animals

Article
Effects of Different Proteases on Protein Digestion In Vitro and
In Vivo and Growth Performance of Broilers Fed Corn–Soybean
Meal Diets
Mengli Zheng 1,2,† , Yan Bai 1,† , Yingxia Sun 1 , Jing An 1,3 , Qinghua Chen 2 and Tieying Zhang 1, *

1 State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural
Sciences, Beijing 100193, China
2 College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China;
[email protected]
3 College of Animal Science, Shanxi Agricultural University, Jinzhong 030801, China
* Correspondence: [email protected]; Tel.: +86-13911288868
† These authors contributed equally to this work.

Simple Summary: With the increase of protease types and products, it is time-consuming and
laborious to evaluate the effect of protease on feed protein utilization with animal experiments, and it
is not conducive to evaluate a large number of samples in a short time. The purpose of this study
was to quickly evaluate the effects of four proteases (acidic, neutral, alkaline and keratinase) on feed
ingredients (corn gluten meal, corn and soybean meal) using an in vitro method to determine the
optimal dosage of each protease for corn gluten meal and corn and soybean meal, and to explore the
factors affecting the effect of proteases. In addition, this research also carried out animal experiments
to verify the effect of protease on the corn–soybean meal diet of 31-day-old broilers.

Abstract: This study was conducted to investigate the effects of different proteases alone or in
combination on protein digestibility of broilers. In vitro, the properties of four proteases in broilers,
including acidic protease (AcP), alkaline protease (AlP), neutral protease (NeP) and keratinase (Ker),
Citation: Zheng, M.; Bai, Y.; Sun, Y.; on endogenous protease activity and their effects on protein digestibility of common ingredients in
An, J.; Chen, Q.; Zhang, T. Effects of broiler diets were investigated using a gut-mimicking model. In vivo, 640 1-day-old male broilers
Different Proteases on Protein were randomly divided into 8 groups of 10 with 8 replicates of 10 birds per replicate cage. Eight dietary
Digestion In Vitro and In Vivo and treatments included a corn–soybean meal basal diet (control), and the basal diet with 1.6 U AcP/g,
Growth Performance of Broilers Fed
0.8 U NeP/g, 0.8 U AlP/g, 0.4 U Ker/g, 1.6 U AcP/g + 0.8 U NeP/g, 1.6 U AcP/g + 0.8 U AlP/g,
Corn–Soybean Meal Diets. Animals
or 1.6 U AcP/g + 0.4 U Ker/g added. The experiment lasted for 31 days. The results showed that
2023, 13, 1746. https://doi.org/
the optimum pH values of AcP, NeP, AlP and Ker were 3.0, 9.0, 11.0 and 11.0 in vitro, respectively.
10.3390/ani13111746
Ker recovery proportion was 37.68% at pH 3.3–6.2. AcP alone or in combination with NeP, AlP or
Academic Editors: Sylwia Ker increased in vitro crude protein digestibility (IVCPD) and decreased ileal apparent digestibility
Szymańczyk, Marek Bienko and
of crude protein in 31-day-old broilers (p < 0.05). All protease supplementation reduced the ileal
Radosław P. Radzki
apparent digestibility of amino acids compared to the control (p < 0.05). Acidic protease had a positive
Received: 16 March 2023 effect on trypsin and chymotrypsin activities, while AlP and Ker showed a negative effect. In vivo,
Revised: 16 April 2023 average daily gain and average daily feed intake were significantly (p < 0.05) increased in broiler
Accepted: 21 April 2023 diets supplemented with AcP compared to the control group. When adding exogenous proteases
Published: 25 May 2023 to broiler diets, their sensitivity to digestive pH and their negative effects on endogenous protease
activity, dosage and combination effects should be taken into account. In addition, the properties and
dosage of proteases and the protein level in the feed should be considered.

Copyright: © 2023 by the authors.

Keywords: protease; broiler; in vitro; in vivo; protein digestibility; pH
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
1. Introduction
creativecommons.org/licenses/by/ The digestion and utilization of dietary nutrients by broilers can be fully considered in
4.0/). combination with their physiological structure, gastrointestinal digestion and absorption

Animals 2023, 13, 1746. https://doi.org/10.3390/ani13111746 https://www.mdpi.com/journal/animals

Animals 2023, 13, 1746 2 of 15

characteristics and dietary structure characteristics [1–3]. After digestion of feed nutrients
in the crop and stomach of broilers, the chyme is transferred into the small intestine to
continue digestion. In this process, the endogenous protease secreted by the body plays an
important role in the degradation of protein macromolecules.
Studies on the digestibility of crude protein (CP) and amino acid (AA) in broilers
showed that valuable proteins passed through the gastro-intestinal tract (GIT) without
complete digestion [4,5]. Soybean meal (SBM), as one of the most commonly used protein
sources for broilers globally, has the advantages of a higher digestibility and good amino
acid balance [6]. However, soybean protein is rich in a variety of anti-nutritional factors,
such as soybean protease inhibitors, particularly the Kunitz trypsin inhibitor (KTI) of
soybean, which is a β-sheet protein with abnormal thermal denaturation stability and
the ability to be easily reduced to natural forms after cooling [7]. Some undigested SBM
proteins were identified as protease inhibitors in chicken digestive fluids fed SBM diets [8].
Two storage proteins, glycine and β-glycine, were considered to be the main soybean
allergens [9,10]. Compared with other plant proteins, they revealed greater resistance
to digestive enzymes [8]. Antinutritional factors, such as trypsin inhibitor, glycine and
β-conglycinin in SBM, might be the most important factors leading to the failure of broilers
to completely digest crude protein and amino acids [11]. About a quarter of the protein in
broiler diets was provided by corn, which was mainly composed of gliadin, accounting
for more than 60% of all protein in whole grains [12]. The amino acid composition was
rich in glutamine and hydrophobic amino acids, which helped the gliadin part of the
protein insoluble in water. The relative abundance of zein was highly resistant to protein
hydrolysis [13] because the cysteine in grain γ-gliadin was highly conserved [14].
The changes in digestive enzyme levels in GIT affected the digestibility of nutrients to
a certain extent. The development of digestive enzyme secretion after hatching may be a
limiting factor for digestion and subsequent food intake and growth [15]. Some proteins in
SBM and corn had a certain degree of resistance to endogenous proteases, which provided
an opportunity to use exogenous proteases in feed to improve protein digestibility.
Protease hydrolysis in vitro has been proven to be an effective method to reduce
or eliminate the anti-nutritional factors in SBM, increase the apparent ileal digestibility
and improve broiler performance [16]. Additional addition of exogenous proteases could
improve amino acid digestibility, feed conversion rate and intestinal integrity of broilers
fed normal [17] or low-protein diets [18,19]. In addition, one study demonstrated that
the addition of AlP (optimal pH 9.0) increased ileal energy digestibility in 35-day-old
broilers [20].
Microbial proteases had the advantages of animal and plant proteases, and could
improve the disadvantages of high production cost, low enzyme production efficiency
and difficulty achieving large-scale production. Protein-producing microorganisms mainly
included bacteria, molds and actinomycetes. Microbial proteases were commonly used
in feed to improve animal protein utilization and reduce nitrogen excretion. Based on
different optimal pH, proteases can be divided into acidic protease (AcP), alkaline protease
(AlP) and neutral protease (NeP). It was reported that the average pH values of the crop,
gizzard, proventriculus, proximal and distal intestinal contents were 4.0–7.8, 0.3–4.1, 0.4–5.4,
5.2~7.6 and 5.5~7.7, respectively [21]. The pH of the contents of different digestive organs
in broilers is different, and might be too low in the gizzard or proventriculus and too high
in the small intestine, which is not conducive to the maximum effect of exogenously added
enzymes. Many studies have shown that the enzyme activity in feed is affected by the
pH value of the digestive tract. For example, xylanase had high activity at pH 6.0–7.0
and no activity at pH 3.0 [21,22]; β-glucanase had high activity at pH 3.0~7.0 [21,23]; the
amylase had high activity at pH 6.0 and 6.5, but no activity or very low activity at pH 3.0,
7.0 and 7.5; α-galactosidase had high activity at pH 6 and no activity at other pH levels.
The protease had no or very low activity at all levels except pH 3.0 [21]; and phytases from
Aspergillus oryzae and Aspergillus niger confirmed the best activity at pH 4.0 and nearly pH
Animals 2023, 13, 1746 3 of 15

