Effect of supplemental serine-protease from Bacillus licheniformis on growth performance and physiological change of broiler chickens

ABSTRACT

An experimental study was conducted to examine the effects of adding serine-protease from Bacillus licheniformis on performance and physiological parameters of broiler chickens under Egyptian condition. A total of 600 one-day-old chicks were randomly divided into four experimental treatments. The treatments consisted of the control diet with 0, 100, 200 and 300 mg/kg serine-protease. Protease supplementation increased (P < 0.05) body weights (BW). Feed conversion ratio (FCR) was improved (P < 0.05) due to 200 and 300 mg/kg protease supplementation. The dry matter and crude protein digestibilities were enhanced (P < 0.05) by both 200 and 300 mg/kg protease supplementation. Plasma albumin and high-density lipoprotein (HDL) concentrations were increased (P < 0.05), while plasma total cholesterol (CHO) and low-density lipoprotein (LDL) concentrations were decreased (P < 0.05) at 100, 200, and 300 mg/kg of protease. Liver malondialdehyde (MDA) levels were declined (P < 0.05) due to 200 and 300 mg /kg protease supplementation. Supplementing 100, 200, and 300 mg/kg of protease increased (P < 0.05) lysine, methionine, and threonine levels in breast muscle. In conclusion, exogenous serine-protease could be used as a feed additive in broiler nutrition and supplementing 200∼300 mg/kg was sufficient to improve growth performance, probably because of its mechanism to enhance protein digestibility.

KEYWORDS:

• Broiler chickens
• breast muscle
• growth performance
• protease
• physiological change

1. Introduction

Protein is one of the most nutrients and the most expensive component of animal diets. The physiological function of proteases is necessary for all living organisms, and proteolytic enzymes can be classified based on their origin: microbial (bacterial, fungal, and viral). The endogenous intestinal proteases are sufficient to improve protein utilization (Le Heurou-Luron et al. 1993; Niban and Mahgna 1993; Baghban-Kanani et al. 2018). However, digestibility of crude protein (CP) and amino acid is not completed in all animals (Wang and Parsons 1998; Lemme et al. 2004). In addition, it has been reported that activities of digestive enzymes (lipase, amylase, and trypsin) increase with age (Noy and Sklan 1979; Nitsan et al. 1991; Jin et al. 1998). Therefore, supplemental exogenous enzymes could optimize the digestibility of CP and consequently growth performance (body weight gain and feed conversion ratio) of broiler chickens (Jiang et al. 2008; Angel et al. 2011; Kalmendal and Tauson 2012; Yegani and Korver 2013; Saleh et al. 2019a).

Previous experiments conducted on serine protease and threonine and found there benefit on growth performance (Cowieson and Ravindran 2008 Ayaşan et al. 2009; Angel et al. 2011; Fru-Nji et al. 2011; Vieira et al. 2013; Ayaşan and Okan 2014a, 2014b; Ding et al. 2016; Lin-Law et al. 2019; Ndazigaruye et al. 2019) and. In addition, it was reported that supplemental protease reduced nitrogen excretion (Aletor et al. 2000; Bregendahl et al. 2002; Canogullari et al. 2009) and decreased feed costs, and consequently broiler industry's production. However, other experiments have concluded inconsistent results (Simbaya et al. 1996; Marsman et al. 1997; Naveed et al. 1998). Additionally, protease enzymes have several benefits including decreasing undigested proteins in the diet, increasing amino acid availability, reducing protein needs in the diet, maintaining weight gain and feed efficiency, reducing proteolytic fermentation, and decreasing biogenic amines and bacterial toxins (Saleh et al. 2013; Bedford and Walk 2014). Therefore, protease enzymes are of interest to many poultry companies and nutrition supplementation companies for use as an important supplement digestive enzyme in broiler diets. There are few studies investigated the effects of serine protease on physiological change and amino acids composition in breast muscle of broiler chickens. Recently study by Giannenas et al. (2017) reported that using Ronozyme®ProAct (serine protease) on performances of a broiler production system in Greece and they found that growth performance and meat quality were improved.

