Effects of crude protease produced by Bacillus subtilis (MAFIC Y7) on growth performance, immune indices, and anti-inflammatory responses of broilers fed soybean meal- or cottonseed meal-based diets

Abstract

A strain of Bacillus subtilis (MAFIC Y7) was isolated from the intestine of Tibetan pigs and was able to express high protease activity. The aim of this study was to characterize the proteases produced by MAFIC Y7, and to investigate the effects of protease addition on growth performance, ileal amino acid digestibility, and serum immunoglobulin and immune factors of broilers fed SBM-based diets, or on growth performance, carcass characteristics, and intestinal morphology of broilers fed CSM-based diets. B. subtilis (MAFIC Y7) expressed protease showed its optimal enzyme activity at 50 °C and pH 7.0. The coated crude enzyme (CCE) showed greater stability at pH 3.0 than its uncoated counterpart. Experiment 1 was conducted with six diets based on three levels of crude protein (CP)—CP_low, CP_medium, and CP_high—with or without CCE. In CP_low, CCE increased gain:feed (G:F) (days 1 to 21, days 1 to 42) by 8%, 3%, respectively, and enhanced apparent ileal digestibility (AID) of crude protein and lysine (on day 42) by 8.8%, 4.6%, respectively, compared with diets containing no CCE (P < 0.05). CCE increased G:F from days 1 to 21 from 0.63 to 0.68, improved G:F and average daily gain (ADG) during days 1 to 42, and enhanced AID of crude protein, lysine, cysteine, and isoleucine on day 42 compared with the unsupplemented treatments (in CP_medium, P < 0.05). CCE increased serum IgA (on day 21), serum IgA and IgG and increased serum IL-10 (on day 42), but decreased serum tumor necrosis factor-α (TNF-α; on day 21), and serum IL-8 and TNF-α (on day 42) compared with unsupplemented treatments. At CP_high, CCE decreased serum levels of IL-6 and TNF-α (on day 21), and IL-8 and TNF-α (on day 42) compared with unsupplemented treatments (in CP_high, P < 0.05). In experiment 2, CSM-based diets with two lysine-to-protein ratios (5.2% or 5.5%) with or without CCE. In the high Lys diet (5.5% Lys:protein), CCE increased ADG and G:F, increased carcass, but decreased abdominal fat compared with the unsupplemented treatment (P < 0.05). In the 5.2% Lys:protein dietary treatment, CCE improved duodenal villus height compared with the unsupplemented treatment (P < 0.05). Supplementation of protease produced by MAFIC Y7 was associated with lower inflammatory responses in SBM diets (CP_medium or CP_high) and improved ADG in broilers fed CP_medium or CP_high. The proteases improved ADG and the efficiency of CSM use when the ratio of Lys to protein was 5.5%.

The aim of this study was to investigate the effects of Bacillus subtilis (MAFIC Y7)-expressed protease on growth performance, G:F and inflammatory responses elicited by soybean meal (SBM) diets and cottonseed meal in broilers.

Introduction

Soybean meal (SBM) is the most common protein source for farmed animals. As a coproduct after oil extraction from whole soybeans, SBM contains 40% to 49% crude protein and balanced amino

acid (AA) profiles for animals (Ibáñez et al., 2020). There are important reasons to reduce the use of SBM in animal feeds. One way to achieve this would be to improve the efficiency of nutrient utilization from SBM in livestock nutrition. Protein digestion in monogastric animals is predominantly accomplished by endogenous proteases released in the gastrointestinal tract. The amount of released endogenous proteases usually is adequate for utilization of dietary proteins in diets (Ndazigaruye et al., 2019). However, there is still a considerable portion of dietary proteins passing the gastrointestinal tract without digestion, which can be as much as 18% to 20% of dietary protein (Bryden et al., 2004). Thus, supplementation with exogenous proteases to broiler feed is important to increase nutritive value of feed and increase utilization of feed proteins. Soybean meal contains a variety of anti-nutritional factors (ANFs) that either decrease its nutritional value or compromise animal health. The major ANFs in SBM include trypsin inhibitors, antigenic factors (glycinin and β-conglycinin), phytic acid, and lectins. Supplementation of exogenous enzymes, such as proteases, phytases, and cellulases can help monogastric animals degrade ANFs. Protease supplementation to broiler diets containing SBM was beneficial for growth performance (Toghyani et al., 2017; Lu et al., 2020), gut morphology, and nutrient utilization (Xu et al., 2017). In SBM, about two-thirds of the phosphorus is bound by phytic acid, which severely limits phosphorus utilization by monogastric animals in SBM-based diets (Nelson et al., 1968). Additionally, phytic acid has high activity in binding proteins and minerals in feed to further reduce the efficiency of nutrient utilization. Supplementing diets with phytase can degrade phytic acid complexes and liberate phosphorus and minerals for use by monogastric animals (Mullaney et al., 2000).

Another approach to reduce the use of SBM is to use alternative protein sources as substitutes for SBM in feed. Cottonseed meal (CSM) is potentially an alternative to SBM due to its relatively high content of protein (Wang et al., 2015; Rahman et al., 2018; Yadav et al., 2021). CSM is a by-product of oil extraction from cottonseed. However, CSM contains gossypol (Gadelha et al., 2014b) that has multiple deleterious effects in chicks (Nagalakshmi et al., 2007; ŚWiĄTkiewicz et al., 2016), pigs (da Silva et al., 2021), and other animals (Zeng et al., 2014; Yu et al., 2022). Typically, CSM contains about 1% gossypol after oil extraction (He et al., 2015). Microbial fermentation can detoxify gossypol. Microbes can produce a variety of enzymes and small substrates that bind free gossypol to form nontoxic gossypol (Zhang et al., 2006, 2007; Wang et al., 2021). Some bacteria, such as Bacillus subtilis, can produce enzymes that break down phenolic compounds (Gadelha et al., 2014a; Hasan and Jabeen, 2015). As a phenolic compound, gossypol was degraded by fermentation with Bacillus spp. which were screened from a high protease-producing strain (Li et al., 2022).

