Effect of keratinase on ileal amino acid digestibility in five feedstuffs fed to growing pigs

Objective This study was conducted to evaluate the effect of keratinase (KE) on the apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of amino acids (AA) in rice bran, cottonseed meal (CSM), rapeseed meal (RSM), corn distillers dried grains with solubles (DDGS), and peanut meal (PNM). Methods Twelve crossbred barrows (Duroc×Landrace×Yorkshire, 50.5±1.4 kg body weight [BW]) fitted with T-cannulas at the terminal ileum were allotted to a 12×6 Youden Square design with 12 diets and 6 periods. The treatment diets included rice bran, CSM, RSM, corn DDGS, PNM, or corn-soybean meal (cSBM) supplemented with 0.05% KE or not. Diets were given to pigs at a level of 3% BW in two equal meals. The endogenous AA losses were the mean results of three previously experiments determined by a same nitrogen-free diet fed to pigs. Pigs had free access to water during the experiment. Results The KE supplementation improved (p<0.05) the AID and SID of Met, Thr, Val, Asp, Cys, and Tyr in rice bran. Inclusion of KE increased (p<0.05) the AID and SID of Met and Val in CSM. The KE supplementation decreased (p<0.05) the AID and SID of His in RSM and all measured AA except for Arg, Met, Trp, Val, Gly, and Pro in corn DDGS. There was an increase (p<0.05) in AID and SID of Leu, Ile, Met, Ala, Cys, Ser, and Tyr in PNM supplemented with KE compared with that without KE. Inclusion of KE increased (p<0.05) the AID and SID of crude protein, Leu, Ile, Phe, Thr, Asp, and Ser in cSBM. Conclusion This study indicated that KE had different effects on ileal AA digestibility of feedstuffs for growing pigs, which can give some usage directions of KE in swine feed containing those detected feedstuffs.


INTRODUCTION
Keratinase (KE), one of the proteases, was first isolated from the culture medium of Bacillus licheniformis PWD-1 [1,2]. Compared with most known proteases, KE displays a higher proteolytic activity [3] and can break down a wide range of proteins such as casein, collagen, elastin, and keratin as well as other proteins containing cysteine disulfide bonds [4][5][6]. Previous studies reported that dietary KE supplementation improved the nutrient digestibility and apparent ileal digestibility (AID) of amino acids (AA) for growing pigs [7]. It was also reported that KE could improve growth performance, breast meat yield, and gut villus structure of broilers fed diets based on corn and soybean meal (SBM) [8][9][10].
Soybean meal is the primary protein source of swine feed in many countries. Some soybean proteins, such as glycinin, β-conglycinin, trypsin inhibitors, lectins and other minor proteins, can negatively impact on intestinal morphology of pigs [11][12][13]. Cottonseed meal (CSM), rapeseed meal (RSM), peanut meal (PNM), rice bran, and corn distillers dried grains feed allotment adjusted accordingly. All pigs had free access to water during the experiment.

Digesta collection and sampling
Each experimental period lasted for 7 d where the first 5 d were considered the adaptation period followed by 2 d of digesta collection. Digesta was collected continuously from 08:00 to 17:00 using procedures described by Stein et al [16]. Digesta was collected in plastic bags attached to the simple T-cannula. Bags were changed when digesta made up no more than 30% of the bag volume. Collected digesta was immediately stored in a -20°C freezer. At the end of the experiment, ileal digesta was thawed and mixed within animal and diet, and a sub-sample of 500 mL was taken. Digesta samples were lyophilized in a vacuum-freeze dryer (Tofflon Freezing Drying Systems, Minhang District, Shanghai, China) and ground through a 1-mm screen for further chemical analysis.