It could be hypothesized that supplementation of exogenous protease to broiler diets might be involved in improving the utilization of proteins in young chickens and consequently enhanced the growth performance of broilers. Therefore, the objective of the current study was to evaluate the effects of Ronozyme®ProAct serine protease from Bacillus licheniformis on blood biochemical permeates, lipid peroxidation, amino acids composition in breast muscle, nutrients utilization, and growth performance in broilers.

2. Material and methods

The study was approved by the Ethics Committee of Local Experimental Animals Care Committee and conducted in accordance with the guidelines of Kaferelsheikh University, Egypt (Number 4/2016 EC).

2.1 Design and diets

Ross 308 broiler chicks (n = 600), one-day-old were divided into four treatments, each included three replicates of 50 chicks (floor pens; ten birds/m²). Chicks were raised in a windowed experimental farm. The experiment was started during February and brooding heat was provided and the temperature inside the barn was maintained at around 32–34°C from day 1 to day 5 post-hatch, and gradually decreased to 24°C at 21d.

The control diet was based on corn, soybean meal, and corn gluten meal (without serine protease, Table 1). Treatments two to four consisted of the control diet supplemented with graded levels of the exogenous serine-protease (Ronozyme® ProAct, DSM; activity 75,000 PROT kg⁻¹ feed) at 100mg, 200mg, and 300mg/kg, respectively. The enzyme activity in all supplemental enzymes diets was about 98%. Ronozyme® ProAct is a preparation of serine protease produced by a genetically modified strain of Bacillus licheniformis. It is produced by fermentation of a sporulation-deficient Bacillus licheniformis strain which expresses a synthetic gene encoding a serine protease (EC 3.4.21.-). Broilers were fed ad-libitum starter, grower and finisher mash diets (day 1–32 of age) and water was available freely.

Table 1. Composition and calculated levels of the basal diets.

	Starter (0–10 days)	Grower (11–24 days)	Finisher (25–32days)
Ingredients, g/kg
Yellow corn	575	615	650
Soybean meal, 44%	320	278	240
Corn gluten meal, 60%	58	50	40
Premix^1	3	3	3
Plant oil	7.5	17.5	30.5
Di-calcium phosphate	15	15	15
Limestone	13.9	13.2	12.5
NaCl	3	3	3
DL- Methionine	1.85	1.8	1.65
L- Lysine. Hcl	2.75	3.5	4.35
Total	1000	1000	1000
Nutrients levels, %
Crude protein (Analyzed)	23	21	19
Digestible Lys	1.19	1.14	1.11
Digestible Meth + Cysteine	0.84	0.78	0.71
Digestible Thr	0.77	0.70	0.63
Calcium	1.0	0.94	0.91
Available phosphorus	0.46	0.45	0.44
ME, MJ/Kg	12.43	12.85	13.15

¹Vitamins and minerals premix / 3Kg: A (6,000,000, IU), D3(900,000 IU), E (40,000 mg), K (2000 mg), B1 (2000 mg), B2 (4000 mg), B6 (2000 mg), B12 (10 mg), Niacin (50,000 mg), Pantothenic acid (10,000 mg), Biotin (50 mg), Folic acid (3000 mg), Choline (250,000) mg, 50,000 mg(Zn), 8500 mg (Mn), 50,000 mg (Fe), 50,000 mg (Cu), 200 mg (I), 100 mg (Se) and 100 mg (Co).

²Digestible AAs based on Rostagno et al. [25].

2.2 Growth performance

Mortality was registered daily through experiment period, while pen body weight (BW) and feed consumption were registered every week. Feed conversion ratio (FCR) was considered by dividing feed intake to the weight gain.

2.4 Blood and liver sampling

At day 32 of age, broiler chickens (n = 48) based on average BW were selected (12 birds/ treatment). They were weighed individually, slaughtered, and dissected to collect visceral organs. Weights of hot carcass, breast muscle, liver and abdominal fat were recorded. Blood samples were collected into heparinized test tubes and centrifuged (3000×g) for 20 min at 5^oC to separate plasma and it was aspirated by pipette and stored in Eppendorf tubes at −20^oC pending analysis. The livers from 12 broiler chickens were immediately collected after slaughter and were snap-frozen with liquid nitrogen, and stored at −80^oC until analysis.

