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Supplemental protease improves in vitro disappearance of dry matter and crude protein in feather meal and copra meal for pigs

ABSTRACT

The aim of the present study was to determine the effects of supplemental protease on in vitro disappearance of dry matter (DM) and crude protein (CP) in feed ingredients for pigs. The test ingredients were three sources of feather meal (FM 1, FM 2, and FM 3), meat and bone meal, soybean meal, corn dried distillers grains with solubles, copra meal (CM), palm kernel meal, wheat, and corn. In experiment 1, in vitro ileal disappearance (IVID) of DM and CP were determined to represent the digestion and absorption of the stomach and small intestine of pigs. In experiment 2, in vitro total tract disappearance (IVTTD) of DM was determined to represent the digestion and absorption of the stomach, small intestine, and large intestine of pigs. The control group had 99% of a test ingredient and 1% of wheat bran. Protease originating from Bacillus spp. was supplemented to the control diet at the expense of wheat bran to make a treatment group (100,000 U/kg of sample). Protease supplementation increased the IVID of DM in FM 1 (39.8 vs. 37.5%) and CM (45.0 vs. 42.2%). In the protease supplementation group, the IVID of CP also increased in FM 1 (48.0 vs. 43.8%) and CM (85.3 vs. 77.5%). The supplemental protease increased the IVTTD of DM in FM 1 (43.9 vs. 42.1%), FM 3 (55.6 vs. 52.7%), and meat and bone meal (85.9 vs. 84.3%). Therefore, nutrient utilization of FM and CM can be improved by supplemental protease originating from Bacillus spp. based on in vitro assays.

Keywords:
digestibility; enzyme; feed additive; pig

1. Introduction

Proteins are expensive nutritional elements that are essential for maintenance and productive functions of pigs (NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.). The use of alternative protein sources such as byproducts has increased due to the price fluctuations of conventional protein sources. However, the digestibility of alternative protein sources is generally lower than the conventional protein sources (Son et al., 2019Son, A. R.; Park, C. S.; Park, K. R. and Kim, B. G. 2019. Amino acid digestibility in plant protein sources fed to growing pigs. Asian-Australasian Journal of Animal Sciences 32:1745-1752. https://doi.org/10.5713/ajas.19.0037
https://doi.org/10.5713/ajas.19.0037...
).

In the gastrointestinal tract of pigs, proteins need to be hydrolyzed by endogenous protease into small peptides and amino acids to be utilized by the host. The digestibility of crude protein (CP) varies depending on ingredients for pigs (NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.; Stein et al., 2016Stein, H. H.; Lagos, L. V. and Casas, G. A. 2016. Nutritional value of feed ingredients of plant origin fed to pigs. Animal Feed Science and Technology 218:33-69. https://doi.org/10.1016/j.anifeedsci.2016.05.003
https://doi.org/10.1016/j.anifeedsci.201...
; Son et al., 2019Son, A. R.; Park, C. S.; Park, K. R. and Kim, B. G. 2019. Amino acid digestibility in plant protein sources fed to growing pigs. Asian-Australasian Journal of Animal Sciences 32:1745-1752. https://doi.org/10.5713/ajas.19.0037
https://doi.org/10.5713/ajas.19.0037...
). The capacity of digestive protease may be insufficient when a large quantity of protein exists in the digesta due to a high-protein ingredient used, such as feather meal (FM; approximately 80% CP), a poultry byproduct (Grazziotin et al., 2006Grazziotin, A.; Pimentel, F. A.; de Jong, E. V. and Brandelli, A. 2006. Nutritional improvement of feather protein by treatment with microbial keratinase. Animal Feed Science and Technology 126:135-144. https://doi.org/10.1016/j.anifeedsci.2005.06.002
https://doi.org/10.1016/j.anifeedsci.200...
; NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.).

