in domestic cassava starch production. In 2021, China’s cassava starch production was 211,800 tons, a year-on-year decrease of 19.23% [
5]. In addition, native cassava contains anti-nutritional factors (hydrocyanic acid, tannin) that reduce animal intake and the digestion and absorption of nutrients (such as protein, etc.), limiting the use of cassava in animal husbandry [
3]. In the feed industry, methods such as mechanical crushing (MC), steam conditioning (SC), puffing conditioning (PU), or conditioning temperature are often used to process feed raw materials, which can not only change the nutritional value of the feed but also change the digestion rate and digestibility of starch, thereby improving its utilization value.
The starch content in feed has always been a concern of animal husbandry scientists. The starch content in fresh cassava roots is as high as 32%, amylose generally accounts for 20–30% of the total starch content, and amylopectin accounts for 70–80% of the total starch content [
6]. Amylopectin has a larger surface area than amylose and can bind to more amylase, while the glucose chains of amylose are mostly bound together by hydrogen bonds, which makes amylose more difficult to be hydrolyzed by amylase than branched starch [
7]. The rate and site of starch digestion in vivo depend on the content of amylose and amylopectin [
8]. Cassava starch is more quickly digested because it contains less amylose than most grains, legumes, and potato starches [
9]. Generally, starch be classified as rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) according to the digestion time of starch in the small intestine [
10]. The rate and extent of starch digestion regulate the postprandial metabolism of broilers. The RDS releases glucose at a faster rate at the front of the small intestine, leading to a decrease in the energy supply of glucose in the jejunum and ileum, an increase in fecal nitrogen excretion, and a decrease in ileum nitrogen retention, thereby improving the digestion and absorption function of animals [
11].
Different processing methods and conditioning temperatures vary and affect the extent and rate of starch digestion, which may influence the balance between nutritional effectiveness and diet quality [
12,
13]. MC is the first step in feed production, mainly through physical effects such as friction, collision, and shear force to change the structure as well as physicochemical properties of starch granules, thereby affecting the digestibility of starch [
14,
15,
16]. The crushing disrupts the crystalline regions of starch and increases the effective contact between digestive enzymes and starch granules to promote the digestion rate of starch [
16,
17]. The SC induces the starch to absorb water and expands to a gelatinization state during heating [
12]. By breaking the hydrogen bonds in starch granules, starch molecules become single molecules, protein denaturation, starch gelatinization, or anti-nutritional factors such as trypsin inhibitors were inactivated to improve starch digestibility [
18,
19]. The puffing conditioning destroys the crystalline structure of native starch [
20], reduces the crystallinity of starch, and increases the gelatinization temperature, which has better thermal stability [
21]. Changes in starch internal structure and starch granule size caused by puffing conditioning increased starch digestion rate and starch digestibility [
22]. Compared with MC, PU increased the digestion rate of feed starch by increasing the content of RDS in grains and obtained a higher starch digestion coefficient [
23]. The SC and PU destroyed the endosperm cell wall of the feed material and the intermolecular hydrogen bond that maintained the starch crystal structure and improved the apparent digestibility of starch in the ileum and feed conversion rate of animals [
24,
25]. Appropriate conditioning temperature not only effectively kills potential pathogens in feed materials [
26] but also improves starch digestibility and the digestion and utilization of nutrients in feed by animals by destroying the crystal structure and gelatinization degree of native starch [
27,
28].
Based on the fact that SC, PU, and appropriate conditioning temperature could alter starch digestibility and had a positive effect on animal growth, we hypothesized that the combined application of processing methods and conditioning temperatures would increase the digestibility of cassava starch in vitro and improve the broiler growth performance. Therefore, the purpose of this study is to analyze the effects of different processing methods and conditioning temperatures and their interaction on the digestibility of cassava starch, the growth performance of broilers, and the digestibility and utilization of nutrients.