2.5 Nutrients digestibility

Forty-eight broiler chickens based on average BW were transferred into digestibility cages at day 33 to conduct a digestibility trial until day 36 of age (12 birds/treatment; 4/replicate). Excreta were collected, weighted, and dried by the drying oven at 60^oC for 24 h from each replicate. The whole dried samples were then homogenized after drying. The proximate analysis in diets and excreta was conducted according to AOAC (2003) for determination of dry matter, crude protein, and ether extract as described by Abdel-Moneim et al. (2019) the calculation was as follows: Nitrogen digestibility (%) = (total nitrogen intake − total nitrogen excreted)/total nitrogen intake × 100.

2.6 Biochemical analysis

Total protein, albumin, total cholesterol (CHO), triglycerides (TG), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, glucose, creatinine, glutamic oxalacetic transaminase (SGOT), and glutamate pyruvate transaminase (SGPT) were measured by using kits from (Diamond Diagnostics, Egypt).

2. 7 amino acids levels in breast muscle

Lysine, methionine, and threonine contents in breast muscle were analysed according to (Ceylan and Mİ 2011). Ten-grams of breast meat and 40 ml of 0.1 N HCl were homogenized for 45 s at 4°C then centrifuged 15,000 × g for 50 min at 4°C. The supernatants were filtered and were analyzed using (GC-4 CM-PFE, Shimadzu gas chromatograph, Tokyo, Japan) equipped with a flame ionization detector (FID). The amino acids determined were lysine, methionine, and threonine. The amino acids determined were lysine, methionine, and threonine. The values were expressed in grams of amino acid per 100 grams of breast meat.

2.8 Assaying malondialdehyde

Lipid peroxidation was evaluated by assaying the level of malondialdehyde (MDA) in the liver by using kits from Cell Biolabs Inc. (San Diego, CA, USA).

2.9 Statistical analysis

Data were analyzed using SAS software 9.2 (SAS Institute, Cary, North Carolina) and the GLM procedure was used in the analysis. Dunnett's t Test was used to compare each protease level to the nun-supplemented control. Direct regression was utilized to examine if there are any linear or quadratic responses of the dependent variables to the supplemental protease.

3. Results

Dietary protease supplementation at any level increased (P < 0.05) the final body weights of broiler chickens compared to the non-supplemented chickens (Table 2). FCR was significantly improved due to dietary 200 and 300 mg/kg protease supplementation. The addition of protease enzyme had no significant influence on the average daily feed intake.

Table 2. Effect of dietary serine protease supplementation on broiler performance at day 32 of age^1,2,3.

Protease (mg/kg)					P-value
items	0	100	200	300	SEM	Protease	Linear	Quadratic
Initial BW, g	41	41	41	41	0.68	0.72	0.29	0.73
Final BW, g	1755^b	1770^a	1805^a	1807^a	5.67	0.0001	0.0001	0.11
BWG, g	1714^b	1729^a	1764^a	1766^a	5.85	0.0001	0.0001	0.11
ADFI, g	3128	3132	3120	3125	8.43	0.56	0.42	0.89
FCR	1.83^a	1.81^a	1.77^b	1.77^b	0.008	0.0001	0.0001	0.22

¹n = 150 birds/ treatment (50 birds/replicate);

²Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

³No morality through the experiment period.

Digestibility of dry matter was enhanced (P < 0.05) by the addition of both 200 and 300 mg/kg of protease compared to the control and a linear response was observed (Table 3). Similarly, a linear increase in crude protein digestibility was observed with increasing dietary protease levels. Ether extract digestibility was the highest when protease was supplemented at 100 mg/kg.

Table 3. Effect of dietary serine protease supplementation on nutrients digestibility^1,2.