The use of exogenous protease potentially improves the utilization of protein and lowers the fecal excretion of nitrogen (Tactacan et al., 2016Tactacan, G. B.; Cho, S.; Cho, J. H. and Kim, I. H. 2016. Performance responses, nutrient digestibility, blood characteristics, and measures of gastrointestinal health in weanling pigs fed protease enzyme. Asian-Australasian Journal of Animal Sciences 29:998-1003. https://doi.org/10.5713/ajas.15.0886
https://doi.org/10.5713/ajas.15.0886...
; Pan et al., 2017Pan, L.; Shang, Q. H.; Ma, X. K.; Wu, Y.; Long, S. F.; Wang, Q. Q. and Piao, X. S. 2017. Coated compound proteases improve nitrogen utilization by decreasing manure nitrogen output for growing pigs fed sorghum soybean meal based diets. Animal Feed Science and Technology 230:136-142. https://doi.org/10.1016/j.anifeedsci.2017.05.014
https://doi.org/10.1016/j.anifeedsci.201...
). Pearlzyme® is a serine protease originating from Bacillus spp. The supplementation of this protease into poultry diets improves the growth performance (Kim et al., 2015Kim, J.; Kang, S.; Yoon, J. Y.; Yang, Y. R.; Kim, W.; Jang, J. and Choi, Y. H. 2015. Effects of dietary pearlzyme on growth performance and development of digestive organs in broilers. Korean Journal of Poultry Science 42:291-297. https://doi.org/10.5536/KJPS.2015.42.4.291
https://doi.org/10.5536/KJPS.2015.42.4.2...
; Ndazigaruye et al., 2019Ndazigaruye, G.; Kim, D.; Kang, C.; Kang, K.; Joo, Y.; Lee, S. and Lee, K. 2019. Effects of low-protein diets and exogenous protease on growth performance, carcass traits, intestinal morphology, cecal volatile fatty acids and serum parameters in broilers. Animals 9:226. https://doi.org/10.3390/ani9050226
https://doi.org/10.3390/ani9050226...
). To our knowledge, the effects of this protease on the nutrient digestibility of swine feed ingredients have not been documented.

In vitro experiments are a time-saving and inexpensive method compared with in vivo assays. The in vitro procedures have been extensively used to determine the effects of supplementation of enzymes on nutrient digestibility of feed ingredients and diets (Kong et al., 2015Kong, C.; Park, C. S. and Kim, B. G. 2015. Effects of an enzyme complex on in vitro dry matter digestibility of feed ingredients for pigs. SpringerPlus 4:261. https://doi.org/10.1186/s40064-015-1060-1
https://doi.org/10.1186/s40064-015-1060-...
; Park et al., 2016aPark, C. S.; Park, I. and Kim, B. G. 2016a. Effects of an enzyme cocktail on digestible and metabolizable energy concentrations in barley, corn, and wheat fed to growing pigs. Livestock Science 187:1-5. https://doi.org/10.1016/j.livsci.2016.02.003
https://doi.org/10.1016/j.livsci.2016.02...
) based on the high correlation between in vitroin vivo data (Noblet and Jaguelin-Peyraud, 2007Noblet, J. and Jaguelin-Peyraud, Y. 2007. Prediction of digestibility of organic matter and energy in the growing pig from an in vitro method. Animal Feed Science and Technology 134:211-222. https://doi.org/10.1016/j.anifeedsci.2006.07.008
https://doi.org/10.1016/j.anifeedsci.200...
; Park et al., 2012Park, C. S.; Son, A. R. and Kim, B. G. 2012. Prediction of gross energy and digestible energy in copra meal, palm kernel meal, and cassava root fed to pigs. Journal of Animal Science 90(suppl 4):221-223. https://doi.org/10.2527/jas.53954
https://doi.org/10.2527/jas.53954...
; Son et al., 2017Son, A. R.; Park, C. S. and Kim, B. G. 2017. Determination and prediction of digestible and metabolizable energy concentrations in byproduct feed ingredients fed to growing pigs. Asian-Australasian Journal of Animal Sciences 30:546-553. https://doi.org/10.5713/ajas.16.0607
https://doi.org/10.5713/ajas.16.0607...
). Therefore, in the present study, we determined the effects of a novel source of protease supplementation on in vitro disappearance of dry matter (DM) and CP for swine feed ingredients.

2. Material and Methods

Pearlzyme® (EC 3.4.21.14), a commercial protease product, was provided by Solton Biochem Inc. (Seoul, Republic of Korea). Pearlzyme® with a molecular weight of 28 kDa was purified and characterized from Periserrula leucophryna. This protease maintains activity at a high temperature of 50 to 60 °C and is stable at pH 4-12 (Joo et al., 2001Joo, H.; Park, G.; Kim, K.; Paik, S. R. and Chang, C. 2001. Novel alkaline protease from the polychaeta, Periserrula leucophryna: Purification and characterization. Process Biochemistry 36:893-900. https://doi.org/10.1016/S0032-9592(00)00290-9
https://doi.org/10.1016/S0032-9592(00)00...
).