4. Discussion
Since in vitro studies could be used to measure digestibility without sacrificing animals, many scholars have used the in vitro starch degradation method of the digestive tract model to simulate the in vivo hydrolysis of starch, and the in vitro time could be used to represent different parts of the small intestine [
7,
10,
11,
40]. It is generally believed that RDS refers to the starch that can be digested and absorbed in the small intestine within 20 min, and SDS refers to the starch that can be completely digested and absorbed in the small intestine within 20 to 120 min. RS cannot be digested and absorbed in the small intestine within 120 min but can reach the colon and be fermented by colonic microorganisms [
10]. However, this definition applies to food nutrition but has limitations in animal nutrition. Weurding et al. (2001 b) [
41] found that starch digestibility after 2 and 4 h incubation in vitro correlated well with starch digestibility in jejunum and ileum in broilers. Therefore, we adopted the regression equation proposed by Weurding et al. (2001 b) [
41] and took the incubation for 2 and 4 h as the dividing points for the calculation of RDS, SDS, and RS.
For most native starches, the in vitro digestion profile reaches an asymptotic level after 6 h [
31]. In the present study, the digestibility of cassava starch reaches asymptotic levels after 4 or 2 h when the conditioning temperature was 90 °C or PU, and the digestibility within 0.25–2 h was higher than other treatments, indicating that the increase of the conditioning temperature destroyed the hydrogen bonds between starch molecules, which increased the gelatinization degree of starch and promoted digestion of starch [
28]. And PU also changes the physical and chemical structure of starch, increasing its digestibility during enzymatic hydrolysis [
42]. Moreover, compared with MC, SC and PU, increased digestion rate and starch digestibility for 0.25–1 h, which is similar to the previous reports [
22,
40].
Starch digestion is mainly driven by amylase, a glycolytic enzyme that degrades starch into glucose, making it easier to absorb in the small intestine, and amylase activity is not only affected by external factors but also by the structure of starch itself (such as amylose, amylopectin, RDS, SDS, DS, and RS) [
20,
43]. The PU decreased the content of amylose, amylose/amylopectin, and SDS and increased the content of amylopectin and RDS, indicating that PU caused the starch structure to lose its integrity, the size of starch granules decreased, and the increase in the surface area led to an increase in its sensitivity to amylase, which promoted starch digestion [
7,
20].
The temperature required to promote starch digestion is not uniform but related to the characteristics of the starch varieties and processing methods. In vitro digestibility and digestion rate of cassava starch increase with increasing conditioning temperature, which is consistent with the findings obtained by Ali et al. (2020) [
44]. Interestingly, MC had the lowest in vitro starch digestibility (0.25–2 h) and digestion rate at a conditioning temperature of 60 °C, while PU had the highest starch digestibility (0.5–1 h) at a conditioning temperature of 75 °C. Ali et al. (2020) [
44] reported that potato starch was PU at a conditioning temperature of 100 °C had the lowest in vitro starch digestibility, while corn starch was PU at 160 °C had the highest digestibility. This may be due to the fact that no moisture was added during the PU process in this study. If in the presence of moisture, high temperature leads to high gelatinization of starch, thereby increasing the sensitivity of starch to enzymatic hydrolysis [
44]. Compared with SC, the RS content of wheat was similar at 20, 60, and 75 °C conditioning temperatures after MC, and the RS content was the highest at 90 °C [
45]. However, in our study, SC and PU had the lowest RS content at the conditioning temperature of 75 °C, not 90 °C. Moreover, cassava starch had lower amylose content and amylose/amylopectin at a conditioning temperature of 60 °C in combination with PU or SC. It shows that different types of starch use different processing methods, and the optimum temperature for promoting starch digestion is different. Furthermore, the amylose content, amylose/amylopectin, and RDS content of cassava at conditioned 60 °C were lower than 75 °C or 90 °C, whereas the amylopectin and RS content were the highest, which is in agreement with the results of Wang et al. (2019) [
27], who found the conditioning temperature increased from 65 °C to 85 °C and the RS content in sorghum increased. This may be because amylopectin was more sensitive to high temperature than amylose, and many of its branch points were easily broken, which led to the formation of linear fragments similar to amylose [
7]. Additionally, high temperature leads to high gelatinization of starch, increasing the susceptibility of starch to enzymatic hydrolysis [
44]. The gelatinization of starch has a direct impact on the digestibility of starch, as in which crystal structure is changed and the glycosidic bonds are destroyed, and therefore the molecules become easily accessible to digestive enzymes, the content of amylopectin is increased, and the amylose/amylopectin was decreased, thereby leading to increased digestibility [
20,
44].