			Protease (mg/kg)				P-value
Items, %	0	100	200	300	SEM	Protease	Linear	Quadratic
Dry matter	70.13^b	71.52^b	71.82^a	72.11^a	0.67	0.02	0.005	0.25
Crude protein	62.21^b	64.61^a	65.84^a	67.93^a	0.82	0.0001	0.0001	0.79
Ether extract	46.32^b	49.32^a	48.24^b	47.82^b	1.10	0.07	0.33	0.03

¹n = 12 birds/treatment (4 birds/ replicate).

²Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

Plasma total protein was elevated (P < 0.05) in a quadratic fashion with an increase of 200 mg of supplemental protease then a further increased at 300 mg (Table 4). Similarly, plasma albumin levels were increased quadratically by protease supplementation at all levels. Plasma GOT was declined (P < 0.05) at 300 mg/kg of protease, while plasma GPT was declined at 200 and 300 mg protease /kg. Plasma creatinine concentrations were decreased linearly (P < 0.05) as protease supplementation increased from 100 to 300 mg/kg. Plasma cholesterol and LDL-cholesterol concentrations were decreased in a quadratic manner with increasing protease supplementation. However, plasma HDL- cholesterol concentration was increased linearly (P < 0.05) with increasing protease supplementation. In addition, plasma triglycerides and glucose concentrations were decreased at only 200 or 300 mg of protease and their response to the enzyme was linear.

Table 4. Effect of dietary serine protease supplementation on blood biochemical indices^1,2.

		Protease (mg/kg)				P-value
Items	0	100	200	300	SEM	Protease	Linear	Quadratic
Protein metabolism
Total protein, mg/dl	3.01^b	3.14^b	3.42^a	3.92^a	0.088	0.0001	0.0001	0.005
Albumin, mg/dl	1.69^b	1.82^a	1.89^a	1.88^a	0.045	0.0001	0.0001	0.03
GOT, IU/I	177.50^a	179.67^a	166.33^a	154.83^b	5.23	0.000	0.000	0.07
GPT, IU/I	38.33^a	43.67^a	31.33^b	29.00^b	2.18	0.0001	0.0001	0.01
Creatinine, mg/dl	0.25^a	0.21^b	0.18^b	0.17^b	0.01	0.0001	0.0001	0.12
Lipid metabolism
CHO, mg/dl	152.50^a	137.83^b	133.33^b	136.33^b	3.38	0.0001	0.0001	0.001
TG, mg/dl	27.17^a	26.67^a	21.50^b	20.33^b	1.93	0.001	0.0001	0.80
HDL, mg/dl	68.17^b	73.83^a	70.33^a	75.17^a	2.07	0.006	0.01	0.77
LDL, mg/dl	79.17^a	62.17^b	61.33^b	59.83^b	4.24	0.0001	0.0001	0.01
GLU, mg/dl	193.83^a	188.08^a	181.17^b	182.17^b	3.06	0.0001	0.0001	0.12

¹n = 12 birds / treatment (4 birds/ replicate).

²Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t test. GOT: glutamic oxaloacetic transaminase; GPT: glutamic pyruvic transaminase; CHO: cholesterol; TG: triglycerides; HDL: high density lipoprotein; LDL: low density lipoprotein; GLU: glucose.

Hot carcass and breast weights were increased (P < 0.05) when protease was included at any level compared to the control group (Table 5). Whereas, thigh muscle weight was increased only at 200 and 300 mg of protease. A significant reduction in abdominal fat was observed due to the addition of 300 mg protease. Regarding the internal organ weights, liver weight was increased by supplementation of 200 and 300 mg protease, while heart and gizzard were not significantly affected by protease supplementation.

Table 5. Effect of dietary serine protease supplementation on carcass, cuts, internal organ and abdominal fat (g/100g BW)^1,2.

