Novel sfericase protease improves amino acid digestibility of soybean meal and rapeseed meal in broiler chickens

ABSTRACT 1. Two experiments were conducted to explore the effects of an exogenous sfericase protease on the apparent ileal nutrient digestibility of soybean meal (SBM) and rapeseed meal (RSM) in broiler chickens. 2. In each experiment, a total of 256 sixteen-day-old male Cobb 500 broilers were fed one of four semi-purified experimental diets, comprising two different batches (A and B) of samples for either SBM (Exp. 1) or RSM (Exp. 2) without or with an exogenous sfericase (0 or 30,000 NFP/kg). Each experimental diet was fed to eight replicate pens of broiler chickens from 16 to 21 d of age (eight birds per cage), and ileal digesta were collected for measuring the digestibility coefficients. 3. In Exp. 1, the amino acid digestibility was greater (P < 0.05) in SBM B compared with SBM A for Arg and Val, and a similar trend (P < 0.1) was observed for Tyr, Leu and Thr. Exogenous sfericase increased (P < 0.10) digestibility of most of amino acids except Gly, His and Trp. There was an interaction between SBM source and sfericase, whereby digestibility of P, N and Asp was increased by sfericase for SBM B but not for SBM A. In Exp. 2, there was no interaction (P > 0.05) between RSM source and sfericase for ileal nutrient digestibility. Digestibility was greater in RSM A compared to RSM B for all non-essential AA and most essential AA (except for Trp), while the reverse was noted for Ca and P (P < 0.05). Exogenous sfericase increased (P < 0.1) digestibility for all amino acids except Cys and Met. 4. In conclusion, the current studies showed that both SBM and RSM batches influenced amino acid digestibility. Sfericase protease supplementation increased amino acid digestibility for both SBM and RSM. The digestibility effects were greater in the SBM batch with low digestibility for N and Asp which was in line with an increase in P digestibility.


Introduction
Among the plant-origin protein ingredients, soybean meal (SBM) and rapeseed meal (RSM) are the predominant sources of amino acids (AA) in poultry diets due to their high protein content and desirable AA profile.Compared to SBM, the inclusion of RSM in poultry diets is limited by the presence of anti-nutritional factors and fibre (Kocher et al. 2000).In recent years, there has been an increasing interest in using RSM in poultry diets which has been driven by increasing demand for vegetable oils and advances in breeding and processing technologies (Khajali and Slominski 2012;Olukomaiya et al. 2019).It has been reported that the nutritional value of SBM and RSM is variable and affected by agronomical and environmental conditions, feed composition and processing as well as the response of the target animal species (Bell 1993;Cheng et al. 2022;Ibáñez et al. 2020;Karr-Lilienthal et al. 2004;Kasprzak et al. 2016;Parsons et al. 1992;Salazar-Villanea et al. 2022).Such variability in nutritional value could potentially affect the performance and profitability of animal production.
With increasing economic and environmental pressure on the animal production industry, several measures are being examined to reduce feed cost without detrimental effects on animal performance and environmental emissions.The use of exogenous enzymes, like phytase and xylanase, has become a standard practice in animal feed.The use of mono-component protease has become more popular since 2011 (Angel et al. 2011;Freitas et al. 2011;Fru-Nji et al. 2011).Animals synthesise and secrete endogenous proteases in the gastrointestinal tract, and these are generally considered to be sufficient to optimise feed protein utilisation (Le Heurou-Luron et al. 1993).However, despite the presence of endogenous protease activity, nitrogen and amino acid digestibility reported for poultry indicated that a substantial amount of dietary protein passes through the GIT without being completely digested (Cowieson et al. 2020a;Parsons et al. 1997).This undigested protein represents an opportunity to improve protein digestibility.
Measuring and predicting the nutrient value of feed ingredients and their response to exogenous enzymes are essential for precision feed formulation and optimal enzyme application.To date, there are limited studies to evaluate the efficacy of a novel sfericase protease in feedstuffs for broilers.The tested protease is a novel sfericase endopeptidase expressed in Bacillus licheniformis production strain which was genetically modified to produce a novel subtilisin from a sub-family of the S8 proteases.More details of the product have been reported by Cupi et al. (2022).Therefore, the objective of the study was to evaluate the effect of sfericase supplementation on apparent ileal nutrient digestibility of SBM and RSM in broiler chickens.To estimate reproducibility of sfericase effect, two divergent batches each of SBM and RSM were used in the study.