Broilers fed PU diets had higher ADFI than those fed MC diets. Broilers fed diets conditioned at 90 °C or PU diets had higher F/G in both the starter and grower periods. These results agree with those of Liermann et al. (2019) [
46], who demonstrated that broilers fed PU diets had higher ADG, final weight (day 35), and ADFI compared to those fed MC diets. In fact, the puffing cassava starch granules were disintegrated into small molecular substances that were easier to digest and absorb, and the viscosity was reduced, the structure was loose, the volume was increased, and the protein was denatured and more easily degraded by proteases [
42]. Moreover, the denatured starch and protein reduced the stress of the broiler intestinal flora and promoted the development of the digestive system and the establishment of the intestinal micro-ecosystem [
47], which further affects the growth performance of broilers. Broilers fed SC diets had higher ADG and lower F/G compared to MC diets; this finding is in line with the concluding remarks of Naderinejad et al. (2016) [
48], who reported that broilers fed MC diets had lower FI and body weight gain than those fed SC diets. Furthermore, compared with MC corn diets, SC diets increased ADG and in vitro, true digestibility, and decreased F/G in yaks [
40]. In our study, the PU diets had the highest ADFI and F/G, indicating that the PU diets were more palatable. Although the SC diets did not increase ADFI, it had the lowest F/G, the highest AME, and relatively high ileum starch digestibility, indicating that the rapid digestion of starch in the ileum is conducive to improving the AME and feed conversion rate of broilers, thereby promoting growth performance [
31].
Broilers fed diets conditioned at 90 °C had higher ADFI than those fed diets conditioned at 75 or 60 °C in the starter period. In the grower period, broilers fed diets conditioned at 60 °C had higher ADG than those fed diets conditioned at 90 °C, which is consistent with the findings of Abdollahi et al. (2011) [
45], who found that the growth performance (consumed more feed) of broilers decreased with increased conditioning temperature. The F/G of broilers increased with the increase in conditioning temperature. In addition, Abdollahi et al. (2020) [
49] also found that broilers fed diets conditioned at 90 °C had higher F/G and consumed more feed than the unconditioned diets, but similar to the 60 °C conditioned diets. A study on nursery pigs also found that the F/G of high-temperature conditioned (88 °C) diets was higher than low temperature (54 °C) diets [
24]. A possible explanation was that high-temperature conditioning could make starch gelatinization and anti-nutritional factors degrade, thereby improving the performance of nutrients and improving the nutritional value of poultry diets, but high-temperature conditioning would also inactivate enzymes and vitamins in the diet and reduced the utilization of protein and starch [
49].
It is worth noting that the effective utilization of starch is not only related to the digestion rate but also depends on the passage time of starch in the digestive tract; the longer the starch stays in the small intestine, the more complete the starch digestion [
50]. Broilers fed PU diets had higher starch apparent digestibility of ileum in the starter and grower periods than those fed MC diets. There was also an increase in starch digestibility of ileum if corn, wheat, and sorghum diets were PU [
51]. This increase is likely the result of starch gelatinization, which breaks down the intermolecular bonds in the starch granules so that the gelatinized starch is more readily available to intestinal enzymes [
52]. The puffing increases RDS content in barley diets, peas diets, or diets containing potato starch and wheat bran, reducing RS content and thus increasing ileum starch digestibility in pigs [
53]. In our study, the benefits observed in RDS, amylopectin, and RS were also caused by starch gelatinization. Broilers fed PU diets had higher ileum digestibility of CP and nitrogen retention in the starter period than those fed MC diets. However, the PU had no effect on the apparent digestibility of DM in the total tract and the nitrogen retention in the grower period, which indicated that the puffing promoted the digestion of nutrients in the small intestine. It may be that the heat generated by puffing changes the three-dimensional structure of the protein, thereby increasing the contact of the digestive enzymes with the peptide bonds [
54]. Additionally, Rodriguez et al. (2020) [
51] found that puffing corn increased the standardized ileum digestibility of CP and all amino acids except lysine and proline, but CP and amino acids digestibility in wheat and sorghum diets were not affected by PU. This may be related to the types of proteins present in corn, wheat, and sorghum, which differ in their susceptibility to puffing [
51].