5.0, respectively [24]. The pH level in the digestive tract of broilers might be an important
limiting factor for the maximum activity of exogenous proteases.
The efficacy of exogenous proteases was influenced by commercial sources of fungi or
bacteria [25–28], optimum pH or temperature characteristics [25,27,28], addition levels [20]
and combination levels [18]. Most proteases in published studies were single-component
proteases or commercial products, but the effects of different protease combinations on
broiler growth and protein digestibility have not been widely reported. The AcP, NeP
and AlP selected in this study are serine proteases such as pepsin and chymotrypsin, and
have similar action sites, while keratinase (Ker) has the characteristics of hydrolyzing
hydrophobic amino acids. The purpose of this study was to investigate the effects of single
addition of AcP, NeP, AlP and Ker and their combination on in vitro and in vivo protein
digestibility of broilers fed corn–soybean meal diets from the perspective of insufficient
secretion of endogenous enzymes in broilers and the effect of exogenous enzymes on
endogenous enzymes.

2. Materials and Methods

The experiment was conducted in the State Key Laboratory of Animal Nutrition. The
experimental protocols used in the current study were reviewed and approved by the
Animal Care and Use Committee of Institute of Animal Science, Chinese Academy of
Agricultural Sciences (2017-007).

2.1. Animals and Proteases

A total of 640 one-day-old Arbor Acres+ (AA+) male broilers were purchased from
Beijing Huadu Chicken Co., Ltd. and vaccinated. They were randomly divided into
8 groups of 10 with 8 replicates of 10 birds per replicate cage. The temperature of broilers
was increased to 35 ◦ C in the early stage, and then decreased by 2 ◦ C per week until being
maintained at 25 ◦ C. The broilers were immunized according to the routine immunization
degree, and the chicken house was regularly disinfected and well-ventilated during the
broiler feeding process. All broilers were free to feed and drink throughout the experiment.
The proteases used in this research were described as AcP from Aspergillus niger, AlP
and NeP from Bacillus subtilis (Jinan Baisijie Biological Engineering Co., Ltd., Jinan, China)
and Ker from Bacillus licheniformis (our own laboratory) for the in vitro and in vivo studies.

2.2. Experimental Design

Corn–soybean meal-based diets were recommended to meet the nutritional require-
ments of AA+ 1~21- and 22~31-day-old broilers, either alone or in combination with
proteases, as shown in Table 1. The detailed process was as follows. According to the
addition of different proteases in the diet, these broilers were divided into the control group,
0.8 U/g NeP, 0.8 U/g AlP, 0.4 U/g Ker, 1.6 U/g AcP alone or together with 0.8 U/g NeP,
0.8 U/g ALP or 0.4 U/g Ker groups, and these proteases of four different microorganisms
were evaluated in broilers in vitro and in vivo. Dietary chromium oxide (Cr2 O3 ) was added
as an exogenous indicator to evaluate the digestibility of nutrients. Diet composition and
nutritional levels are shown in Table 2.
Animals 2023, 13, 1746 4 of 15

Table 1. The experiment design and treatments.

Treatments Levels (U/g Diet)

Control 0
AcP 1.6
NeP 0.8
AlP 0.8
Ker 0.4
AcP + NeP 1.6 + 0.8
AcP + AlP 1.6 + 0.8
AcP + Ker 1.6 + 0.4
AcP, acidic protease; NeP, neutral protease; AlP, alkaline protease; Ker, keratinase.

Table 2. Composition and nutrient profile of the basal diet (%).

Ingredients Days 0–21 Days 22–31

Corn 58.75 61.45
Soybean meal 44% CP 34.50 31.00
Soya oil 2.70 3.80
CaHPO4 1.90 1.60
Limestone 1.20 1.30
NaCl 0.35 0.30
Micro-mineral Premix a 0.20 0.20
Vitamin Premix b 0.02 0.02
DL-Met 0.18 0.10
L-Lys 0.1 0.15
Choline chloride 0.10 0.08
Total 100.00 100.00
Calculated nutritive value
Metabolizable energy (kcal/kg) 3040.00 3100.00
Crude protein 20.46 19.76
Calcium 0.98 0.90
Available phosphorus 0.45 0.40
Lysine 1.12 1.11
Methionine 0.52 0.40
Threonine 0.78 0.78
a.The premix provided the following per kg of diets: Cu, 8 mg; Fe, 80 mg; Zn, 75 mg; Mn, 100 mg; Se, 0.30 mg; I,
0.35 mg. b. The multi-vitamin provided the following per kg of diets: vitamin A, 10,000 IU; vitamin D3, 2600 IU;
vitamin B1, 2 mg; vitamin B2, 3. 6 mg; pantothenic acid, 12 mg; vitamin B6, 3 mg; vitamin B12, 0.024 mg; vitamin E,
20 IU; vitamin K3, 2 mg; nicotinic acid, 10 mg; folic acid, 0.8 mg; biotin, 0.12 mg.

2.3. Enzyme Activity In Different pH Buffers

The concentrations of citrate buffer, sodium phosphate, Tris-HCl and glycine-NaOH
were 0.2 mol/L, 0.03 mol/L, 0.05 mol/L and 0.05 mol/L, respectively. The buffers were
configured and stored at room temperature. The proteolytic activity of 4 proteases under
various pH values (citrate buffer, pH 2–6; sodium phosphate, pH 6–8; Tris-HCl, pH 8–9;
glycine-NaOH, pH 10–12) was measured by hydrolyzing casein substrate at 40 ◦ C for
30 min according to the Folin phenol reagent method. One unit of protease activity was
defined as the amount of enzyme liberated 1 g of tyrosine at 40 ◦ C under the optimal pH.
The activity of the highest enzyme detected in the experiment was defined as 100% to
evaluate the activity of the remaining enzymes. All reactions were carried out in triplicate.