Ethical statement
The animal handling and management procedures were reviewed and approved by the Animal Welfare Committee of DSM China Animal Nutrition Research Centre.

Experimental design and bird management
Two experiments were conducted to evaluate the apparent ileal nutrient digestibility of SBM (Exp. 1) and RSM (Exp.2) with two batches of samples for each, as well as their response to exogenous sfericase in broiler chickens.
Bird management and feed preparation followed the same procedures in both experiments, in which day-old Cobb 500 male broilers, obtained from a commercial hatchery, were randomly allocated to pre-study battery cages, where they received a common starter diet (Exp.1: AME: 12.55 MJ/kg , CP: 21.2%; Exp.2: AME: 12.57 MJ/kg, CP: 21.5%) based on maize and soybean meal for 7 d.On d 8, for each experiment, 256 birds were selected, individually weighed, randomly distributed to the experimental cages and received the common starter diet for adaptation to the new battery cage environment until d 16.On d 16, birds were introduced to one of the four semi-purified experimental diets, comprising two different batches of samples (A and B) for either SBM (Exp. 1) or RSM (Exp.2) without or with an exogenous sfericase (0 or 30,000 NFP/kg).There were eight birds per cage and eight replicate cages per diet.The diets in both experiments were provided in mash form from 16 to 21 d of age and formulated according to the recommendations of Ravindran et al. (2017) for the measurement of amino acid digestibility of feedstuffs in broilers.Titanium dioxide (TiO 2 ) was added at 0.3% of feed as the indigestible marker to enable the calculation of ileal digestibility coefficients.
Birds were reared in an environmentally controlled room and received 23 L:1D of lighting program during the first week and 20 L:4D afterwards until the end of the study.The temperature of the room was adjusted according to breed guidelines.Birds were allowed ad libitum access to feed and water.
Samples of SBM and RSM used in the two experiments were collected from different vendors in China.The novel protease product (ProAct360 TM , DSM Nutritional Products AG, Kaiseraugst, Switzerland) was a sfericase endopeptidase expressed in Bacillus licheniformis.One protease unit (NFP) unit was defined as the amount of enzyme that releases 1 µmol p-nitroaniline from 1 mM substrate (N-succinyl-Ala-Ala-Pro-Phe p-nitroaniline) per min at pH 9.0 and 37°C (Cupi et al. 2022).The enzyme activity was measured photometrically at a wavelength of 405 nm with the amount of released yellow pNA being proportional to the protease activity of the enzyme.The enzyme was added at 50 mg/kg to provide 30,000 NFP/kg of feed.

Ileal digesta collection
On d 21, all birds from each cage were euthanised by carbon dioxide asphyxiation for collection of ileal digesta content.
The ileum was defined as the region from the Meckel's diverticulum to 4 cm proximal to the ileo-caecal junction.The ileal content was collected by gently flushing with distilled water and pooled per cage before immediately freezing on ice.Ileal digesta samples from birds within each cage were pooled, frozen at − 20°C and lyophilised, and the dried samples were ground to pass through a 0.5 mm sieve and were stored at 4°C in airtight bags for further analysis.
For feed preparation, each basal diet was prepared in one batch and subdivided into two portions; an appropriate amount of sfericase product was pre-mixed with a small quantity of the basal diet which was then added to the feed to achieve the target concentration.