In our previous study, a strain of B. subtilis (named MAFIC Y7) was isolated from the gut of Tibetan pigs. Fermentation of MAFIC Y7 under specific conditions showed dominant protease activity and minor activities of phytase, amylase, lipase, xylanase, and cellulase. The purpose of the present study was to investigate the effects of crude protease produced by MAFIC Y7 fermentation on growth performance of broilers fed diets containing different levels of protein. Additionally, we evaluated the efficiency of the crude protease in diets containing CSM at various concentrations.

Materials and Methods

This present study was conducted at the National Feed Engineering Technology Research Center at the Ministry of Agriculture Feed Industry Center Animal Testing Base (Hebei, China). All experimental procedures involving sacrificing and sampling broilers were conducted in accordance with the Chinese Guidelines for Animal Welfare and approved by the China Agricultural University Institutional Animal Care and Use Committee (AW70600825).

Fermentation of MAFIC Y7

In this study, high-density fermentation of MAFIC Y7 was optimized to achieve high protease activity in fermentation broth. MAFIC Y7 was cultured in 20 L of liquid medium (7.5% soluble starch, 6.0% enzyme-treated soybean, 0.43% KH₂PO₄, 0.10% NaCl) for 60 h. During fermentation, pH of the medium was adjusted to 7 using 30% NaOH and 25% HCl. Dissolved oxygen concentration was maintained above 20% by adjusting rotation speed and airflow, and temperature was controlled at 37 °C. Fermentation was stopped when proteolytic activity in the medium reached 3,400 U/mL. Proteolytic activity of the medium was measured using a colorimetric method every 2 h (Caldas et al., 2002). The fermentation supernatant was collected by centrifugation of fermentation medium at 10,000 × g for 15 min. The fermentation supernatant was lyophilized for 34 h at −20 °C, and the product was named crude enzyme (CE). The CE solution (10 L) produced 400 g of lyophilized CE. CEs provided 85,000 U protease activity per gram of product as is.

Assay of enzyme activities of CE

Activities of protease, xylanase, amylase, phytase, and lipase in CE were determined. For assay of protease activity, 2% casein solution (1 mL) and the CE solution (1 mL) were mixed and incubated at 40 °C for 10 min, then mixed with trichloroacetic acid (2 mL of 0.4 M) to terminate the reaction. The mixture was centrifuged at 1,900 × g for 10 min. The supernatant (1 mL) was reacted with sodium carbonate (5 mL of 0.4 M) and Folin-phenol reagent (1 mL) for 20 min at 40 °C. The absorbance was measured at 680 nm. Protease activity was expressed as tyrosine equivalent in the supernatant. One unit of enzyme activity was defined as the amount of enzyme (μg/mg) that produces 1 μg of tyrosine per minute. A blank was assayed in the same manner as the experimental samples, except that enzyme solution was added after the addition of trichloroacetic acid. All samples were measured in triplicate.

Xylanase activity was assayed according to procedures described by Yang et al. (2017). The substrate was xylan (Sigma-Aldrich, St. Louis, MO, USA) dissolved in water at 0.8% (w/v). Liberated reducing sugar was measured using the 3,5-dinitrosalicylic acid method (Miller, 1959). Each assay was performed in triplicate. One unit of xylanase activity was defined as the amount of enzyme that released 1 μmol of xylose per minute under assay conditions. Amylase and cellulase were analyzed using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Phytase activity was determined by measuring the amount of inorganic phosphate liberated from sodium phytate (Haros et al., 2001). The reaction mixture consisted of sodium acetate/acetic acid buffer (0.1 M, pH 5.5, 400 μL), containing sodium phytate (1.2 mM) and CE solution (200 μL). After incubation at 50 °C for 30 min, the reaction was stopped by adding trichloroacetic acid solution (20%, 100 μL).

For the assay of lipase activity, pH-stat titration was used (Valivety et al., 1992). Briefly, sodium phosphate buffer (2 mL, 50 mM, pH 9.0), olive oil (0.5 mL), and enzyme solution (0.1 mL) were mixed and incubated at 37 °C for 10 min. The reaction was terminated by the addition of methylbenzene (4.0 mL). After the mixture was left for 5 min, the supernatant (4.0 mL) was mixed with copper acetate reagent (1.0 mL, 5%, pH 6.1). Liberated fatty acids were measured by absorbance at 710 nm. Oleic acid was used as the standard reference. One unit of lipase activity was defined as the amount of enzyme needed to produce 1 μmol fatty acid per min per mg of soluble protein in the enzyme solution at 37 °C (U/mg protein).

Optimal conditions for total protease activity of CE

The optimal temperature of protease activity in CE was assayed by incubating CE with substrate in buffers (pH 7) at 30, 40, 50, 60, and 70 °C, respectively. The optimal pH for proteolytic activity of CE was determined in buffers at pH of 4, 5, 6, 7, 8, 9, and 10. Casein was used as the substrate. Total protease activity was assayed using the method described as above. Maximal protease activity was defined as 100% of relative enzyme activity. To determine the thermal stability of total protease activity in CE, activity was measured after heating CE buffer to 30, 40, 50, and 60 °C for 30, 60, 90, and 120 min. The pH stability of proteolytic enzymes in CE was assayed by pre-incubating CE in buffers at different pH (3, 4, 5, 6, 7, 8, 9) at 40 °C for 30, 60, 90, and 120 min followed by assay of protease activity.

2.4. Coating of crude protease

CE powder (1 kg) was mixed with phosphatidylcholine (1 kg), and vitamin E (0.2 kg) in n-hexane solution. The mixture was heated to 50 °C for 3 h a monolayer of coated CE was formed by vacuum drying. Lactose (4 kg) and gum arabic (3.75 kg) were emulsified in water (25 L) at 50 °C to act as a carrier solution (9,600 g). The coated CE was emulsified with the carrier solution for 30 min and homogenized at 50 MPa three times which produced CE with two layers of coating. Finally, a mixture of cornstarch and maltodextrin (1:2, wt/wt) was spray-dried on the two-layer coated CE to produce three-layer coated crude enzyme (CCE).