2.4. The Restorability of Four Proteases Treated by Different pH Buffers Was Measured In Vitro
The stability of 4 proteases at gastro (acidic pH 3.3) and small intestine (neutral pH 6.2)
pH conditions of broiler was determined by incubating 300 U of each protease with 2 mL
of citrate buffer (pH 3.3) for 50 min, or followed by adjusting pH to 6.2 by NaOH for an
additional 86 min at 40 ◦ C without substrate. The residual activity of the protease at two
stages was assayed at each optimal pH, respectively. The highest enzyme activity was
Animals 2023, 13, 1746 5 of 15

defined as 100% to evaluate the remaining enzyme activities. All reactions were performed
in triplicate.

2.5. Effects of Four Proteases on Trypsin and Chymotrypsin Activity Were Detected In Vitro
Subsequently, the effects of 4 proteases on trypsin and chymotrypsin activity were
conducted in this experiment. The stock solutions of trypsin (about 230,000 U/mL; Sigma,
St. Louis, MO, USA) and chymotrypsin (about 25,000 U/mL; Sigma) were prepared
in 0.2 M Tris-HCl buffer (pH 7.8) with 20 mM CaCl2 according to the manufacturer’s
definition of units of activity. The activities of trypsin and chymotrypsin were determined
according to Borda-Molina [29] with some modifications, and the benzoyl-DL-arginine-p-
nitroanilide (BAPNA) and N-glutary-L-pH-enylalanine-p-nitroanilide (GPPNA) were used
as substrates, incubating with or without 4 proteases at final concentrations of 0, 0.2, 0.4,
0.8 and 1.6 U/mL at pH 7.8 and 40 ◦ C for 60 min.

2.6. Effects of Different Doses of Four Proteases on Crude Protein Digestibility of Corn Gluten
Meal and SBM In Vitro
Under conditions in vitro, the effects of four different doses of proteases on CP di-
gestibility of SBM and corn gluten meal (CGM) in the whole digestive tract (including
crop, stomach and intestines) were studied to determine whether the crop of broilers was
the relatively optimal position for the four protein hydrolysis feed proteins under weak
acid conditions.
According to the in vitro digestion process of the GIT of 31-day-old broilers described
by Bryan et al. [30], the following modifications were made. In the process of in vitro
digestion, 1.00 g matrix (CGM, soybean meal or a mixture of corn and soybean meal
in 7:3) was ground and screened by 60-mesh, and then placed in 50 mL of centrifuge
tubes. The pH value was adjusted to 4.91 with 3.5 mL of 1.1 mol/L of HCl, and 0.5 mL of
chloramphenicol (30 µg/mL) was added. Subsequently, each protease was dissolved in
0.25 M sodium acetate buffer with the best pH value to different final concentrations, and
the final concentrations of protease in the digestive system were ensured to reach 0, 0.2, 0.4,
0.6 and 0.8 U/g feed. Finally, these centrifuge tubes were incubated in an air-bath agitator
at 40 ◦ C and shaken at 120 rpm for 50 min to simulate the process of crop digestion.
During the crop digestion period, 1 g of substrate was weighed, 3.5 mL of phosphate
buffer (0.1 M, pH = 4.9) was added, 0.5 mL of chloramphenicol solution was added to inhibit
microbial growth and the substrate was digested in an incubator at 40 ◦ C at 120 r/min
for 50 min. During the gastric digestion period, the pH was adjusted to 3.3 with 0.1 M
hydrochloric acid, digested with pepsin (P7012, ≥2500 U/mg)–salt solution and cultured
under the same conditions for 61 min.
Subsequently, the pH value was adjusted to 6.02 by adding 1 mL of NaHCO3 with
a certain concentration, and 26 mL of sodium acetate buffer (pH 6.20) and trypsin from
porcine pancreas (P3292, 4× USP; Sigma-Aldrich, St. Louis, MO, USA) was mixed and
cultured for 163 min at the same temperature and shaking speed to replicate the digestive
process in the small intestine.
At the end of digestion, 5 mL of 20% sulfosalicylic acid was added to precipitate
the digestive juice for 30 min, and then centrifuged at 8000 r/min for 30 min. Then, the
supernatant was collected in the new centrifuge tube to analyze the amount of CP. The
IVDCP was calculated using the following formula:

IVDCP (%) = (CPsample − (CPResidue − CPBlank ))/CPSample × 100

2.7. Effects of Four Proteases Alone or in Combination on IVDCP of Corn–Soybean Meal Mixture
Further, the effects of adding 4 kinds of protease alone or in combination on the in vitro
CP digestibility of corn–soybean meal mixture (7:3) were investigated. The digestion
procedure was completely consistent with that in Section 2.6 in vitro.
Animals 2023, 13, 1746 6 of 15

2.8. Effects of Four Proteases on Crude Protein and AA Digestibility of Broilers Fed Corn–Soybean
Meal Diet
An animal trial was carried out at the State Key Laboratory of animal nutrition to
explore the effects of the supplementation of 4 proteases individually or in combination on
the growth, the CP and AA apparent digestibility in broiler-fed corn–soybean meal diet.

2.9. Effects of Four Proteases on Growth Performance of Broilers Fed Corn–Soybean Meal Diet
Broilers were weighed on days 1, 22 and 31 of the experiment, and fasted for up to 12 h
before weighing. The body weight of each stage was recorded, the addition and loss of feed
and the feed intake of each group were recorded in detail and the average daily feed intake
(ADFI), average daily gain (ADG) and feed-to-conversion ratio (FCR) were calculated.

2.10. Sampling and Analysis of Ileal Digesta

After the broilers were fed normally for 1 h at the age of 31 days, two broilers were
randomly selected from each treatment every 2 h. After intravenous injection of anesthetics
under the wings, the abdominal cavity of broilers was opened, and the middle 1/3 segment
of ileal chyme was taken. The chyme samples of each replicate group were mixed and
stored in −20 ◦ C refrigerator quickly.
The contents of CP, AA and Cr in freeze-drying sub-samples of ileal digesta and diets
were analyzed. The CP content was analyzed using the Kjeldahl method, and Cr content
was determined according to Williams et al. [31]. Additionally, detection of amino acid
content was performed as described in Zhou et al. [32].
The digestibility coefficients (DCs) of CP or AAs for each diet were calculated on a
pen basis, according to the following formula:

DCAA(CP)diet (%) = 100 − [(Crdiet ×AA(CP)digesta )/(Crdigesta ×AA(CP)diet )]

Among them, Crdiet and Crdigesta represented the concentration of Cr (g/kg) in diet
and digesta samples, respectively. AA(CP)diet and AA(CP)chyme were the respective concen-
trations (g/kg) of AA (CP) in diet and chyme samples.

2.11. Statistical Analysis

SPSS 19.0 (IBM SPSS, Armonk, NY, USA) was used for statistical analysis. All the
results were expressed as mean ± mean square error (SEM). Three independent experi-
ments were conducted for each experiment. The significance of the difference between
the two groups was analyzed by a double-tailed Student’s t-test unless special attention
was paid to paired comparison. More than 2 groups were statistically compared through
one-way ANOVA, and multiple comparisons were performed with Tukey–Kramer cor-
rection. All statements of significance were considered on a p-value less than 0.05 unless
otherwise specified.