The AME was lower for MC diets than for SC and PU diets during both the starter and grower periods. Rodriguez et al. (2020) [
51] reported that PU improved the total tract apparent digestibility of energy from corn diets and sorghum diets in finishing pigs but failed to improve the metabolizable energy of wheat diets in pigs. Another study found that although feed particle size had no effect on broiler AME, SC diets (70 °C) increased the apparent digestibility of starch in the ileum [
48]. The increased AME of PU may be a consequence of the increased apparent ileum digestibility of starch and CP that we observed, but more studies are needed to verify this hypothesis.
High temperature during the puffing process leads to the denaturation of proteins or inhibits the action of digestive enzymes. Meanwhile, an increased conditioning temperature increases water-soluble non-starch polysaccharides in the intestine, resulting in an increased viscosity of the digesta and influencing the digestion of starch [
55]. Therefore, the digestibility of nutrients varies with the conditioning temperature of the diets. Our study found that the ileum digestibility of DM and starch in the diets was not influenced by the conditioning temperature. Nevertheless, the CP digestibility of diets conditioned at 60 °C was higher than that conditioned at 75 °C or 90 °C. This result is different from the results of Wang et al. (2019) [
27], who evaluated the influence of the different conditioning temperatures (65, 70, 75, 80, and 85 °C) of sorghum-based diets on nutrition digestibility in pigs and observed that the ileum digestibility of starch and CP was higher at 75 and 80 °C than at other temperatures. Lundblad et al. (2012) [
56] found that the ileum starch digestibility of wheat diets increased with increasing conditioning temperature but had no effect on DM and CP digestibility. On the one hand, during the conditioning process, high temperature and interaction between water and sugar lead to the Maillard reaction, inhibiting starch gelatinization and thereby reducing nutrient absorption [
44,
46]. On the other hand, high temperature caused the formation of a disulfide oligomer complex between sulfur-containing amino acids, resulting in a change of the protein’s secondary structure.
The conditioning temperature of SC diets at either 60 °C or 90 °C decreased the ileum digestibility of DM, compared with those fed nonconditioned MC diets. Moreover, broilers fed diets conditioned at 60 °C had higher ileum digestibility of DM and starch compared to those conditioned at 90 °C [
49]. Similarly, Abdollahi et al. (2011) [
45] compared the wheat mash diets of unconditioned MC or SC at 60, 75, or 90 °C and reported that SC resulted in slightly improved N and starch digestibility at 60 °C. However, increasing conditioning temperature from 60 to 75 and 90 °C both decreased N and starch digestibility. These results may at least partly explain the reduced digestibility of diets starch under high temperature conditions. This was inconsistent with our results that the ileum digestibility of starch in broilers fed SC diets was higher than in MC diets regardless of conditioning temperature, which may also result in higher ADG and lower F/G in SC diets than in MC diets, part of the reason. The PU diets had the highest AME at a conditioning temperature of 60 °C and the lowest at 90 °C. This is similar to the results of Abdollahi et al. (2011) [
45] that increasing the conditioning temperature from 60 to 90 °C with SC diets decreased the AME of the diets. Once again, it is clear that the processing methods and conditioning temperatures had an effect on the biochemical properties of the diets, which leaded to a change in digestibility. Therefore, the potential nutritional value of diets could be improved through an effective combination of conditioning temperatures and processing methods.