Ten test ingredients were three sources of FM (FM 1, FM 2, and FM 3), meat and bone meal, soybean meal (SBM), corn dried distillers grains with solubles (DDGS), copra meal (CM), palm kernel meal (PKM), wheat, and corn. Before the in vitro analysis, the test ingredients were finely ground (<1 mm), and each one was supplemented with either protease mixed evenly with wheat bran or only wheat bran at 1.0% to make up the enzyme group or the control group, respectively. The concentration of supplemental Pearlzyme® in the mixed feed sample was 100,000 unit/kg. All experiments and analyses were conducted in a biosafety level 1 containment facility in Seoul, Republic of Korea (37°32'24.8" N 127°04'27.5" E). The test ingredients were analyzed for DM (method 930.15), ash (method 942.05), CP (method 990.03), neutral detergent fiber (NDF, method 2002.04), and acid detergent fiber (ADF, method 973.18) as described in AOAC International (2016)AOAC International - Association of the Official Analytical Chemists. 2016. Official methods of analysis. 20th ed. AOAC International, Gaithersburg, MD, USA..

In experiment 1, the two-step in vitro assay was conducted to determine in vitro ileal disappearance (IVID) of DM as described in Boisen and Fernández (1995Boisen, S. and Fernández, J. A. 1995. Prediction of the apparent ileal digestibility of protein and amino acids in feedstuffs and feed mixtures for pigs by in vitro analyses. Animal Feed Science and Technology 51:29-43. https://doi.org/10.1016/0377-8401(94)00686-4
https://doi.org/10.1016/0377-8401(94)006...
; Figure 1). Briefly, 1 g of a ground ingredient sample was transferred into 100-mL conical flasks. Twenty-five milliliters of sodium phosphate buffer solution (0.1 M, pH 6.0) and 10 mL of HCl (0.2 M, pH 0.7) were added. To simulate digestion conditions in the stomach, the pH was adjusted to 2.0, and 1 mL of freshly prepared pepsin solution (10 mg/mL; ≥250 units/mg solid, P7000, pepsin from porcine gastric mucosa, Sigma-Aldrich, St. Louis, MO, USA) was added to the samples. Test flasks were incubated in a shaking incubator at 39 °C for 6 h as the final procedure of the first step. After the incubation, 10 mL of sodium phosphate buffer solution (0.2 M, pH 6.8) and 5 mL of NaOH (0.6 M, pH 13.8) were added. Then, the pH was adjusted to 6.8. Thereafter, 1 mL of freshly prepared pancreatin solution (50 mg/mL; 4 × USP, P1750, pancreatin from porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA) was added. After incubation in a shaking incubator at 39 °C for 18 h, 5 mL of 20% sulfosalicylic acid solution were added, and samples were left for 30 min at room temperature to precipitate the indigestible protein. The samples were then filtered through pre-dried and pre-weighed glass filter crucibles (Filter Crucibles CFE Por. 2, Robu, Hattert, Germany) containing 400 mg of Celite. Test flasks were rinsed twice with 1% sulfosalicylic acid solution, and 20 mL of 95% ethanol and 99.5% acetone were added twice to the glass filter crucibles. Glass filter crucibles with undigested residues were dried at 80 °C for 24 h. After conducting the two-step procedure, undigested residues on filter crucibles were collected for analysis of CP concentration to determine the IVID of CP.

Figure 1
In vitro ileal disappearance (IVID) procedure and in vitro total tract disappearance (IVTTD) procedure (Boisen and Fernández, 1995Boisen, S. and Fernández, J. A. 1995. Prediction of the apparent ileal digestibility of protein and amino acids in feedstuffs and feed mixtures for pigs by in vitro analyses. Animal Feed Science and Technology 51:29-43. https://doi.org/10.1016/0377-8401(94)00686-4
https://doi.org/10.1016/0377-8401(94)006...
; Boisen and Fernández, 1997Boisen, S. and Fernández, J. A. 1997. Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses. Animal Feed Science and Technology 68:277-286. https://doi.org/10.1016/S0377-8401(97)00058-8
https://doi.org/10.1016/S0377-8401(97)00...
; Recharla et al., 2019Recharla, N.; Kim, D.; Ramani, S.; Song, M.; Park, J.; Balasubramanian, B.; Puligundla, P. and Park, S. 2019. Dietary multi-enzyme complex improves in vitro nutrient digestibility and hind gut microbial fermentation of pigs. PLoS One 14:e0217459. https://doi.org/10.1371/journal.pone.0217459
https://doi.org/10.1371/journal.pone.021...
).