Animals, experimental design, and diets

Experiment 1

Arbor Acres male broilers (n = 216, 1 d of age) with an initial body weight of 43 ± 0.4 g were allocated to six dietary treatments with six replicate pens/treatment and six broilers per pen. The experiment was designed as a 3 × 2 factorial arrangement of treatments. The factors were dietary protein levels (CP_low, CP_medium, and CP_high) and protease inclusion levels (0 or 4,000 U CCE/kg diet). Broilers were fed for 42 d with a two-phase feeding plan which included a starter phase (days 1 to 21) and a grower-finisher phase (days 22 to 42). Nutrient composition of feed was based on NRC (1994). The basal diets satisfy or exceed nutritional requirements of broilers as described by NRC (1994) (Table 1). From days 35 to 42, chromic oxide (0.25%) was used as an indigestible marker in diets to determine digestibility of dietary nutrients. Broilers were housed in an environmentally controlled room with free access to feed and water. Room temperature was maintained at 33 °C initially, and was reduced 3 °C/wk to 24 °C by 21 d of age then was held constant to the end of the experiment. Broilers were raised under continuous lighting.

Table 1.

Composition and nutrient levels of basal diets (%, as-fed basis, experiment 1)

Item	Protein in diets
	Days 1 to 21			Days 22 to 42
	CPlow	CPmedium	CPhigh	CPlow	CPmedium	CPhigh
Corn	70.64	66.14	62.01	74.68	69.57	69.40
Soybean meal	16.75	18.21	19.38	13.23	15.35	11.02
Corn gluten meal	6.32	10.55	13.58	3.96	9.11	12.32
Fish meal	1.24	0.00	0.00	2.41	0.00	2.72
Soybean oil	0.00	0.00	0.00	1.18	1.29	0.00
Dicalcium phosphate	1.67	1.73	1.67	1.48	1.66	1.37
Limestone	1.37	1.46	1.48	1.12	1.26	1.17
Salt	0.30	0.30	0.30	0.30	0.30	0.30
l-Lysine HCl, 98%	0.49	0.54	0.61	0.47	0.50	0.73
dl-Methionine, 98%	0.20	0.14	0.09	0.15	0.07	0.04
l-Threonine, 98%	0.28	0.19	0.12	0.27	0.16	0.16
Tryptophan, 98%	0.00	0.00	0.00	0.00	0.00	0.00
Chromic oxide	0.25	0.25	0.25	0.25	0.25	0.25
Vitamin–mineral premix1	0.50	0.50	0.50	0.50	0.50	0.50
Total	100.00	100.00	100.00	100.00	100.00	100.00
Calculated nutrient content
Metabolizable energy, kcal/kg	3,050	3,050	3,050	3,150	3,150	3,150
Crude protein	19.00	21.00	23.00	17.00	19.00	21.00
Calcium	1.00	1.00	1.00	0.90	0.90	0.90
Total phosphorus	0.68	0.68	0.68	0.65	0.65	0.65
Lysine	0.99	1.10	1.2	0.88	0.99	1.10
Methionine	0.50	0.50	0.50	0.40	0.40	0.40
Threonine	0.81	0.81	0.81	0.72	0.72	0.72
Tryptophan	0.20	0.21	0.23	0.18	0.19	0.19

¹The vitamin and mineral premix provided per kilogram of diets as following nutrients per kilogram of diet: Vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K₃, 2.2 mg; thiamin, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 102 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg.

Experiment 2

From days 1 to 21, broilers were fed a corn and SBM-based diet containing 3,050 kcal/kg diet and 21% CP according to NRC (1994). On day 22, Arbor Acres male broilers (n = 144) with initial body weight of 588 ± 3.1 g were divided into four dietary treatments with six replicate pens of six broilers per pen in each experimental diet. The experiment was designed as a 2 × 2 factorial arrangement of diets. The primary factors were lysine-to-protein ratio (Lys:protein; 5.2% and 5.5%) and protease levels (0 and 4,000 U CCE/kg diet) in the diet. CSM replaced 50% of SBM in all experimental diets. Diets were formulated to meet or exceed nutrient requirements of broilers outlined by the NRC (1994) (Table 2). Dietary treatments lasted from days 22 to 42. Broilers were housed in an environmentally controlled room with free access to feed and water. Room temperature was maintained at 33 °C initially and reduced 3 °C/wk to 24 °C by 21 d of age. Room temperature was held at 24 °C throughout the experimental period. Broilers were raised under continuous lighting.

Table 2.

Composition and nutrient levels of basal diets (%, as-fed basis, experiment 2)

Item	Days 1 to 21	Days 22 to 42
Lysine-to-protein ratio (%)		5.2	5.5
Ingredients
Corn	66.13	64.77	64.88
Soybean meal	18.21	11.50	11.50
Cottonseed meal	0.00	11.50	11.50
Corn gluten meal	10.55	3.73	3.49
Soybean oil	0.00	3.85	3.84
Dicalcium phosphate	1.73	1.37	1.38
Limestone	1.46	1.38	1.37
Salt	0.30	0.30	0.30
l-Lysine HCl, 98%	0.54	0.51	0.65
dl-Methionine, 98%	0.14	0.12	0.12
l-Threonine, 98%	0.19	0.22	0.22
Chromium oxide	0.25	0.25	0.25
Vitamin–mineral premix1	0.50	0.50	0.50
Total	100.00	100.00	100.00
Calculated nutrient content
Metabolizable energy, kcal/kg	3,050	3,150	3,150
Crude protein	21.00	19.00	19.00
Calcium	1.00	0.9	0.90
Total phosphorus	0.68	0.65	0.65
Lysine	1.10	0.99	1.05
Methionine	0.50	0.40	0.40
Threonine	0.81	0.72	0.72
Tryptophan	0.21	0.20	0.20

¹The vitamin and mineral premix provided per kilogram of diets as following nutrients per kilogram of diet: Vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K₃, 2.2 mg; thiamin, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 102 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg.