3. Results
3.1. The Activity of Four Exogenous Proteases in Different pH Buffers
The activity of four proteases hydrolyzing casein substrate under different pHs is
shown in Figure 1. The optimal pH values of AcP, NeP, AlP and Ker were 3.0, 9.0, 11.0 and
11.0, respectively. The activities of acidic protease can be maintained at more than 50% in
the range of pH 2.2 to 5.0, and little activity was detected as pH was raised to above 7.0.
 3.1. The
TheActivity
activityofof
Four Exogenous
four proteases Proteases in Different
hydrolyzing caseinpHsubstrate
Buffers under different pHs is
shown Thein activity
Figure 1.
ofThe
fouroptimal
proteasespHhydrolyzing
values of AcP, NeP,substrate
casein AlP and under
Ker were 3.0, 9.0,
different pHs11.0
is
and
shown11.0,
in respectively.
Figure 1. TheThe activities
optimal of acidic
pH values protease
of AcP, NeP,canAlPbeand
maintained
Ker wereat3.0,
more9.0,than
11.0
50%
and in therespectively.
11.0, range of pH 2.2
Thetoactivities
5.0, and little activity
of acidic was detected
protease can be as pH was raised
maintained at moreto above
than
Animals 2023, 13, 1746 7.0.
50% in the range of pH 2.2 to 5.0, and little activity was detected as pH was raised to above 7 of 15
7.0.

Figure 1. The proteolytic activity of AcP, NeP, Ker and AlP in different pH buffers. a p < 0.05 or d p <
Theproteolytic
proteolytic activity a p < 0.05
Figure
0.05
Figure 1.1.The
compared with NeP, activity
AlP ofof
or Ker AcP,
group;
AcP, NeP,
NeP, Ker
b p < 0.05
Ker andand AlP
compared
AlP inwith
different
AcP,
in different pH
pHNeP buffers.
or AlP
buffers. 0.05 orc pd <p or
group;
ap < <
d
0.05
p< compared with AcP,
0.05 compared withNeP
NeP,orAlP group. b p compared
NeP, AlP Keror Ker group;
group; b p < 0.05 < 0.05 compared with
with AcP, AcP,
NeP orNeP
AlP or AlP group;
group; cp <
c p <compared
0.05 with AcP,
0.05 compared with NeP
AcP,or Kerorgroup.
NeP Ker group.
Less than 50% of NeP activity was reserved between pH 6.0–7.0 close to the jejunum
Less
pH ofLess than50%
broilers,
than 50%of
and ofNeP
NeP
only 10% activity
activity
activity was reserved
atreserved
was between
pH 3.0, between
which pH
was
pH 6.0–7.0
close closeto
to gastric
6.0–7.0 close topH.
theAlP
the jejunum
and
jejunum
pH
Ker of broilers,
pH exhibited
of broilers, and
similar only 10%
properties
and only activity
to NePat
10% activity at
atpHpH 3.0,
different which was
pH values,
3.0, which close
withto
was close to gastric
lower pH.
activity
gastric AlP and
at acidic
pH. AlP and
Ker
pH exhibited
Ker (3.0) similar
and neutral
exhibited properties
similar pH (6.0–7.0).
properties to NeP at different
Theseatresults
to NeP different pH values,
suggested that
pH values, with lower
thelower
with activity
crop and gastro
activity at acidic
were
at acidic
pH(3.0)
pH (3.0)sites
suitable andfor
and neutral
acidic
neutral pH
pH (6.0–7.0).
proteases
(6.0–7.0). These
toThese results
hydrolyze
resultsfeedsuggested thatthe
protein, that
suggested and thecrop
crop crop and
andand
small gastro were
intestines
gastro were
suitable
for sitesand
NeP, sites
suitable AlP foracidic
for acidicproteases
Ker. proteasesto tohydrolyze
hydrolyzefeedfeedprotein,
protein,and
andcrop
cropandandsmall
smallintestines
intestines
for NeP, AlP and
for NeP, AlP and Ker. Ker.
3.2. The pH Values on Recoverability of Four Exogenous Proteases
3.2. The pH Values on Recoverability of Four Exogenous Proteases
3.2. The
ThepH Values on Recoverability
recoverability of Four Exogenous
of four proteases at pH 3 and Proteases
6.2 are shown in Figure 2. In this
The recoverability of four proteases at pH 3 and 6.2 are shown in Figure 2. In this
research,
The AcP was stableof
recoverability infour
pH 3.3 buffer atat37pH
proteases °C3for 50 6.2
and minarewith little in
shown loss of activity,
Figure but
2. In this
research, AcP was stable in pH 3.3 buffer at 37 ◦ C for 50 min with little loss of activity,
was sensitive
research, AcP towasneutral
stable pH
in pH6.2,3.3
retaining
buffer atonly 15%
37 °C for of
50 the
minprotein hydrolyzate
with little from pH
loss of activity, but
but was sensitive to neutral pH 6.2, retaining only 15% of the protein hydrolyzate from
6.2.
was NeP, AlP to
sensitive and Ker were
neutral sensitive
pH 6.2, to low
retaining onlypH,15%with negligible
of the protein residual
hydrolyzateactivity
fromafter
pH
pH 6.2. NeP, AlP and Ker were sensitive to low pH, with negligible residual activity after
incubation
6.2. NeP, AlPin acidic
and KerpH were
3.3 at 37 °C for
sensitive 50
to min.
low The
pH, recoverable
with activities
negligible of
residual AcP, NeP,
activity AlP
after
incubation in acidic pH 3.3 at 37 ◦ C for 50 min. The recoverable activities of AcP, NeP, AlP
and Ker at in
incubation pHacidic
3.3 topH
6.23.3
were 15.31%,
at 37 0%,min.
°C for 50 0.48%
Theand 37.68%, respectively.
recoverable Additionally,
activities of AcP, NeP, AlP
and Ker at pH 3.3 to 6.2 were 15.31%, 0%, 0.48% and 37.68%, respectively. Additionally, Ker
Ker
and showed
Ker at pHbetter recoverability
3.3 to 6.2 were 15.31%, from0%,
pH 0.48%
3.3 to and
pH 37.68%,
6.2. It was revealed that
respectively. the small
Additionally,
showed better recoverability from pH 3.3 to pH 6.2. It was revealed that the small intestine
intestine
Ker maybetter
showed be therecoverability
main site of action
from of
pHNeP,
3.3 AlP
to pHand Ker.
6.2. It was revealed that the small
may be the main site of action of NeP, AlP and Ker.
intestine may be the main site of action of NeP, AlP and Ker.

Figure 2. Storability of AcP NeP, AlP and Ker incubated at pH 3.3 for 50 min (treatment A) or from
Figure 2. Storability of AcP NeP, AlP and Ker incubated at pH 3.3 for 50 min (treatment A) or from
◦ C. Enzyme activity
pH3.3
pH 3.3atat50
50min
minfollowed
followedby byincubation
incubationatatpH
pH6.2
6.2for
for68
68min
min(treatment
(treatment B)atat40
40°C.
Figure 2. Storability of AcP NeP, AlP and Ker incubated at pH 3.3 for 50B)min Enzyme
(treatment A) activity
or from
without
without
pH incubation
3.3 atincubation wasconsidered
was
50 min followed considered 100%.
100%.
by incubation Significant
Significant
at pH differences
6.2 for 68differences areshown
are
min (treatment shown by
B) at 40by
°C. bars
bars labeled
labeled
Enzyme with
with
activity
various letters. a p < 0.05 compared with AcP, NeP or AlP group in treatment B; b p < 0.05 compared
without incubation was considered 100%. Significant differences are shown by bars labeled with
with Ker group in treatment B.