In experiment 2, the in vitro total tract disappearance (IVTTD) of DM was determined using the three-step procedure (Boisen and Fernández, 1997Boisen, S. and Fernández, J. A. 1997. Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses. Animal Feed Science and Technology 68:277-286. https://doi.org/10.1016/S0377-8401(97)00058-8
https://doi.org/10.1016/S0377-8401(97)00...
; Figure 1). The first and second steps were similar to the IVID procedure. In the third step of the IVTTD procedure, 10 mL of 0.2 M EDTA solution were added to the samples. The pH was then adjusted to 4.8. Samples were supplemented with 0.5 mL of multi-enzyme (V2010, Viscozyme®, Sigma-Aldrich, St. Louis, MO, USA) as a substitute for microbial enzymes and incubated in a shaking incubator for 18 h at 39 °C. After incubation, the samples were then filtered, and the undigested residues were collected and dried as previously described in the IVID procedure except for the drying condition (at 130 °C for 6 h).

The IVID or IVTTD of DM was calculated using the following equation (Park et al., 2016bPark, K. R.; Park, C. S. and Kim, B. G. 2016b. An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs. SpringerPlus 5:598. https://doi.org/10.1186/s40064-016-2194-5
https://doi.org/10.1186/s40064-016-2194-...
):

IVID or IVTTD of DM ( % ) = ( DM TI DM RS ) ÷ DM TI × 100

in which DMTI (g) is the amount of test ingredient as DM basis and DMRS (g) is the amount of residue after in vitro digestion procedures.

After the two-step in vitro assay, the residues of test ingredient and Celite were collected and analyzed for CP. Then, IVID (%) of CP was calculated using the following equation (Akonjuen et al., 2019Akonjuen, M. B.; Hong, B.; Choi, H. and Kim, B. G. 2019. Digestibility of energy and crude protein in Korean rice wine residues fed to pigs. American Journal of Animal and Veterinary Sciences 14:183-189. https://doi.org/10.3844/ajavsp.2019.183.189
https://doi.org/10.3844/ajavsp.2019.183....
):

IVID of CP ( % ) = [ ( DM TI × CP TI ) ( DM RS × CP RS ) ] ÷ ( DM TI × CP TI ) × 100

in which CPTI and CPRS (% DM) are the CP concentrations (%) expressed as DM basis in the test ingredient and the residue, respectively.

Data were analyzed using the GLM procedures of SAS (Statistical Analysis System, version 9.4). The model included the protease supplementation as a fixed variable. Least squares means for IVID and IVTTD of DM and IVID of CP for each ingredient were calculated. Each flask was considered as the experimental unit. The statistical significance of each treatment effect was declared at P<0.05 and tendency at P<0.10. Variables were analyzed according to the following mathematical model:

Y i j = μ + T i + ε i j '

in which Yij = in vitro disappearance of nutrients from j-th experimental unit of the i-th treatment, μ = general constant, Ti = fixed effects of protease supplementation, and ɛij = random error associated with the j-th experimental unit of the i-th treatment.

3. Results

Dry matter, ash, CP, NDF, and ADF concentrations in the test ingredients ranged from 88.1 to 97.0%, 1.5 to 36.9%, 9.1 to 88.0%, 9.8 to 65.5%, and 6.0 to 47.1%, respectively (Table 1). In experiment 1, supplemental protease increased (P<0.05) the IVID of DM in FM 1 and CM (Table 2). The protease supplementation also increased (P<0.05) the IVID of CP in FM 1 and CM (Table 3). In addition, supplementation of protease tended to increase (P = 0.081) the IVID of CP in FM 2.