Chemical analysis
Dry matter and crude ash were determined according to the AOAC 934.01 (1990) andAOAC 942.05 (1990), respectively.Crude fat was determined by submerging samples in petroleum ether in a fat extractor (FOSS Analytical Co. Ltd.China) according to method AOAC 960.39 (1990).Nitrogen was determined by the Dumas method using a combustion analyser (Leco Model FP-528, Leco Corp., St. Joseph, MI, U.S.A.).The concentration of Ti, Ca and P was measured by an inductively coupled plasma optical emission spectrophotometer (Optima 8000, PerkinElmer, Shelton, CT, U.S.A.) after HNO 3 /NH 4 F/H 2 O 2 mineralisation.Amino acids (except Trp) were analysed by Ultra Performance Liquid Chromatograph (Acquity UPLC CH-A, Waters Corporation, Milford, MA, U.S.A.) after the samples were hydrolysed for 24 h in 6 N hydrochloric acid at 110°C, while the samples were oxidised with performic acid before the hydrolysis for Met and Cys measurement.Tryptophan was analysed by High Performance Liquid Chromatograph (Agilent 1200 series, Agilent Technologies, Santa Clara, CA, U.S.A.) whereby the samples were hydrolysed for 24 h in 4 N lithium hydroxide at 110°C.Solubility in KOH and urease activity of the two SBM samples were determined according to the EN ISO 14244:2014 andAraba andDale (1990), respectively.Phytate P was calculated as the difference between total P and free P according to McKie and McCleary (2016).

Calculations and statistical analysis
The concentrations of nutrients in feed and ileal digesta samples were used to calculate the apparent ileal digestibility (AID) coefficient of nutrient according to the following formula: CM f = concentration of marker in feed (g/kg dry matter); CM e = concentration of marker in ileal digesta (g/kg dry matter); CN f = concentration of nutrient in feed (g/kg dry matter); CN e = concentration of nutrient in ileal digesta (g/kg dry matter).
Data from the two experiments were analysed separately using the Fit mode platform in JMP version 16.0 (SAS Institute, Cary, NC, US).Cage served as the experimental unit.The statistical model included effects of ingredient batch, sfericase supplementation and their interactions.
Significance was set at P < 0.05, with trends assigned where P < 0.1.

Chemical composition of meal samples and feeds, and sfericase recovery
The analysed chemical composition of the tested meal samples and corresponding feeds used in the two experiments is presented in Tables 1 and 2, respectively.
Solubility in KOH and CP content in both protein meal samples and feeds was higher for SBM A than SBM B, while the opposite was the case for crude fat content.Other measurements were similar between the two SBM samples and feeds.The two RSM samples and feeds had similar chemical composition except for crude fat content, which was almost doubled in RSM B than in RSM A (39.5 vs. 20 g/kg).The analysed sfericase activities in the supplemented feeds were 31,541, 36,234, 33,863 and 35,953 NFP/kg for the SBM A, SBM B, RSM A and RSM B diets, respectively, whereas sfericase activity was not detected in the control diets.The initial body weight (d 16) was not different among treatments in both experiments.

Nutrient digestibility (experiment 1)
The effect of SBM batch and exogenous sfericase on the apparent ileal digestibility of Ca, P, N and non-essential AA is presented in Table 3. Digestibility tended to be higher (P < 0.1) for Tyr and lower for Gly in SBM B compared with SBM A. Sfericase supplementation increased (P < 0.05) digestibility for Ala, Cys, Glu, Pro, Ser and Tyr.However, increases in the digestibility of P, N and Asp associated with sfericase supplementation were detected only for SBM B. This resulted in significant interactions between SBM batch and sfericase for these measurements.
The effect of SBM batch and exogenous sfericase on the apparent ileal digestibility of essential AA is summarised in Table 4.There was no interaction (P > 0.05) between SBM batch and sfericase on essential AA.The digestibility of Arg and Val was greater (P < 0.05) in SBM B than in SBM A, and a similar trend (P < 0.1) was observed for Leu and Thr.Exogenous sfericase increased the digestibility of Arg and Met numerically (P < 0.10) and for other essential AA significantly (P < 0.05), with the exception of His and Trp.

Nutrient digestibility (experiment 2)
The effects of RSM source and exogenous sfericase on the apparent ileal digestibility of Ca, P, N and non-essential AA are presented in Table 5.
There was no interaction (P > 0.05) between RSM source and sfericase on non-essential AA.The digestibility was greater (P < 0.05) for all non-essential AA in RSM A compared with RSM B, but the reverse was noted for Ca and P. Sfericase supplementation increased the digestibility of Asp, Ser and Tyr numerically (P < 0.1) and the rest of non-essential AA significantly (P < 0.05), except for Cys.
The effects of RSM source and exogenous sfericase on apparent ileal digestibility of essential AA are presented in Table 6.
No interaction between RSM batch and sfericase was observed for essential AA (P > 0.05).Digestibility was either numerically (only for Met) or significantly greater for most of essential AA in RSM A than RSM B, except for Trp.Supplementation of sfericase increased the digestibility of Arg and Val numerically (P < 0.1) and of other essential AA significantly (P < 0.05), except for Met.