3.3. Four Exogenous Proteases on Trypsin and Chymotrypsin Activities

The effects of proteases on the activity of trypsin and chymotrypsin are presented in
Figures 3 and 4, respectively. The activity of trypsin was improved by all levels of AcP,
and increased significantly by 0.2, 0.4, 0.8 and 1.6 U/g at 23.5%,18.7%, 21.8% and 6.8%,
respectively, but was decreased by AlP and Ker dramatically. NeP did not affect trypsin
activity. The effect of AcP on chymotrypsin activity was similar to trypsin, but NeP, AlP
and Ker had little influence on the activity of chymotrypsin.
 The effects of proteases on the activity of trypsin and chymotrypsin are presented in
Figures 3 and 4, respectively. The activity of trypsin was improved by all levels of AcP,
and increased significantly by 0.2, 0.4, 0.8 and 1.6 U/g at 23.5%,18.7%, 21.8% and 6.8%,
respectively, but was decreased by AlP and Ker dramatically. NeP did not affect trypsin
Animals 2023, 13, 1746 activity. The effect of AcP on chymotrypsin activity was similar to trypsin, but NeP, 8AlP
of 15
and Ker had little influence on the activity of chymotrypsin.

Figure 3. Effects of four proteases incubated with trypsin at different final concentrations (0, 0.2, 0.4,
Figure 3. Effects of four proteases incubated with trypsin at different final concentrations (0, 0.2, 0.4,
0.8and
0.8 and1.61.6U/g)
U/g)ononthe
therelative
relativeactivity
activityofoftrypsin
trypsin(%).
(%).Significant
Significantdifferences
differencesare
areshown
shownby bybars
bars
labeledwithwithvarious
variousletters. a
letters.a p <p < 0.05 compared with AlPororKer
Kergroup
groupincubated
incubatedwith
with0.2,
0.2,0.4,
0.4,0.8
0.8
labeled a 0.05 compared with AlP
oror1.6
1.6U/g
U/g ofoftrypsin; bb pb <p 0.05
trypsin; < 0.05 compared
compared withwith
AlPAlP or Ker
or Ker group
group incubated
incubated with
with 0.8 0.8 or 1.6
or 1.6 U/gU/gof
trypsin; cc p <c 0.05 compared with AlP, NeP or Ker group incubated with 0.2, 0.4, 0.8 or 1.6 U/g of
of trypsin; p < 0.05 compared with AlP, NeP or Ker group incubated with 0.2, 0.4, 0.8 or 1.6 U/g
trypsin.
of trypsin.

Figure 4. Relative activity of chymotrypsin (%) incubated with or without four proteases in final
Figure 4. Relative activity of chymotrypsin (%) incubated with or without four proteases in final
concentrationsofof0,0,0.2,
concentrations 0.2,0.4,
0.4,0.8
0.8and
and1.6
1.6U/g.
U/g.Significant
Significantdifferences
differencesare
areshown
shownby
bybars
barslabeled
labeledwith
with
various letters. a p < 0.05 compared with AcP group incubated with 0 U/g of trypsin; b p < 0.05
various letters. a p < 0.05 compared with AcP group incubated with 0 U/g of trypsin; b p < 0.05 com-
a b

compared
pared withgroup
with AcP AcP group incubated
incubated with0.4,
with 0.2, 0.2,0.8
0.4,
or0.8
1.6or 1.6of
U/g U/g of trypsin.
trypsin.

3.4. Effects of Different Doses of Exogenous Proteases on Crude Protein Digestibility of CGM and
3.4.
SBM Effects of Different Doses of Exogenous Proteases on Crude Protein Digestibility of CGM
In Vitro
and SBM In Vitro
The effects of four different doses of protease on the in vitro crude protein digestibility
The effects
(IVCPD) of CGM of four
anddifferent
SBM aredoses
shown of in
protease
Tables on the in
3 and 4. vitro crude protein
In general, digestibil-
the effects of AcP,
ity (IVCPD)
NeP, AlP and of CGM
Ker onand SBM of
IVCPD areSBM
shownandinCGM
Tables 3 and
were 4. In general,
roughly the effects(pof<AcP,
dose-dependent 0.05),
NeP, AlP and Ker on IVCPD of SBM and CGM were roughly dose-dependent
but the optimal enzyme activity addition of each enzyme was different. Except for Acp (p < 0.05),
but
andtheKeroptimal enzymeenzyme
(the optimum activityactivity
addition
was of 1.6
each enzyme
U/g), was different.
the optimum enzymeExcept for of
activity Acp
the
and Ker (the optimum enzyme activity was 1.6 U/g), the optimum enzyme
other two enzymes to improve the digestibility of SBM crude protein was 0.8 U/g (Table activity of the3).
other two enzymes
Compared with the tocontrol
improve the digestibility
group, of SBM
the proportions of crude
IVCPD protein was by
increased 0.8 the
U/goptimal
(Table
3). Compared
amount of Acp,with the Alp
Nep, control
andgroup,
Ker werethe 14.34%,
proportions of IVCPD
14.37%, 14.81%increased by the
and 10.65%, optimal
respectively.
amount of Acp,suggested
These results Nep, Alpthat anddifferent
Ker weresources
14.34%,of14.37%,
enzymes 14.81% and 10.65%,
and different respectively.
doses of enzymes
These results suggested
had different effects on that different
the IVCPD ofsources
SBM. of enzymes and different doses of enzymes
had different effects on the IVCPD of SBM.
The improvement effect of four proteases with different enzyme activities on IVCPD of
CGM was different. The optimum enzyme activities of AcP, NeP, AlP and Ker were 0.8 U/g,
1.6 U/g, 0.4 U/g and 0.2 U/g, respectively (Table 4). Compared with the control, AcP,
NeP, AlP and Ker increased the relative levels of IVCPD of SBM by 42.24%, 47.31%, 91.38%
and 64.83%, respectively. The above results revealed that the improvement effect of four
exogenous enzymes on IVCPD of CGM was better than that of SBM, and the improvement
effect of Ker and AlP was relatively obvious.
Animals 2023, 13, 1746 9 of 15

Table 3. The IVCPD (%) of SBM with or without four proteases in different doses.