Sample collection and processing

Experiment 1

Feed consumption was recorded on a pen basis and broilers were weighed individually on days 1, 21, and 42. Average daily gain (ADG), average daily feed intake (ADFI), and gain:feed (G:F) were calculated during days 1 to 21, and days 22 to 42.

On days 21 and 42, one broiler closest to the average body weight of the pen was selected for blood collection. Blood was collected from the precaval vein and centrifuged at 1,300 × g for 10 min at 4 °C. Serum was harvested and stored at −20 °C for later analysis. After blood collection, the selected broilers were euthanized by cervical dislocation. Chyme from the proximal ileum was collected and frozen immediately at −80 °C for storage until analysis.

Experiment 2

On days 22 and 42, body weight and feed were weighted on a pen basis to calculate ADG, ADFI, and G:F from 22 to 42 d, respectively. On day 42, one broiler with BW closest to the pen average BW was selected and killed by cervical dislocation. Chyme in the proximal ileum was collected immediately, stored at −80 °C, and used to determine AID of AA as described below. Following euthanasia, broilers were defeathered, decapitated, declawed, and eviscerated to obtain carcasses, breast meat, leg quarters (drum), and abdominal fat pads. Tissues of duodenum (2 cm sections from the pylorus to the ligament of Treitz) were separated, cleaned with saline solution (0.9%), and fixed in neutral-buffered formalin (10%) for morphological analysis.

AID of amino acid

AA composition of ileal chyme and experimental diets were determined using high-performance liquid chromatography (Hitachi L-8800 Amino Acid Analyzer, Tokyo, Japan). Ileal chyme was hydrolyzed with 6 N HCl at 110 °C for 24 h. Cysteine (Cys) and methionine (Met) were analyzed as cysteic acid and methionine sulfone using the performic acid oxidation method (AOAC, 2016) after being hydrolyzed with 7.5 N HCl at 110 °C for 24 h (Sedgwick et al., 1991; Cowieson and Ravindran, 2008; AOAC, 2016). Chromium concentrations were determined using an atomic absorption spectrophotometer (Hitachi Z-5000, Tokyo, Japan) according to procedures reported by Dansky and Hill (1952). Apparent ileal digestibility (AID) of AA was calculated as

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{I}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{A}(\mathrm{\%})=[1-\frac{\mathrm{A}\mathrm{A}\mathrm{i}}{\mathrm{A}\mathrm{A}\mathrm{d}}\times \frac{\mathrm{C}\mathrm{R}\mathrm{d}}{\mathrm{C}\mathrm{R}\mathrm{i}}]\times 100}\end{array}}$$

where AAi is the AA content in ileal digesta, AAd is the AA content in the diet, CRd is the chromium content in the diet, and CRi is the chromium oxide content in ileal digesta.

Ileal digesta samples were dried at 60 °C for 72 h and the digestibility of CP was calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{I}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{C}\mathrm{P}(\mathrm{\%})=[1-\frac{\mathrm{N}\mathrm{i}}{\mathrm{N}\mathrm{d}}\times \frac{\mathrm{C}\mathrm{R}\mathrm{d}}{\mathrm{C}\mathrm{R}\mathrm{i}}]\times 6.25\times 100}\end{array}}$$

where Ni is the nitrogen content in ileal digesta, Nd is the nitrogen content in the diet, CRd is the chromium content in the diet, and CRi is the chromium content in ileal digesta.

Immune and biochemical measurements

Immunoglobulins (IgA, IgM, and IgG) in serum were measured using an automatic biochemical analyzer (Hitachi 7600, Hitachi High-Technologies Corporation, Tokyo, Japan) following the instructions of commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), and tumor necrosis factor-α (TNF-α) in serum were determined using ELISA kits (Beijing Sino-UK Institute of Biological Technology, Beijing, China).

Intestinal morphology

Intestinal morphology was characterized according to a previously described method (Wang et al., 2019). Formalin-fixed intestinal tissues were dehydrated in ethanol and xylene and embedded in paraffin. Tissues were sectioned (6 μm thick) and stained with hematoxylin-eosin for examination under a light microscope at 40 × power (Nikon, Tokyo, Japan). Villus height (VH) was defined as the distance from the villus tip to the villus-crypt junction. Crypt depth (CD) was defined as the distance from the villus–crypt junction to the base of the crypt (Youssef et al., 2021). The ratio of VH to CD was calculated.

Data analysis

Data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). Significant differences among treatment means were declared at P < 0.05 using Duncan’s multiple tests.

Results

Protease activity in CE

CEs contained predominantly protease activity (85,000 ± 2,250 U/g) compared with other enzyme activities related to nutrient digestion. CEs contained minor activities of xylanase (1.25 ± 0.11 U/g), cellulase (16 ± 11 U/g), amylase (51 ± 24 U/g), phytase (32 ± 14 U/g), and lipase (101 ± 35 U/g). The coated form of CE contained 12,200 ± 310 U/g of protease activity.

Optimal temperature and pH of CCE

Proteases had higher enzymatic activity at 30 to 60 °C compared with 70 °C. The optimal temperature of total protease activity was 50 °C (Figure 1A). The optimal pH for total protease activity was 7.0 at 50 °C (Figure 1B). Protease activity was at least 60% at pH ranging from 6.0 to 9.0. Less than 10% of protease activity was retained at pH 4.0, which indicated that total protease activity of CE was not stable at pH lower than 6.0 (Figure 1B). Total protease activity of CE showed stable activity at 30 °C after 60 min. At 40 °C, more than 60% of protease activity was retained for up to 90 min (Figure 1C). At 50 °C, about 48% of protease activity was retained for up to 60 min. At 60 °C, about 45% of protease activity was maintained for 30 min. Seventy percent or more of total protease activity was maintained at pH from 6.0 to 9.0 for 90 min at 40 °C (Figure 1D).

Figure 1.