Table 1
Analyzed chemical composition of feed ingredients (% as-is basis)
Table 2
In vitro ileal disappearance of dry matter (%) in feed ingredients with or without protease supplementation in experiment 11 1 Each least squares mean represents three observations except for the control group in copra meal (two observations).
Table 3
In vitro ileal disappearance of crude protein (%) in feed ingredients with or without protease supplementation in experiment 11 1 Each least squares mean represents three observations except for the control group in copra meal (two observations).

In experiment 2, the supplemental protease increased (P<0.05) the IVTTD of DM in FM 1, FM 3, and meat and bone meal (Table 4). Supplemental protease tended to increase the IVTTD of DM in corn DDGS (P = 0.077), PKM (P = 0.058), and corn (P = 0.083).

Table 4
In vitro total tract disappearance of dry matter (%) of feed ingredients with or without protease supplementation in experiment 21 1 Each least squares mean represents three observations.

4. Discussion

The analyzed concentrations of DM, ash, CP, NDF, and ADF in the test ingredients were mostly within the values in the literature (NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.). The NDF and ADF concentrations in coconut byproducts, palm kernel byproducts, and corn DDGS vary among the studies (Stein et al., 2009Stein, H. H.; Connot, S. P. and Pedersen, C. 2009. Energy and nutrient digestibility in four sources of distillers dried grains with solubles produced from corn grown within a narrow geographical area and fed to growing pigs. Asian-Australasian Journal of Animal Sciences 22:1016-1025. https://doi.org/10.5713/ajas.2009.80484
https://doi.org/10.5713/ajas.2009.80484...
; Park et al., 2012Park, C. S.; Son, A. R. and Kim, B. G. 2012. Prediction of gross energy and digestible energy in copra meal, palm kernel meal, and cassava root fed to pigs. Journal of Animal Science 90(suppl 4):221-223. https://doi.org/10.2527/jas.53954
https://doi.org/10.2527/jas.53954...
; Stein et al., 2016Stein, H. H.; Lagos, L. V. and Casas, G. A. 2016. Nutritional value of feed ingredients of plant origin fed to pigs. Animal Feed Science and Technology 218:33-69. https://doi.org/10.1016/j.anifeedsci.2016.05.003
https://doi.org/10.1016/j.anifeedsci.201...
). The deviations of fiber concentrations among the previous studies were likely due to the processing methods for oil extraction in coconut byproducts and palm kernel byproducts (Lee and Kim, 2017Lee, S. A. and Kim, B. G. 2017. Classification of copra meal and copra expellers based on ether extract concentration and prediction of energy concentrations in copra byproducts. Journal of Animal and Plant Sciences 27:34-39.) and bioethanol production in corn DDGS (Singh and Cheryan, 1998Singh, N. and Cheryan, M. 1998. Extraction of oil from corn distillers dried grains with solubles. Transactions of the American Society of Agricultural Engineers 41:1775-1777. https://doi.org/10.13031/2013.17322
https://doi.org/10.13031/2013.17322...
).

The lower IVID of DM in the plant-derived feed ingredients compared with the IVTTD of DM observed in the present work coincides with the previous experiments (Kong et al., 2015Kong, C.; Park, C. S. and Kim, B. G. 2015. Effects of an enzyme complex on in vitro dry matter digestibility of feed ingredients for pigs. SpringerPlus 4:261. https://doi.org/10.1186/s40064-015-1060-1
https://doi.org/10.1186/s40064-015-1060-...
; Park et al., 2016bPark, K. R.; Park, C. S. and Kim, B. G. 2016b. An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs. SpringerPlus 5:598. https://doi.org/10.1186/s40064-016-2194-5
https://doi.org/10.1186/s40064-016-2194-...
). The greater values in the IVTTD of DM are attributed partially to the presence of fiber-degrading enzymes in the IVTTD procedure and the substrates in the ingredients.