Protease Dosage (U/g) AcP NeP AlP Ker

0 57.66 c 57.66 c 57.66 c 57.66 c
0.2 60.13 b 62.01 b 63.11 b 58.58 c
0.4 61.05 b 62.16 b 62.50 b 61.85 b
0.8 60.62 b 65.95 a 66.20 a 62.45 b
1.6 65.93 a 63.38 b 64.28 ab 63.80 a
SEM 0.283 0.652 1.138 0.620
p-value <0.001 <0.001 <0.001 <0.001
Means with different superscript letters differ (p < 0.05). AcP, acidic protease; NeP, neutral protease; AlP, alkaline
protease; Ker, keratinase. a p < 0.05 compared with 0, 0.2, 0.4 or 0.8 U/g of AcP or Ker group, 0, 0.2, 0.4 or 1.6 U/g
of NeP group or 0, 0.2 or 0.4 U/g of AlP group; b p < 0.05 compared with 0 or 1.6 U/g of AcP, 0 or 0.8 U/g of NeP
or AlP group or 0, 0.2 or 1.6 U/g of Ker group; c p < 0.05 compared with 0.2, 0.4, 0.8 or 1.6 U/g of AcP, NeP or AlP
group or 0.4, 0.8 or 1.6 U/g of Ker group.

Table 4. The IVCPD (%) of CGM with or without four proteases in different doses.

Protease Dosage (U/g) AcP NeP AlP Ker

0 21.92 c d d 21.92 e
21.92 21.92
0.2 22.32 c 22.52 d 39.05 b 36.13 a
0.4 21.96 c 23.76 c 42.39 a 31.43 b
0.8 31.18 a 30.13 b 39.40 b 28.92 c
1.6 28.90 b 32.29 a 38.61 b 24.24 d
SEM 0.423 0.401 0.727 4.761
p-value <0.001 <0.001 <0.001 0.008
Means with different superscript letters differ (p < 0.05). AcP, acidic protease; NeP, neutral protease; AlP, alkaline
protease; Ker, keratinase. a p < 0.05 compared with 0, 0.2, 0.4 or 1.6 U/g of AcP; 0, 0.2, 0.4 or 0.8 U/g of NeP group,
0, 0.2, 0.8 or 1.6 U/g of AlP group, or 0, 0.4, 0.8 or 1.6 U/g of Ker group; b p < 0.05 compared with 0, 0.2, 0.4 or
0.8 U/g of AcP, 0, 0.2, 0.4 or 1.6 U/g of NeP, 0 or 0.4 U/g of AlP group, or 0, 0.2, 0.8 or 1.6 U/g of Ker group;
c p < 0.05 compared with 0.8 or 1.6 U/g of AcP, 0, 0.2, 0.8 or 1.6 U/g of NeP or 0, 0.2, 0.4 or 1.6 U/g of Ker group;
d p < 0.05 compared with 0.4, 0.8 or 1.6 U/g of NeP, 0.2, 0.4, 0.8 or 1.6 U/g of AlP, 0, 0.2, 0.4 or 1.6 U/g of Ker

group. e p < 0.05 compared with 0.2, 0.4, 0.8 or 1.6 U/g of Ker group.

3.5. Crude Protein Digestibility of a Mixture of Corn and SBM with or without Four Proteases
Alone or in Combination In Vitro
The optimal levels of the four proteases to increase CGM and SBM IVCPD are shown
in Tables 3 and 4. Subsequently, the effects of proteases on corn–SBM (7:3) mixed diets were
screened by comprehensively considering the improvement effects of four proteases on the
IVCPD of corn and SBM. The individual dosages of AcP, NeP, AlP, and Ker were 1.6, 0.8, 0.8,
and 0.4 U/g, respectively, and their combinations were at the same level as AcP together
with NeP, AlP or Ker. The effect of the addition of proteases alone or in combination on the
IVCPD of a 7:3 ratio corn and SBM mixture is shown in Table 5. Compared with the control,
AcP alone or together with NeP, AlP or Ker significantly increased IVCPD (p < 0.05), while
adding NeP, AlP or Ker alone had no significant effect (p > 0.05). Supplementation with
AcP alone was more effective for IVCPD than any other treatment. The combination of
AcP and NeP, AlP or Ker further increased IVCPD compared to NeP, AlP and Ker provided
alone (p < 0.05).

3.6. Growth Performance and Apparent Ileal AA Digestibility In Vivo

Compared with the control group, the addition of ACP or NeP alone, or ACP combined
with NeP or Ker alone, significantly increased the DFI of broilers during the growing period
(day 22 to day 31) (p < 0.05). However, ACP combined with AlP or Ker significantly reduced
the DFI of broilers from day 1 to day 31 (p < 0.05); in addition, compared with the control
group, the addition of AcP, NeP or AlP alone and the combined addition of AcP and
Ker significantly increased the ADG of broilers from day 1 to day 31, while the addition
of AcP combined with AlP significantly decreased the ADG of broilers from day 1 to
day 31 (p < 0.05) (Table 6). The apparent ileal digestibility of CP in a broiler of 31 days was
Animals 2023, 13, 1746 10 of 15

significantly reduced by the addition of four proteases (p < 0.05) whatever the supplemented
individual or combined form (Table 7). Ker alone or together with AcP showed a better
performance than other protease supplementation (p < 0.05), but lower than the control
group (p < 0.05). The addition of Ker alone and AcP combined with NeP supplementation
increased the apparent ileal digestibility of Tyr (p < 0.05) (Table 8). Adding Ker alone
and AcP combined with NeP, AlP or Ker all significantly increased Pro apparent ileal
digestibility (p < 0.05). Compared with the control group, the apparent ileal digestibility of
Asp, Thr, Ser, Glu, Gly, Ala, Cys, Met and Phe were decreased after adding all proteases
(p < 0.05), including alone and in combination.

Table 5. The IVCPD of corn and SBM mixture with or without four proteases individually or in
combination.

Treatments Protease Dosage (U/g) Digestibility (%)

Control 0 77.49 c
AcP 1.6 86.82 a
NeP 0.8 79.33 bc
AlP 0.8 80.21 bc
Ker 0.4 77.71 c
AcP + NeP 1.6 + 0.8 86.36 a
AcP + AlP 1.6 + 0.8 85.68 a
AcP + Ker 1.6 + 0.4 80.63 b
SEM - 0.778
p-value - <0.001
Means with different superscript letters differ (p < 0.05). AcP, acidic protease; NeP, neutral protease; AlP, alkaline
protease; Ker, keratinase. a p < 0.05 compared with control, AcP, NeP, AlP, AcP together with Ker group; b p < 0.05
compared with control, AcP, Ker, AcP together with AlP or Ker group; c p < 0.05 compared with AcP alone or
together with NeP, AlP or Ker group.

Table 6. Growth performance of broilers (1 day to 31 days) with or without four proteases individually
or in combination.