Characterization of enzymatic properties. (A) Effects of temperature on enzyme activity; (B) effects of pH on enzyme activity; (C) temperature effects vs. time; (D) pH effects vs. time; (E) intact protease vs. coated-protease activities at 40 °C and pH 3.

Thermal and pH stability of CCE were assayed compared to that of CE. Protease activity of CCE was higher and more stable over 150 min at 40 °C and pH = 3 than CE (Figure 1E). This result confirmed that coating greatly improved the thermal and pH stability of CE.

Experiment 1

Growth performance

The effects of diet regime and protease supplementation on ADFI, ADG, and G:F of broiler are shown in Table 3. G:F was higher (P < 0.05) in CP_low or CP_medium with CCE supplementation compared with the unsupplemented treatment during days 1 to 21. Broilers fed CP_low had lower ADG than the broilers fed of CP_medium and CP_high during days 22 to 42. Protease supplementation significantly increased (P < 0.05) ADG and G:F of broilers fed CP_medium and G:F of broilers fed CP_low compared to unsupplemented broilers during days 1 to 42. Protein content had a significant effect on the G:F from days 1 to 21, ADG during days 22 to 42, and ADG and G:F from days 1 to 42 (P < 0.05). Proteases had an effect on G:F from days 1 to 21 and ADG and G:F during days 1 to 42 (P < 0.05). No significant interaction between diet protein content and supplementation of protease was observed for growth performance during days 1 to 42 (P > 0.05).

Table 3.

Effects of dietary coated protease on growth performance of broilers fed different crude protein levels (experiment 1)

Item	CPlow		CPmedium		CPhigh		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
1 to 21 d
ADFI, g	42.99	41.00	43.59	41.91	41.35	40.90	0.43	0.32	0.12	0.75
ADG, g	26.53	27.55	27.22	28.46	28.09	28.23	0.23	0.12	0.08	0.57
G:F	0.62b	0.67a	0.63b	0.68a	0.68a	0.69a	0.05	0.04	< 0.01	0.37
22 to 42 d
ADFI, g	105.92	103.40	105.34	108.07	106.93	108.58	1.34	0.65	0.77	0.68
ADG, g	56.91b	58.93ab	58.55ab	62.86a	62.06a	62.37a	0.67	0.02	0.07	0.40
G:F	0.54	0.57	0.56	0.58	0.58	0.57	0.03	0.31	0.12	0.31
1 to 42 d
ADFI, g	74.45	72.20	74.46	75.30	74.14	74.74	0.64	0.63	0.84	0.59
ADG, g	41.72c	43.34abc	42.88bc	45.41a	45.07ab	45.30ab	0.38	< 0.01	0.03	0.28
G:F	0.56c	0.60ab	0.58bc	0.61a	0.61a	0.61a	0.01	0.04	0.01	0.16

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

CP, crude protein; CCE, coated crude enzyme; CP_low, level of dietary crude protein was 19% during days 1 to 21 and was 17% during days 22 to 42; CP_medium, level of dietary crude protein was 21% during days 1 to 21 and was 19% during days 22 to 42; CP_high, level of dietary crude protein was 23% on days 1 to 21 and was 21% during days 22 to 42; CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet.

AID of crude protein and AA

The effects of dietary CP level and CCE supplementation on the AID of CP and AA are shown in Table 4. As dietary CP level increased, AID of CP improved. Compared with the unsupplemented treatment, CCE in CP_low increased (P < 0.05) AID in broilers. CCE had an increased (P < 0.05) AID of lysine in CP_low and CP_medium diets compared with unsupplemented diets. With CCE supplementation, AID of cysteine and isoleucine in broilers fed CP_medium were higher (P < 0.05) than unsupplemented diets independent of protein levels in the diets. The AID of histidine in broilers fed CP_high was higher than that of CP_low regardless of CCE supplementation (P < 0.05). There was no significant interaction between dietary regimens and protease supplementation for AID of CP and AA on day 42.

Table 4.

Effect of crude protein and coated crude enzyme level on and apparent ileal digestibility (AID) of amino acids and crude protein of broilers at the age of 42 d (experiment 1)

Item	CPlow		CPmedium		CPhigh		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
Crude protein, %	67.29c	73.22b	75.54ab	78.11a	78.64a	78.98a	5.01	< 0.01	0.02	0.14
Essential amino acid, %
Arg	76.23	79.99	80.02	81.23	76.96	77.24	4.43	0.14	0.23	0.60
Lys	73.61c	77.05ab	75.65bc	79.79a	77.98ab	78.43ab	3.23	0.03	0.01	0.22
Leu	75.94	76.73	77.28	78.20	78.88	78.19	2.62	0.21	0.74	0.77
Cys	70.32c	72.85bc	73.34bc	78.32a	74.17bc	75.25ab	4.05	0.01	0.03	0.56
Ile	73.41b	74.46b	74.80b	79.23a	76.24ab	76.02ab	3.19	0.03	0.07	0.12
His	72.56b	73.18b	75.01ab	78.91a	77.81a	78.48a	3.91	< 0.01	0.11	0.36
Met	77.00	78.99	80.34	83.86	78.47	78.93	5.09	0.11	0.24	0.75
Phe	70.87	72.69	71.20	73.68	71.69	71.99	4.33	0.91	0.34	0.81
Thr	74.95	77.47	75.39	79.35	76.06	75.79	4.00	0.65	0.13	0.43
Val	74.35	76.63	75.10	78.00	74.58	76.39	3.96	0.76	0.06	0.95
Trp	72.58	76.71	72.81	73.89	73.97	74.96	3.38	0.59	0.07	0.43
Tyr	72.84	74.23	75.28	80.06	75.02	75.81	4.69	0.09	0.13	0.50
Nonessential amino acid, %
Ala	73.25	77.31	73.79	76.53	76.02	76.79	3.98	0.70	0.06	0.59
Asp	76.11	77.06	77.14	77.37	76.95	75.47	4.90	0.88	0.95	0.83
Glu	75.43	77.09	75.93	78.98	76.51	76.60	2.89	0.57	0.10	0.46
Gly	69.76	72.57	70.46	73.41	69.02	69.56	7.26	0.69	0.42	0.91
Pro	70.49	74.30	72.74	75.41	71.57	73.56	4.56	0.63	0.08	0.89
Ser	71.65	73.44	72.39	74.72	71.59	69.30	6.07	0.47	0.77	0.62

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

CP, crude protein; CCE, coated crude enzyme; CP_low, level of dietary crude protein was 19% during day 1 to 21 and was 17% during days 22 to 42; CP_medium, level of dietary crude protein was 21% during days 1 to 21 and was 19% during days 22 to 42; CP_high, level of dietary crude protein was 23% on days 1 to 21 and was 21% during days 22 to 42; CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet.