The IVID of CP in feed ingredients more accurately represents protein and amino acid availability of pigs compared with the IVTTD of CP, because the small intestine of pigs is probably the only section where amino acids are absorbed (Holmes et al., 1974Holmes, J. H. G.; Bayley, H. S.; Leadbeater, P. A. and Horney, F. D. 1974. Digestion of protein in small and large intestine of the pig. British Journal of Nutrition 32:479-489. https://doi.org/10.1079/BJN19740102
https://doi.org/10.1079/BJN19740102...
; Sauer et al., 1980Sauer, W. C.; Just, A.; Jørgensen, H. H.; Fekadu, M. and Eggum, B. O. 1980. The influence of diet composition on the apparent digestibility of crude protein and amino acids at the terminal ileum and overall in pigs. Acta Agriculturae Scandinavica 30:449-459. https://doi.org/10.1080/00015128009435293
https://doi.org/10.1080/0001512800943529...
; Stein et al., 2007Stein, H. H.; Sève, B.; Fuller, M. F.; Moughan, P. J. and de Lange, C. F. M. 2007. Invited review: Amino acid bioavailability and digestibility in pig feed ingredients: Terminology and application. Journal of Animal Science 85:172-180. https://doi.org/10.2527/jas.2005-742
https://doi.org/10.2527/jas.2005-742...
). Furthermore, in the total tract digestibility assays of protein and amino acids, microorganisms may influence the metabolism of amino acids such as deamination, bacterial synthesis, or endogenous secretion that can reduce the accuracy of availability in protein and amino acids. Therefore, the IVID of CP was determined in the present work.

The IVID and IVTTD of DM in plant feed ingredients used in the current study were similar to the values in the literature (Kong et al., 2015Kong, C.; Park, C. S. and Kim, B. G. 2015. Effects of an enzyme complex on in vitro dry matter digestibility of feed ingredients for pigs. SpringerPlus 4:261. https://doi.org/10.1186/s40064-015-1060-1
https://doi.org/10.1186/s40064-015-1060-...
; Park et al., 2016bPark, K. R.; Park, C. S. and Kim, B. G. 2016b. An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs. SpringerPlus 5:598. https://doi.org/10.1186/s40064-016-2194-5
https://doi.org/10.1186/s40064-016-2194-...
) except for corn DDGS. The low DM digestibility of corn DDGS in the present work appears to be mainly due to the relatively high fiber contents compared with values in the literature (Jones et al., 2010Jones, C. K.; Bergstrom, J. R.; Tokach, M. D.; DeRouchey, J. M.; Goodband, R. D.; Nelssen, J. L. and Dritz, S. S. 2010. Efficacy of commercial enzymes in diets containing various concentrations and sources of dried distillers grains with solubles for nursery pigs. Journal of Animal Science 88:2084-2091. https://doi.org/10.2527/jas.2009-2109
https://doi.org/10.2527/jas.2009-2109...
; NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.; Rojas et al., 2016Rojas, O. J.; Vinyeta, E. and Stein, H. H. 2016. Effects of pelleting, extrusion, or extrusion and pelleting on energy and nutrient digestibility in diets containing different levels of fiber and fed to growing pigs. Journal of Animal Science 94:1951-1960. https://doi.org/10.2527/jas.2015-0137
https://doi.org/10.2527/jas.2015-0137...
). The fiber content variability in corn DDGS is largely due to different corn ethanol production processes including corn processing, fermentation method, and heat treatment (Stein and Shurson, 2009Stein, H. H. and Shurson, G. C. 2009. Board-invited review: The use and application of distillers dried grains with solubles in swine diets. Journal of Animal Science 87:1292-1303. https://doi.org/10.2527/jas.2008-1290
https://doi.org/10.2527/jas.2008-1290...
; Kim et al., 2012Kim, B. G.; Kil, D. Y.; Zhang, Y. and Stein, H. H. 2012. Concentrations of analyzed or reactive lysine, but not crude protein, may predict the concentration of digestible lysine in distillers dried grains with solubles fed to pigs. Journal of Animal Science 90:3798-3808. https://doi.org/10.2527/jas.2011-4692
https://doi.org/10.2527/jas.2011-4692...
; Stein et al., 2016Stein, H. H.; Lagos, L. V. and Casas, G. A. 2016. Nutritional value of feed ingredients of plant origin fed to pigs. Animal Feed Science and Technology 218:33-69. https://doi.org/10.1016/j.anifeedsci.2016.05.003
https://doi.org/10.1016/j.anifeedsci.201...
). High dietary fiber concentrations in swine diets are known to negatively affect nutrient digestibility (Noblet and Jaguelin-Peyraud, 2007Noblet, J. and Jaguelin-Peyraud, Y. 2007. Prediction of digestibility of organic matter and energy in the growing pig from an in vitro method. Animal Feed Science and Technology 134:211-222. https://doi.org/10.1016/j.anifeedsci.2006.07.008
https://doi.org/10.1016/j.anifeedsci.200...
; Choi and Kim, 2019Choi, H. and Kim, B. G. 2019. A low-fiber diet requires a longer adaptation period before collecting feces of pigs compared with a high-fiber diet in digestibility experiments using the inert marker method. Animal Feed Science and Technology 256:114254. https://doi.org/10.1016/j.anifeedsci.2019.114254
https://doi.org/10.1016/j.anifeedsci.201...
; Choi et al., 2020Choi, H.; Sung, J. Y. and Kim, B. G. 2020. Neutral detergent fiber rather than other dietary fiber types as an independent variable increases the accuracy of prediction equation for digestible energy in feeds for growing pigs. Asian-Australasian Journal of Animal Sciences 33:615-622. https://doi.org/10.5713/ajas.19.0103
https://doi.org/10.5713/ajas.19.0103...
).