Days 1–21 Days 22–31 Days 1–31

Treatments
ADG (g) DFI(g) FCR ADG (g) DFI(g) FCR ADG (g) DFI(g) FCR
Control 50.52 75.92 1.51 70.92 de 1.52 68.96 e 101.25 a 1.47
107.91
AcP 51.50 76.95 1.49 75.06 115.08 a 1.53 70.94 a 104.18 a 1.47
NeP 50.29 76.87 1.53 71.60 110.69 bc 1.54 69.41 d 102.68 a 1.48
AlP 50.71 74.86 1.47 71.23 110.23 bcd 1.54 69.57 c 101.27 a 1.46
Ker 49.60 73.69 1.49 69.59 106.77 e 1.53 68.91 e 99.81 a 1.46
AcP + NeP 49.87 75.08 1.50 72.29 113.54 ab 1.57 69.01 e 99.80 a 1.45
AcP + AlP 49.59 74.2 1.49 68.12 109 cde 1.60 67.49 e 89.79 b 1.33
AcP + Ker 51.48 77.44 1.50 73.05 111.3 bc 1.52 70.63 b 93.28 b 1.32
SEM 1.172 2.253 0.074 2.788 1.474 0.072 0.05 2.066 0.147
p-value 0.573 0.640 0.997 0.384 <0.001 0.946 <0.001 <0.001 0.889
Means with different superscript letters differ (p < 0.05). AcP, acidic protease; NeP, neutral protease; AlP, alkaline
protease; Ker, keratinase; ADG, average daily gain; DFI, daily feed intake; FCR, feed-to-conversion ratio. a p < 0.05
compared with DFI in control, NeP, AlP, Ker, AcP together with AlP or Ker group from days 22 to 31, ADG in all
groups except AcP group or DFI in AcP together with AlP or Ker group from days 1 to 31; b p < 0.05 compared
with DFI in control, AcP, Ker, AcP together with AlP group from days 22 to 31, ADG and DFI in all groups from
days 1 to 31; c p < 0.05 compared with DFI in control, AcP, Ker, AcP together with NeP from days 22 to 31, ADG in
all group from days 1 to 31; d p < 0.05 compared with DFI in AcP, NeP, Ker, AcP together with NeP or Ker group
from days 22 to 31, ADG in all group from days 1 to 31; e p < 0.05 compared with AcP, NeP, AlP, AcP together
with NeP or Ker group from days 22 to 31, ADG in AcP alone, NeP alone, AlP alone or together with Ker.
Animals 2023, 13, 1746 11 of 15

Table 7. The crude protein apparent ileal digestibility in broilers fed corn soybean meal diet with or
without four proteases individually or combination.

Treatments Digestibility (%)

Control 79.14 a
AcP 73.10 c
NeP 73.55 c
AlP 72.80 c
Ker 76.21 b
AcP + NeP 73.28 c
AcP + AlP 73.90 c
AcP + Ker 76.90 b
SEM 1.828
p-value 0.027
Means with different superscript letters differ (p < 0.05). AcP, acidic protease; NeP, neutral protease; AlP, alkaline
protease; Ker, keratinase. a p < 0.05 compared with NeP, AlP, Ker, AcP alone or together with NeP, AlP or Ker
group; b p < 0.05 compared with control, AcP, NeP, AlP, AcP alone or together with NeP; c p < 0.05 compared with
control, Ker, AcP together with Ker.

Table 8. The AAs apparent ileal digestibility of corn soybean meal diet with monocomponent or
combination protease (%).

Items Control AcP NeP AlP Ker AcP + NeP AcP + AlP AcP + Ker SEM p-Value
Asp 77.98 a 68.70 d 71.55 c 72.08 c 75.58 b 70.63 cd 68.70 d 72.4 c 2.324 0.012
Thr 73.02 a 59.41 d 64.72 c 64.45 c 69.08 b 63.71 c 60.04 d 63.39 c 3.185 0.011
Ser 78.09 a 68.27 cd 70.40 c 69.71 c 74.26 b 71.57 bc 65.61 d 70.03 c 3.126 0.035
Glu 86.22 a 79.92 cd 81.57 c 81.53 c 84.29 b 81.65 c 78.94 d 81.43 c 1.777 0.020
Gly 75.16 a 65.22 bc 65.85 c 63.62 bc 68.69 cd 62.11 cd 59.58 d 63.29 cd 3.755 0.025
Ala 75.16 a 65.22 bc 65.85 bc 63.62 cd 68.26 b 62.11 cd 59.58 d 63.29 cd 3.748 0.026
Cys 61.44 a 49.36 d 51.32 cd 50.60 cd 54.13 bc 49.07 d 49.39 d 55.29 b 3.663 0.048
Val 73.58 a 63.50 c 67.73 bc 65.71 bc 69.79 ab 67.07 bc 58.87 d 57.23 d 4.104 0.018
Met 96.89 a 95.05 ef 95.95 bc 95.83 cd 96.33 de 95.45 ef 95.46 f 94.60 b 0.613 0.043
Ile 78.55 ab 70.48 d 75.75 bc 75.82 bc 80.06 a 76.65 bc 74.54 c 69.88 d 2.989 0.040
Leu 82.78 a 74.62 c 78.49 b 78.49 b 82.80 a 80.91 a 77.70 b 78.51 b 2.009 0.013
Tyr 68.78 bc 59.37 d 65.38 c 68.93 bc 76.23 a 74.59 a 67.32 bc 68.80 b 3.292 0.004
Phe 80.22 a 70.69 c 74.43 c 74.08 c 78.48 b 76.35 c 73.63 c 74.99 b 2.139 0.012
Lys 83.14 a 75.61 c 78.36 b 79.44 b 82.76 a 79.73 b 77.86 b 79.70 b 2.057 0.036
His 81.17 a 72.43 c 75.45 b 74.70 bc 79.03 a 75.52 b 72.83 bc 75.41 b 2.559 0.050
Arg 88.42 a 81.27 c 84.21 b 84.14 b 86.95 a 83.62 b 81.86 c 83.90 b 1.657 0.009
Pro 72.97 b 53.45 d 67.24 c 70.25 bc 77.86 a 79.61 a 78.17 a 79.77 a 3.075 <0.001
Means with different superscript letters differ (p < 0.05). AcP, acidic protease; NeP, neutral protease; AlP,
alkaline protease; Ker, keratinase; Asp, Asparticacid; Thr, Threonine; Ser, Serine; Glu, Glutamicacid; Gly, Glycine;
Ala, Alanine; Cys, Cysteine; Val, Valine; Met, Methionine; Ile, Isoleucine; Leu, Leucine; Tyr, Tyrosine; Phe,
Phenylalanine; Lys, Lysine; His, Histidine; Arg, Arginine; Pro, Proline. a p < 0.05 compared with all AAs apparent
ileal digestibility in NeP, AlP, Ker, AcP alone or together with NeP, AlP or Ker group; b p < 0.05 or c p < 0.05
compared with all AAs apparent ileal digestibility in control, NeP, AlP, Ker, AcP alone or together with NeP,
AlP or Ker group; d p < 0.05 compared with Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Tyr or Pro apparent
ileal digestibility in control, AlP, Ker, AcP alone or together with NeP, AlP or Ker group; e p < 0.05 or f p < 0.05
compared with Met apparent ileal digestibility in control, NeP, AlP, AcP together with AlP or Ker group.