Serum biochemical indices

On day 21, CCE increased serum IgA in CP_medium (P < 0.05) (Table 5). On day 42, at all dietary treatments (CP_low, CP_medium, and CP_high) CCE increased serum IgA and IgG (P = 0.01). No significant interactions were found between dietary CP regimens and protease supplementation.

Table 5.

Effect of crude protein and coated crude enzyme on serum immunoglobulin of broiler (experiment 1)

Item	CPlow		CPmedium		CPhigh		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
Day 21
IgA, µg/mL	13.23b	13.26b	13.23b	14.45a	13.83ab	14.48a	0.14	0.02	0.05	0.37
IgM, µg/mL	4.42	5.11	3.26	4.24	3.65	4.89	0.31	0.42	0.13	0.94
IgG, µg/mL	8.41	8.08	7.40	8.40	6.77	7.72	0.24	0.22	0.25	0.43
Day 42
IgA, µg/mL	15.19b	16.30ab	16.17b	17.62a	16.14b	16.65ab	0.22	0.07	0.01	0.61
IgM, µg/mL	3.69	4.23	4.39	4.72	4.11	4.91	0.18	0.16	0.07	0.79
IgG, µg/mL	6.39c	7.53abc	6.77bc	8.55a	7.64abc	8.26ab	0.22	0.13	0.01	0.50

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

CP, crude protein; CCE, coated crude enzyme; CP_low, level of dietary crude protein was 19% during days 1 to 21 and was 17% during days 22 to 42; CP_medium, level of dietary crude protein was 21% during days 1 to 21 and was 19% during days 22 to 42; CP_high, level of dietary crude protein was 23% on days 1 to 21 and was 21% during days 22 to 42; CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.

CCE decreased serum IL-6 and TNF-α in broilers fed CP_high compared with unsupplemented diets (P < 0.05) on day 21 (Table 6). On day 42, CCE decreased serum IL-8 and TNF-α, increased serum IL-10 in the broilers of CP_medium, and decreased serum TNF-α in CP_high broilers compared to broilers fed unsupplemented diets (P < 0.05). No significant interaction was found between dietary CP regimens and protease supplementation for serum IL-6, IL-8, IL-10, and TNF-α contents on days 21 and 42.

Table 6.

Effect of crude protein and coated crude enzyme on serum immune indices of broiler (experiment 1)

Item	CPlow		CPmedium		CPhigh		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
Day 21
IL-6, pg/mL	30.58b	29.79b	30.82ab	28.69b	32.78a	30.18b	0.33	0.04	< 0.01	0.40
IL-8, pg/mL	15.79	14.31	15.60	14.74	16.57	15.14	0.89	0.46	0.03	0.88
IL-10, pg/mL	8.38b	9.98a	10.69a	10.79a	10.27a	10.55a	0.24	0.02	0.14	0.32
TNF-α, ng/mL	65.20bc	63.94c	64.24ab	68.96c	71.03a	64.46c	0.68	0.08	< 0.01	0.16
Day 42
IL-6, pg/mL	29.93	29.94	30.67	30.02	29.84	30.70	0.16	0.52	0.82	0.16
IL-8, pg/mL	14.93abc	14.37bc	16.65ab	14.05c	17.10a	14.49bc	0.35	0.34	< 0.01	0.32
IL-10, pg/mL	9.59ab	10.14a	8.89c	10.38a	9.31ab	10.26a	0.17	0.83	< 0.01	0.47
TNF-α, ng/mL	64.32b	63.49b	69.22a	61.75b	70.24a	64.46b	0.74	0.06	< 0.01	0.06

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

CP, crude protein; CCE, coated crude enzyme; CP_low, level of dietary crude protein was 19% during days 1 to 21 and was 17% during days 22 to 42; CP_medium, level of dietary crude protein was 21% during days 1 to 21 and was 19% during days 22 to 42; CP_high, level of dietary crude protein was 23% on days 1 to 21 and was 21% during days 22 to 42; CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet; IL-6, interleukin 6; IL-8, interleukin 8; IL-10, interleukin 10; TNF-α, tumor necrosis factor-α; CD, crypt depth; VH, villus height; VH/CD, villus height to crypt depth.

Experiment 2

Growth performance

At different dietary lysine levels (crude protein) diets, CCE supplementation increased ADG and G:F at 5.5% Lys:protein compared with broilers receiving CCE supplementation (Table 7). In the 5.5% Lys diet, CCE supplementation improved ADG by 2.57%, and improved G:F by 8.47% compared with unsupplemented diets (P < 0.05). CCE in 5.5% Lys:protein diet had higher growth performance than 5.2% Lys:protein level diet.

Table 7.

Effect of protease in containing diets soybean meal and cottonseed meal on growth performance of broilers at day 42 (experiment 2)

Item	5.2% Lys:protein		5.5% Lys:protein		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
ADG, g	70.84ab	71.85ab	72.34b	74.20a	0.53	0.98	0.01	0.17
ADFI, g	120.43	118.57	122.31	116.63	0.77	0.07	0.16	0.67
G:F	0.59b	0.61b	0.59b	0.64a	0.01	0.12	< 0.01	0.16

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet.