In the present study, the IVID and IVTTD of DM and IVID of CP in some FM sources were less than those in other feed ingredients and were increased by the supplemental protease. Although FM contains high CP contents, a large proportion of CP in FM is keratin, which has a great degree of cross-linking by disulfide bonds, resulting in low CP utilization of FM (Grazziotin et al., 2006Grazziotin, A.; Pimentel, F. A.; de Jong, E. V. and Brandelli, A. 2006. Nutritional improvement of feather protein by treatment with microbial keratinase. Animal Feed Science and Technology 126:135-144. https://doi.org/10.1016/j.anifeedsci.2005.06.002
https://doi.org/10.1016/j.anifeedsci.200...
; Brandelli et al., 2010Brandelli, A.; Daroit, D. J. and Riffel, A. 2010. Biochemical features of microbial keratinases and their production and applications. Applied Microbiology and Biotechnology 85:1735-1750. https://doi.org/10.1007/s00253-009-2398-5
https://doi.org/10.1007/s00253-009-2398-...
).

Pearlzyme®, a chymotrypsin-like serine protease originating from Bacillus spp., is activated by trypsin in the gastrointestinal tract and enhances protein and amino acid digestibility of non-ruminants mainly by acting on binding complexes with arginine or lysine at the outside of the helical structure in feed ingredients (Rothberg and Axilrod, 1968Rothberg, S. and Axilrod, G. D. 1968. The action of trypsin on insoluble epidermal keratin and the nature of the epidermal proteins solubilized. Biochemical Medicine 2:1-11. https://doi.org/10.1016/0006-2944(68)90002-1
https://doi.org/10.1016/0006-2944(68)900...
; Joo et al., 2001Joo, H.; Park, G.; Kim, K.; Paik, S. R. and Chang, C. 2001. Novel alkaline protease from the polychaeta, Periserrula leucophryna: Purification and characterization. Process Biochemistry 36:893-900. https://doi.org/10.1016/S0032-9592(00)00290-9
https://doi.org/10.1016/S0032-9592(00)00...
; Kim et al., 2015Kim, J.; Kang, S.; Yoon, J. Y.; Yang, Y. R.; Kim, W.; Jang, J. and Choi, Y. H. 2015. Effects of dietary pearlzyme on growth performance and development of digestive organs in broilers. Korean Journal of Poultry Science 42:291-297. https://doi.org/10.5536/KJPS.2015.42.4.291
https://doi.org/10.5536/KJPS.2015.42.4.2...
). The amounts of arginine and lysine concentrations in FM are relatively greater than those in other feed ingredients, and additionally, the ileal lysine digestibility in FM is less than those in other feed ingredients (NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.). Taken together, the large magnitude of protease effects on CP digestibility in FM and CM is likely due to the large quantity of indigestible arginine and lysine in this ingredient (Sundu et al., 2006Sundu, B.; Kumar, A. and Dingle, J. 2006. Palm kernel meal in broiler diets: effect on chicken performance and health. World's Poultry Science Journal 62:316-325. https://doi.org/10.1079/WPS2005100
https://doi.org/10.1079/WPS2005100...
; Sundu et al., 2009Sundu, B.; Kumar, A. and Dingle, J. 2009. Feeding value of copra meal for broilers. World's Poultry Science Journal 65:481-492. https://doi.org/10.1017/S0043933909000348
https://doi.org/10.1017/S004393390900034...
; NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.). However, in the current study, the responses of supplemental protease were different among the FM sources. A possible explanation is that time and temperature conditions varied by manufacturers can reflect the digestibility and protein composition of FM (Papadopoulos et al., 1986Papadopoulos, M. C.; El Boushy, A. R.; Roodbeen, A. E. and Ketelaars, E. H. 1986. Effects of processing time and moisture content on amino acid composition and nitrogen characteristics of feather meal. Animal Feed Science and Technology 14:279-290. https://doi.org/10.1016/0377-8401(86)90100-8
https://doi.org/10.1016/0377-8401(86)901...
; Zhang et al., 2014Zhang, Y.; Yang, R. and Zhao, W. 2014. Improving digestibility of feather meal by steam flash explosion. Journal of Agricultural and Food Chemistry 62:2745-2751. https://doi.org/10.1021/jf405498k
https://doi.org/10.1021/jf405498k...
). Additionally, protease supplementation increased the IVID of DM and CP in CM, which is also likely due to the large quantity of substrates for protease in CM.