Chemical analyses
The DM, CP, and AA were analyzed in the feedstuffs, diets, and digesta. Feedstuffs were also analyzed for ether extract (EE), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF) and ash. All experimental diets and digesta were analyzed for Cr to calculate the concentration of indigestible index chromic oxide. The DM, CP, and ash in samples were analyzed according to AOAC procedures (method   [18]. The CF, NDF, and ADF were determined using filter bags and fiber analyzer equipment (Fiber Analyzer, Ankom Technology, Macedon, NY, USA) following the procedures described by Van Soest et al [19]. The concentration of NDF was analyzed using heat stable α-amylase and sodium sulfite without correction for insoluble ash. Determination of AA was conducted according to Li et al [20] where samples were hydrolyzed with 6 N HCl at 110°C for 24 h and then analyzed using an Amino Acid Analyzer (Hitachi L-8900, Tokyo, Japan). Sulfur-AA (Met and Cys) content of digesta, diets, and feedstuffs was determined using cold performic acid oxidation overnight and hydrolyzed with 7.5 N HCl at 110°C for 24 h before measurement using an Amino Acid Analyzer (Hitachi L-8900, Japan). Estimates of Trp were made by hydrolyzing samples with LiOH for 22 h at a constant temperature of 110°C and then analyzed using High Performance Liquid Chromatography (Agilent 1200 Series, Santa Clara, CA, USA). Analysis of the Cr concentration in all diets and digesta was conducted using a polarized Zeeman Atomic Absorption Spectrometer (Hitachi Z2000, Japan) after nitric acid-perchloric acid wet ash sample preparation. All chemical analyses were conducted in two duplicates. The analyzed composition of feedstuffs is presented in Table 4.

Calculations
The AID of AA was calculated using the following method described by Stein et al [21]: Where, AA d and Cr d were the concentrations of AA and Cr in the ileal digesta (g/kg of DM) and AA f and Cr f were the concentrations of AA and Cr in the test diets (g/kg of DM). The SID of AA was calculated using the following equation: In which IAA end is the basal endogenous loss of an AA (g/kg of DM intake). The AID and SID of CP were determined using the same two aforementioned equations.

Statistical analysis
Data were checked for normality, and outliers were detected and then removed using the UNIVARIATE procedure of SAS (SAS Inst. Inc., Cary, NC, USA). Outliers were defined as those beyond the range of mean±3 times standard deviation. Data were then analyzed using MIXED procedure of SAS, and the statistical model included the fixed effects of feedstuff and KE supplementation, and their interaction effect, and the random effects of animal and period. Pig was treated as the experimental unit. Means were calculated using the LSMEANS statement, and multiple comparison were adjusted using Tukey's test.
Because the interaction effects between feedstuff and KE were significant for almost all the parameters we tested (AID and SID of AA), data were then analyzed within each feedstuff by one-way analysis of variance using the general linear model procedure of SAS to test the effects of KE within each ingredient. An α value of 0.05 was used to assess statistical significances among treatment means.

RESULTS
The results of AID and SID of CP and AA are shown in Tables 5, 6, respectively. There was a significant interaction effect between feedstuff and KE supplementation for AID and SID of  2) "-" represents diets without keratinase addition, and "+" represents diets with keratinase addition.   1) Data of digestibility were mean of six replicates. Data within a column without common superscripts differ (p < 0.05). 2) "-" represents diets without keratinase addition, and "+" represents diets with keratinase addition.

1951
Huang et al (2018) Asian-Australas J Anim Sci 31:1946-1955 CP and almost all the AAs except for Arg, Ala, Gly, Pro, and Ser. Among the six tested diets, the cSBM diet and PM diet showed the greatest AID and SID for almost all the AAs, while rice bran diet and corn DDGS diet showed the lowest AID and SID for almost all the AAs.
Effects of KE on the AID of AA in feedstuffs analyzed within each diet in our study are shown in Table 7. Inclusion of KE improved (p<0.05) the AID of Met, Thr, Val, Asp, Cys, and Tyr in rice bran. The CSM supplemented with KE had greater AID of Met and Val compared with that in CSM without KE Table 7. Apparent ileal digestibility (AID) of crude protein (CP) and amino acids (AA) in 6 experimental diets with or without keratinase addition fed to pigs analyzed within each diet (%) 1) Effects of KE on the SID of AA in feedstuffs analyzed within each diet in our study are shown in Table 8. Rice bran supple- Table 8. Standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AA) in 6 experimental diets with or without keratinase addition fed to pigs analyzed within each diet (%) 1)