4. Discussion
Microbial proteases are usually used in feed to improve protein utilization and reduce
nitrogen excretion. Proteases were divided into AcP, NeP and AlP according to their
optimum pH for activation. The average pH values of crop, stomach, proximal and distal
small intestine of broilers were 6.5, 3.0, 7.0 and 7.5, respectively [21]. A previous study in
our laboratory demonstrated that the average pH values of crop, stomach and jejunum
contents of broilers at 31 days were 5.0, 3.3 and 6.2 (unpublished), respectively. Factors
such as breed, growth stage, feed, and growth environment of broiler chickens might cause
differences in pH in the contents of different organs. The widespread use of exogenous
Animals 2023, 13, 1746 12 of 15

proteases was limited by the instability of animal stomachs and small intestines at acidic or
neutral pH conditions; thus, well low pH adaptability has become one of the most valuable
properties of proteases in animal feed.
The activity of the enzyme is affected by pH, and the enzyme usually exerts the greatest
effect under optimal pH conditions [33]. To study the effects of different exogenous enzymes
on protein utilization in broiler diets, mainly due to the different pH of the digestive parts
of broiler chickens [33], considering the characteristics of four kinds of exogenous proteases
and the difference in pH environment from crop to the stomach and small intestine during
broiler development, the effects of different exogenous proteases on the protein digestibility
of broilers were studied. AcP, NeP and AlP efficiently hydrolyze animal and plant proteins
under acidic, neutral and alkaline conditions, respectively, and hydrolyze macromolecular
proteins into small molecular peptides or amino acids to facilitate the effective absorption
and utilization of proteins [34]. Ker is a special alkaline serine protease containing disulfide
bond hydrolase and polypeptide hydrolase, which can efficiently open disulfide bonds and
degrade keratin, gliadin, polypeptide and other proteins [35]. The location and degradation
rate of AcP, NeP, AlP and Ker in broiler chickens are also two factors that affect the function
of four proteases. Studies have shown that the main action sites of AcP, NeP and AlP in
broilers are the crop, stomach and small intestine, respectively [7,36]. However, the position
of Ker in broilers has not been extensively studied. The optimum pH of AcP, NeP, AlP and
Ker were 3.0, 9.0, 11.0 and 11.0, respectively. Combined with the previous research results
of our laboratory (unpublished), the average pH of crop, gizzard, glandular stomach and
jejunum contents of broilers at 31 d was 4.91, 3.37, 3.37 and 6.20, respectively. The activities
of AcP, NeP, AlP and Ker may also be inhibited when the pH of the broiler digestive tract
changes. Indeed, our in vitro study confirmed that the refolding rate of acid protease was
15.31% and the refolding rate of keratinase was 37.68% when the acid condition (pH 3.3)
was adjusted to weak acid (pH 6.2), while the refolding rate of the other two enzymes was
negligible. Under the conditions of this study, the selection of proteases with a wide range
of acid resistance is helpful for exogenous enzymes to exert their effects in different parts
of broilers, thereby improving the enzymatic hydrolysis efficiency of proteins.
The protein digestibility of exogenous proteases on CGM and SBM and the mixture
of corn and SBM were the focus of our attention. The results showed that the optimum
addition of AcP, NeP, AlP and Ker to improve the crude protein digestibility of soybean
meal was 1.6, 0.8, 0.8 and 1.6 U/g, and the proportion of IVCPD increased by SBM was
14.34%, 14.37%, 14.81% and 10.65%, respectively. The optimal enzyme activities of AcP, NeP,
AlP and Ker were 0.8, 1.6, 0.4 and 0.2 U/g, respectively, and the proportions of increasing
CGM IVCPD were 42.24%, 47.31%, 91.38% and 64.83%, respectively. These results indicate
that the effect of four exogenous enzymes on the IVCPD of CGM is better than that of SBM,
which may be due to the different protein structures of the two diets. The protein of CGM
is mainly gliadin, glutelin, globulin and albumin. The natural corn protein peptide chain
is curled into a compact sphere, and the structure is relatively stable. By adding protease
to destroy the structure of corn protein, exposing the contact site with the enzyme, and
increasing the action point of the enzyme, the enzymatic hydrolysis rate can be improved.
In addition, we also found that AlP was superior to the other three exogenous proteases
in improving IVCPD of SBM. The enzymes used to hydrolyze SBM can be acid protease,
alkaline protease and neutral protease, and alkaline protease is widely used [37–39].
From our results, the addition of protease had a greater effect on the DFI of broiler
chickens during the brooding period (days 21 to 31), and the effect of AcP and NeP alone
or in combination was more significant. It can be seen that AcP and NeP had a synergistic
effect on improving the feed intake of broiler chickens during the brooding period. In
addition, AcP and Ker had a synergistic effect in improving ADG of broilers, while AcP and
AlP had an antagonistic effect. Considering that the optimum pH span between AcP and
AlP is large, it is necessary to explore the effect of batch treatment on the ADG of broilers.
There were anti-nutritional factors, such as soybean antigen protein, trypsin inhibitor
and plant lectin in soybean meal, which seriously affected the hydrolysis of a soybean
Animals 2023, 13, 1746 13 of 15

meal protein by exogenous protease [40]. Our results showed that the addition of four
proteases alone and in combination significantly reduced the apparent ileal digestibility of
CP in broilers at 31 days, especially the apparent ileal digestibility of Asp, Thr, Ser, Glu,
Gly, Ala, Cys, Met and Phe. This was not consistent with in vitro evaluation results, which
might be due to in vitro evaluation of proteases using a single feed ingredient, or animal
hormones, such as regulatory effects [41]. Although a large amount of evidence showed
that exogenous protease supplementation could make up for the deficiency of endogenous
enzymes in broilers and promote the utilization of protein and amino acids [42,43], the
effect of protease supplementation was related to the dosages [43]. When the dosage
was too high, it might inhibit the secretion of endogenous protease in a feedback manner.
Therefore, the addition of high-dose protease in the experiment might not be conducive to
improving nutrient digestibility.
The best exogenous enzymes and combinations were screened. In short, the evaluation
and screening of protease is still a more complex problem, not only need to consider the
digestibility of crude protein, but also need to consider the digestibility of amino acids,
nitrogen deposition and metabolism in order to accurately reflect the true effect of protease,
protease to achieve scientific evaluation.

5. Conclusions
The application of proteases in broiler diets is complex. Exogenous proteases were
not only sensitive to intestinal conditions (pH and endogenous protease), but also harmed
trypsin and chymotrypsin activities in in vitro studies. In addition, exogenous proteases
could improve the growth performance of broilers, but decrease the ileal digestibility
of crude protein in vivo. Therefore, the characteristics and dosage of protease and the
protein level in feed should be comprehensively considered when providing protease in
animal feed.

Author Contributions: Conceptualization, T.Z.; methodology, M.Z. and Y.B.; software, Y.S.; vali-
dation, M.Z., Y.S. and J.A.; formal analysis, Y.S.; investigation, T.Z.; resources, T.Z.; data curation,
T.Z.; writing—original draft preparation, M.Z. and Y.B.; writing—review and editing, T.Z. and Q.C.;
visualization, Y.S.; supervision, T.Z.; project administration, T.Z.; funding acquisition, T.Z. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Key R&D Program of China (2021YFD1301004), the
Central Public-interest Scientific Institution Basal Research Fund (Y2017CG36), National Natural
Science Foundation of China (31470122) and Special fund for science and technology innovation
project of Chinese Academy of Agricultural Sciences (ASTIP-IAS08).
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Ethics Committee of Chinese Academy of Agricultural Sciences,
Animal Care and Use Committee (protocol code 2017-007 and approved on 1 April 2015).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data used to support the findings of this study are available from
the corresponding author upon request.
Acknowledgments: We thank the Chinese Academy of Agricultural Sciences for kindly providing
the experimental facility.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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