Although SBM contains 49% CP, the digestibility of DM and CP in SBM was not affected by supplemental protease in the present work. This is likely due to the fact that most proteins in SBM are easily digested and absorbed (ileal CP digestibility ranges from 88 to 90%) without additional protease (NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.; Son et al., 2019Son, A. R.; Park, C. S.; Park, K. R. and Kim, B. G. 2019. Amino acid digestibility in plant protein sources fed to growing pigs. Asian-Australasian Journal of Animal Sciences 32:1745-1752. https://doi.org/10.5713/ajas.19.0037
https://doi.org/10.5713/ajas.19.0037...
). The lack of effects of supplemental protease on CP digestibility in corn DDGS may be partially explained by the fermentation process during the bio-ethanol production procedure that involves diverse microbial enzymes including proteases (Stein and Shurson, 2009Stein, H. H. and Shurson, G. C. 2009. Board-invited review: The use and application of distillers dried grains with solubles in swine diets. Journal of Animal Science 87:1292-1303. https://doi.org/10.2527/jas.2008-1290
https://doi.org/10.2527/jas.2008-1290...
; Johnston and McAloon, 2014Johnston, D. B. and McAloon, A. J. 2014. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production. Bioresource Technology 154:18-25. https://doi.org/10.1016/j.biortech.2013.11.043
https://doi.org/10.1016/j.biortech.2013....
). The reason for the lack of responses by exogenous protease in PKM is unclear as the indigestible CP content in PKM is similar to CM (Son et al., 2014Son, A. R.; Hyun, Y.; Htoo, J. K. and Kim, B. G. 2014. Amino acid digestibility in copra expellers and palm kernel expellers by growing pigs. Animal Feed Science and Technology 187:91-97. https://doi.org/10.1016/j.anifeedsci.2013.09.015
https://doi.org/10.1016/j.anifeedsci.201...
). In addition, the indigestible arginine and lysine concentrations in these ingredients are also similar (NRC, 2012NRC - National Research Council. 2012. The nutrient requirements of swine. 11th ed. National Academies Press, Washington, DC, USA.). Palm kernel meal may have some amino acid linkages or factors that potentially inhibit the action of protease. A possible explanation is that the protein in PKM is bound to the fiber matrix, resulting in poor accessibility to protease. Further research is warranted to identify the reason for the lack of protease effects on PKM. The lack of responses of supplemental protease in wheat and corn was expected as there is very limited amount of substrates (12 and 9% CP, respectively; Table 1) for exogenous protease, and CP digestibility in these ingredients is high (89 and 84%, respectively; Table 3) even without exogenous protease.

In the current study, the effect of supplemental protease alone was tested. Further research on the interaction between supplemental protease and other supplemental enzymes on nutrient digestibility for pigs would also be an interesting topic.

5. Conclusions

Nutrient utilization of feather meal and copra meal can be improved by supplemental protease originating from Bacillus spp. based on the present in vitro assays.

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Publication Dates

  • Publication in this collection
    16 Nov 2020
  • Date of issue
    2020

History

  • Received
    08 May 2020
  • Accepted
    07 Sept 2020
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