DISCUSSION
In our study, the chemical composition of the rice bran was within the ranges reported by Shi et al [22]. The chemical composition of the CSM was within the ranges reported by Li et al [23]. The chemical composition of the RSM was within the ranges reported by Li et al [24]. The chemical composition of the corn DDGS was comparable to those values from NRC [25] (corn DDGS containing greater than 6% oil but less than 9% oil) and were within the ranges reported by Li et al [26]. The chemical composition of the PNM was comparable with the values reported by Li et al [27] and NRC [25]. The concentration of CP (47.46% as fed) in SBM was little higher than that of 45.1% reported by Lagos and Stein [28]. The concentration of individual AA in each tested feedstuff was within the relative standard deviation presented in NRC [25]. The values of endogenous CP and AA losses presented in Table 3 were comparable with the values from previous studies that also conducted in our lab [20,24,27]. The KEs are mainly serine and metalloproteases, which could hydrolyze protein without being denatured or broken down by the gastrointestinal tract in broiler chickens [9]. It may also need the presence of live cells for activating the sulfitolysis or reduction in disulfide bonds and proteolysis effect of KE [29,30]. Though the proteolysis effection of most proteases may begin when the animals consumed the feed, most digestive enzymes are not stable at the low pH encountered in the stomach and upper small intestine. As a result, these enzymes may become inactivated, or a portion of the enzyme is inactivated. This process of inactivation is the likely cause of the variable results of other enzymes reported previously. Microbial KEs are predominantly extracellular when grow on keratinous substrates and can be stable over a wide range of pH from 5 to 13 [31]. The optimal pH for KE produced by Bacillus licheniformis PWD-1 was about 7.5 [2], which is like the conditions in the lower jejunum and ileum of pigs [32]. Therefore, the KE included in the current study was likely exerting its effect on protein digestion in the lower small intestine.
The significant interaction effect between feedstuff and KE supplementation indicated that the effect of KE on AA digestibility can be influenced by treatment diets. Considering the more abundant AA contents in cSBM diet and PM diet, and the relative poor AA contents in rice bran diet and corn DDGS diet, it is reasonable to observe the greatest AID and SID of almost all the AAs in cSBM and PM diets, while the lowest AID and SID of almost all the AAs in rice bran and corn DDGS diets. The improved AID of CP and AA in the KE supplemented cSBM diet agreed with Wang et al [7] where the same level of KE (0.05%) supplementation in a cSBM basal diet significantly increased the dietary AID of CP, Arg, His, Leu, Phe, and Thr. About 40% and 30% of the total soybean globulin proteins were glycinin and β-conglycinin, respectively [11,12]. The ratio of CP provided by SBM vs corn was 2.1:1 in cSBM diet used in our trial. Therefore, the improved digestibility of CP and AA in the KE added cSBM-based diet is most likely due to the digestion of glycinin and β-conglycinin, which were done via protein-disulfide reductase break down of the cystine disulfide bond and the peptidohydrolase hydrolyzation of the denatured protein into peptides and AA [31,33]. The KE has been used to improve digestibility of feather meal in livestock diets [34] and in poultry diets [8,10,35]. However, no previous studies reported the utilization of KE on the other feed ingredients.
The KE significantly improved the AID and SID of Met in rice bran, CSM, and PNM but not in RSM and cSBM. We supposed that this is related to the relative lower concentration of Met in the former three feedstuffs, considering relative less disulfide bonds that KE may need to break down. In rice bran, both the AID and SID of Met and Cys improved more than 20%, which can also primarily attribute to break down effect of KE on disulfide bonds of Met and Cys. In CSM, KE only affected the AID and SID of Met and Val, but not the dispensable AA. In RSM, KE supplementation only improved the AID and SID of His. In addition to Met, KE also improved the AID and SID of branched AA of Leu and Ile and some dispensable AA in PNM.
Although KE supplementation significantly improved the AID and SID of some AA in the five detected feedstuffs, it only significantly increased the CP digestibility in cSBM. In the current study, the CP levels in the detected diets except for rice bran diet were similar to those reported by Wang et al [7], who also found that there was no interaction effect between the dietary KE (0.05% vs 0.10%) and CP (22.0% vs 20.0%). Therefore, the various CP levels may not explain the discrepancies of the KE supplementation effects on CP digestibility in different feedstuffs. Moreover, the relative lower digestibi lity of Lys in corn DDGS compared with that in the other feedstuffs was reasonable, because of the heat damage during fermentation or drying during the processing of DDGS [36,37]. However, the reasons for the reduction of AID and SID of most AA in corn DDGS supplemented with KE were not known. This is a new finding that has never been reported before. The possible hypothesis may be that some components