Carcass parameters

There was no significant interaction between diet regimens and protease supplementation for carcass parameters. In the 5.5% Lys:protein dietary treatment, CCE supplementation increased the carcass proportion of live body weight (P < 0.05) (Table 8). CCE decreased abdominal fat of broilers fed 5.5% Lys:protein (P < 0.05), but this effect of diet was not significant in 5.2% Lys:protein treatment. There was no significant difference between the proportions of breast meat and drums in 5.2% Lys:protein and 5.5% Lys:protein treatments.

Table 8.

Effect of soybean meal and cottonseed meal on carcass traits (% of live body weight) at day 42 (experiment 2)

Item	5.2% Lys:protein		5.5% Lys:protein		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
Carcass	65.65b	66.96b	67.18b	69.10a	0.35	< 0.01	0.01	0.56
Breast	21.79	22.64	23.61	23.48	0.78	0.43	0.83	0.77
Drums	7.25	7.47	7.65	7.61	0.27	0.64	0.87	0.83
Abdominal fat	2.72a	2.53ab	2.67a	2.39b	0.05	0.24	0.01	0.58

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version 27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet.

Intestinal morphology of duodenum

Both diet regimens and protease supplementation did not influence crypt or villus height to CD ratio in the duodenum. Supplementation of CCE in the different diets (5.2% or 5.5% Lys:protein diet) improved VH (Table 9). VH was greater for broilers fed 5.5% Lys:protein compared to that of 5.2% without CCE supplementation (P < 0.01). Supplementation of CCE altered VH between supplemented and unsupplemented birds. Broilers fed 5.2% Lys diets supplemented with CCE significantly increased VH of the duodenum compared to those without CCE. There was no interaction between diet type and protease supplementation for duodenal morphology (P > 0.05).

Table 9.

Effect of soybean meal and cottonseed meal on intestinal morphology of the duodenum at day 42 (experiment 2)

Item	5.2% Lys:protein		5.5% Lys:protein		SEM	P value
	CCE0	CCE4,000	CCE0	CCE4,000		Diet	Protease	Diet*Protease
VH, μm	1,329.13c	1,407.74b	1,425.69ab	1,460.49a	12.04	< 0.01	< 0.01	0.07
CD, μm	122.21	113.56	123.46	117.45	2.67	0.64	0.19	0.81
VH/CD	11.04	12.53	11.61	12.53	0.27	0.56	0.03	0.59

^a,bMeans within rows with different superscripts differ (P < 0.05). The data were analyzed by two-way analysis of variance (ANOVA) to determine the main effects (diet and protease) and their interaction using the General Linear Models procedure of SPSS (version27.0). The significant differences among the treatment mean were analyzed with a P < 0.05 statistical level using Duncan’s multiple tests.

VH, villus height; CD, crypt depth; VH/CD, villus height to crypt depth; CCE₀, 0 U CCE per kg of diet; CCE_4,000, 4,000 U CCE per kg of diet.

Discussion

Strains of B. subtilis are used widely for protease production. These bacteria have a short fermentation period and secrete extracellular proteins into their culture medium (Ward et al., 2009; dos Santos Aguilar and Sato, 2018). Proteases secreted by B. subtilis have wide applications in feed industries and are recognized for their nutritional, environmental, and economic benefits (Razzaq et al., 2019). Proteases can improve growth performance of broilers (Cho et al., 2020; Hu et al., 2020). Among proteases, serine proteases are most commonly used in commercial applications because they are stable at high pH and high temperatures in the presence of surfactants and oxidizing agents (Tomar et al., 2008; Bhunia et al., 2012).

Gastric acid and pepsin lead to degradation of protease in the stomach, which reduces available exogenous protease in the small intestine (Gheorghe et al., 2015). Coating is a technology applied to protect oral medications and feed additives from environmental damage (Shen et al., 2014). Enzymes can be coated using various substrate materials and techniques (Zdarta et al., 2018). For instance, physical entrapment, adsorption through noncovalent interactions, and covalent binding to the substrate are common approaches used to coat enzymes (Betancor and Luckarift, 2008; Zhang et al., 2017). Exogenous protease can regulate release of endogenous protease and play a synergistic role with endogenous protease (Yuan et al., 2015). In the current study, we evaluated pH and temperature stability of coated protease in simulated gastrointestinal media in vitro compared with uncoated protease. We demonstrated that coating protease significantly improved pH and temperature stability. We speculated that coating technology reduced flexibility of the polypeptide backbone, thereby increasing stability of the enzyme (Souza et al., 2019).