Discussion
In both experiments, diets were formulated containing the test meals as the sole source of protein with the rest of the ingredients unchanged.This allowed direct comparison of the samples, as well as their responses to exogenous sfericase.In Exp. 1, supplementation with sfericase showed a promising effect on AA digestibility, which agreed with previous studies on the effect of exogenous protease on AA digestibility in SBM (Cowieson et al. 2020a;Salazar-Villanea et al. 2022;Stefanello et al. 2016).The data showed that SBM A had slightly inferior digestibility for certain AA compared with SBM B. This could have been associated with chemical composition and protein quality.Solubility in KOH has been considered as a good indicator of protein quality in SBM (Parsons et al. 1991).However, it was unlikely related to the differences in AA digestibility in the current study, as the values for both SBM samples were within or close to the optimal range of 78-84%.It should be noted that only SBM B benefitted from sfericase supplementation in terms of digestibility for P, N and Asp, whereas this was not seen for SBM A. This discrepancy could be partly attributed to the N and Asp digestibility, which was generally lower in SBM B compared with SBM A. This finding was supported by Liu et al. (2013) and Cowieson and Roos (2014), who noted that the effect of exogenous protease on AA digestibility was negatively associated with the inherent digestibility of AA in the control diet or ingredient.Similar effects have been demonstrated for phytase by Cowieson et al. (2017).However, given the fact that P digestibility was higher in SBM B, the beneficial effect of sfericase on this parameter could not be explained by inherent digestibility.The reason for this was unclear.It may have been possible that sfericase enhanced P digestibility by interrupting the phytate/protein matrix in SBM during proteolysis, releasing phytate-bound P and, hence, increased P digestibility.Another possible mechanism could be that addition of exogenous sfericase complemented endogenous protease and enhanced the ability of protease in hydrolysing peptide chains.It is possible to reduce the degree of protein in chelating phytate in the small intestine.It's worth noting that improvement in P digestibility occurred with an increase in N and Asp digestibility following sfericase supplementation in birds fed SBM B. Cowieson et al. (2020b) observed synergistic effects of protease and phytase on ileal digestibility of certain AA.However, there are few research that have evaluated the relationship between phytate hydrolysis and AA digestibility in response to single protease supplementation, and this topic warrants further elucidation.In the present study, digestibility of Gly, His and Trp was not affected by sfericase supplementation.The lack of responses for these three AA can be attributed to their high inclusion concentrations in SBM.In Exp.2, the AA digestibility of RSM B was inferior to that of RSM A. The reason for this was not fully clear, as AA digestibility can be affected by cultivars, growing conditions and processing technologies.The current results demonstrated that supplementation with sfericase was effective in improving digestibility for all AA, except Cys and Met.
According to current published data, there is no comparable finding for the effect of single protease on AA digestibility in RSM.As RSM is high in sulphur-containing AA, it was logical that Met and Cys were least-affected by supplementation with sfericase.
Comparing the results of the two experiments, AA digestibility was generally lower in RSM than in SBM, which agreed with previous studies (Adedokun et al. 2008;Jondreville et al. 2000).Moreover, differences in nutrient digestibility response to added sfericase were noted for the two meals in the current study, and this corroborated previous reports by Simbaya et al. (1996) and Cowieson et al. (2016).
In conclusion, the current study showed that both SBM and RSM batches influenced AA digestibility.Sfericase protease supplementation increases AA digestibility for both SBM and RSM.Moreover, these effects were greater in SBM batches with low digestibility for N and Asp, which was associated with an increase in P digestibility.

Table 2 .
Ingredient and composition of the experimental diets (as fed basis).

Table 4 .
Effect of sfericase on apparent ileal digestibility of essential amino acids (EAA) for soybean meal (SBM) samples in broilers (Exp. 1, n =8). 1 1Means with different subscripts in a column differ significantly.

Table 6 .
Effect of sfericase on apparent ileal digestibility of essential amino acids (EAA) for rapeseed meal (RSM) samples in broilers (Exp.2, n = 8). 1 1Means with different subscripts in a column differ significantly.