In experiment 1, three levels of crude protein (CP_low, CP_medium, and CP_high) were selected, 19%, 21%, and 23% (days 1 to 21). The protein level of 21% was based on NRC (1994) recommendation, and lower (19%) or higher (23%) levels of protein were designed for comparison. Accordingly, during days 22 to 42, the corresponding levels of protein were 17%, 19%, and 21%. In experiment 1, no significant interaction between diet regimens and protease supplementation was found for broilers’ growth performance, AID of crude protein and AA, and immune and biochemical parameters, but both diet regimens and CCE independently affected those responses. Average daily weight gain decreased with decreasing dietary protein concentration irrespective of the effect of proteases. This occurred because a lack of AAs inhibits the growth of broilers at low dietary protein levels (Lourenco et al., 2020). We found that CCE supplementation improved G:F in CP_low and CP_medium compared to unsupplemented treatments on days 1 to 21 (P < 0.05). Daily weight gain of birds fed CP_medium with CCE was improved significantly compared to birds fed unsupplemented diets in days 1 to 42. Growth performance data indicated that the improvement of G:F was achieved by increasing average daily weight gain. Consistent with our research, supplementing protease to low-protein feeds improved growth performance of broilers by improving gain efficiency from days 1 to 21 and days 1 to 42 (Wang et al., 2022). In accordance with our study results, protease supplementation increased body weight and improved feed conversion (Duque-Ramírez et al., 2023). In addition, we also found that the AID of crude protein digestibility and some AAs (lysine, cysteine, and isoleucine) were also improved significantly. Reasonably, exogenous protease increased endogenous peptidase production which released more AAs and short peptides from dietary proteins that were absorbed through the intestinal tract (Wang et al., 2022). In other words, protease increased growth and protein utilization by increasing AA availability. Inconsistent with our findings, protease in the diet had a positive effect on AID of phenylamine and tyrosine (Salazar-Villanea et al., 2022). This may be due to the different affinities of different proteases for the substrates. Our data reported herein demonstrated that protease supplementation to diets with high-protein concentration (CP_high) did not significantly improve growth performance of broilers over the entire experimental period. We surmise the high-protein diet provided sufficient available AAs to satisfy growth requirements of broilers, but in low-protein diets exogenous protease made more AAs available for growth which resulted in a positive protease effect. Animal’s immune status is related to serum immunoglobulins, which can enhance specific immune mechanisms in broiler to protect them against infections (Long et al., 2020a,b). The increase in IgA, and IgM represents an increase in the animal’s immune capacity (Silva et al., 2023). Inflammatory cytokines such as IL-6, IL-8, and TNF-α are generally believed to be rapidly released during infection. Reduced expression of pro-inflammatory factors in the body helps improve the anti-inflammatory response and regulate the immune function of the body (Huang et al., 2022). As an anti-inflammatory factor, IL-10 expression in the body is beneficial. The IL-10 could enhance or activate the body’s immune function (Moore et al., 2001). In the present study, protease supplementation significantly reduced serum IL-6, IL-8, and TNF-α levels at 23% dietary protein in SBM-based diets compared with the unsupplemented treatment. Higher levels of soybean meal contain a higher content of allergenic proteins, such as glycinin and conglycinin (Wang et al., 2014). In vitro, protease produced by MAFIC Y7 degraded trypsin inhibitor, β-conglycinin, lectin, and glycinin (data not shown). The reduction of serum inflammatory cytokines by protease supplementation may be due to protease hydrolysis of allergenic proteins (Park et al., 2020b), or the combination of proteases with ANFs (Park et al., 2020a). Proteases hydrolyze sensitized proteins or bind to ANFs which indirectly shows their anti-inflammatory properties. The increase in immunoglobulins and the decrease in pro-inflammatory factors are both favorable factors for improved growth performance.

In experiment 2, protease supplementation in diets containing both SBM and CSM improved growth performance of broilers and had a more pronounced effect in diets with 5.5% Lys:protein. Protease increased G:F by increasing ADG in the 5.5% Lys:protein diet. In the current study, we also found that increasing the proportion of lysine or adding proteases could increase the proportion of carcass, which was a consistent improvement in growth performance. Broilers had elevated abdominal fat when fed diets with low lysine levels (Qiu et al., 2023). In agreement with these findings, elevating dietary lysine levels can also improve protein retention, gain efficiency, and minimize fat retention in broiler carcasses (Maqsood et al., 2022). Therefore, lysine deficiency decreases protein utilization, which increases body fat deposition. The protease supplementation significantly improved duodenal VH, which presumably increased digestion and absorption of nutrients (Cowieson and Roos, 2016). The increase in VH may be attributed to two factors. First, proteases promote the release of nutrients such as AAs and small peptides, which may have enhanced villus development. Second, proteases may have reduced ANFs in SBM and CSM. Protease can hydrolyze protein to free AAs and peptides which would be available for absorption in the small intestine (Liu et al., 2013; Adebiyi and Olukosi, 2015). Moreover, improved VH and increased VH/CD ratio in the duodenum may have led to increased absorption of digested nutrients, which contributed to increased weight gain of broilers compared with unsupplemented broilers. Previous researchers have reported that improved intestinal morphology was associated with higher nutrient digestibility (Montagne et al., 2003; Tran, 2017; Wang et al., 2018).

Conclusions

In summary, the optimal catalytic activity of protease harvested from B. subtilis (MAFIC Y7) was achieved at 50 °C and pH 7.0. Compared to uncoated protease, coated protease showed greater stability at a higher temperature and pH. The protective effects of coated protease supplemented with broiler diets were evidenced by enhancement in nutrient digestibility, immune status, intestinal morphology, and carcass traits. Proteases produced by MAFIC Y7 improved growth performance and decreased inflammatory responses of broilers fed SBM-based diets (CP_low, CP_medium, and CP_high) and improved the efficiency of CSM use in CSM-based diets when the ratio of lysine-to-protein was 5.5%.

Glossary

Abbreviations

AA

amino acid

ADFI

average daily feed intake

ADG

average daily gain

AID

apparent ileal digestibility

ANFs

anti-nutritional factors

CCE

coated crude protease

CCE₀

0 U CCE per kg of diet

CCE_4,000

4,000 U CCE per kg of diet

CD

crypt depth

CE

crude enzyme

CP

crude protein

CP_high

level of dietary crude protein was 23% on days 1 to 21 and was 21% during days 22 to 42

CP_medium

level of dietary crude protein was 21% during days 1 to 21 and was 19% during days 22 to 42

CP_low

level of dietary crude protein was 19% during days 1 to 21 and was 17% during days 22 to 42

CSM

cottonseed meal

G:F

gain:feed

IgA

immunoglobulin A

IgG

immunoglobulin G

IgM

immunoglobulin M

IL-6

interleukin 6

IL-8

interleukin 8

IL-10

interleukin 10

SBM

soybean meal

TNF-α

tumor necrosis factor-α

VH

villus height

VH/CD

villus height to crypt depth ratio

Acknowledgments

This study was supported by The National Key Research and Development Program of China (2021YFC2103001) and The 2020 Research Program of Sanya Yazhou Bay Science and Technology City (SYND-2021-01).

Conflict of interest statement. The authors declare that they have no competing interests.

Author Contributions

Xiangyue Guo (Methodology, Validation, Investigation, Formal analysis, Writing—original draft), Qianxi Li (Grammar, Validation, Investigation), Lixue Wang (Validation, Investigation, Formal analysis), Ying Zhang (literature collection), Lee J. Johnston and Crystal L. Levesque (critically edited the presentation of ideas and the text), Yunhe Cao (Methodology, Validation, Investigation), and Bing Dong (Supervision, Writing—review & editing, Project administration, Funding acquisition)