			Protease (mg/kg)				P-value
Items	0	100	200	300	SEM	Protease	Linear	Quadratic
Carcass and Cuts
Hot carcass weight	69.08^b	71.72^a	73.71^a	74.74^a	0.50	0.0001	0.0001	0.02
Breast muscle	20.12^b	20.72^a	22.42^a	23.32^a	0.23	0.0001	0.0001	0.36
Thigh muscle	15.12^b	15.21^b	15.94^a	16.05^a	0.19	0.0001	0.0001	0.97
Abdominal fat	0.59^a	0.63^a	0.54^a	0.44^b	0.03	0.0001	0.0001	0.001
Organ weight
Heart	0.50	0.51	0.52	0.51	0.01	0.74	0.49	0.45
Gizzard	1.78	1.78	1.89	1.79	0.06	0.27	0.55	0.29
Liver	2.12^b	2.20^b	2.45^a	2.87^a	0.08	0.0001	0.0001	0.007

¹n = 12 birds / treatment (4 birds/ replicate).

²Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

It is noteworthy to mention that, liver MDA concentrations were declined (P < 0.05) due to dietary supplementation of 200mg and 300 mg protease /kg (Figure 1).

Figure 1. Effect of serine protease on the levels of MDA in the liver. Values are means ± standard SEM. Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

Interestingly, dietary supplementation of 100, 200, and 300 mg/kg protease increased (P < 0.05) lysine, methionine, and threonine levels in breast muscle compared with the control group (Figures 2–4).

Figure 2. Effect of serine protease on lysine content in the breast muscle. The values (means ± standard SEM) were expressed on a gram of amino acid per 100 grams of breast meat. Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

Figure 3. Effect of serine protease on methionine conten in the breast muscle. The values (means ± standard SEM) were expressed on a gram of amino acid per 100 grams of breast meat. Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

Figure 4. Effect of serine protease on threonine content in the breast muscle. The values (means ± standard SEM) were expressed on a gram of amino acid per 100 grams of breast meat. Means not sharing a common superscript letter with the control differ (P < 0.05) as indicated by Dunnett's t Test.

4. Discussion

The protease used in the present study was a serine protease (EC 3.4.21.-) produced by the bacterial species Bacillus licheniformis and its activity is 75,000 protease/g and is stable in poultry feed manufacturing in Egypt. Supplemental protease in diets has been applied as a zootechnical feed additive to enhance digestibility and feed utilization. Results of the present study indicated that protease supplementation improved growth and FCR. These results are in coincidence with previous studies illustrated the positive effects of exogenous proteases added to poultry diets on growth performance (Cowieson and Ravindran 2008; Saleh et al. 2018). In the present study, the digestibility of dry matter and crude protein were significantly increased with protease supplementation. Therefore, the improvement of the growth performance in the present study might be clarified in the light of enhancements in digestibility of crude protein and dry matter. Previous studies found that protease supplementation improved true nitrogen digestibility (Ghazi et al. 2003; Bertichini et al. 2009; Sorbara 2009; Saleh et al. 2014, 2018). In addition, the improvement of the performance might be associated with blood biochemical parameters in the present study. In the present study, plasma total protein was elevated at 200 and 300 mg of supplemental protease, while albumin level in the plasma was elevated by supplement graded levels of protease 100, 200, and 300 mg/kg. It has been showed that the concentration of plasma albumin related to improving the immunity status in birds (Laborde et al. 1995; Ding et al. 2016; Saleh 2017; Ndazigaruye et al. 2019). Our results are in harmony with Allouche et al. (2015) who found that plasma protein concentrations were enhanced by dietary protease supplementations. Plasma GOT was decreased by the addition of 300 mg protease/kg, while plasma GPT was declined at levels of 200 and 300 mg protease/kg. Elevation in plasma GOT activity indicates hepatic injury. More GOT and GPT are released into the bloodstream when the liver or heart cells are impaired (Saleh 2014; Choi et al. 2018). It has been reported that the alteration of GPT might be attributed to the synthesized fat level (Dongare et al. 2013; Saleh 2016). Results presented in Table 3 showed that plasma total cholesterol concentration was decreased in connection with protease supplementation. In addition, the impaired liver induces lipid peroxidation, which causes hepatotoxicity (Wang et al. 2011). As graphically presented in Figure 1, the levels of liver MDA were declined in 200 and 300 mg of protease/kg diet treatments. These findings might be attributed to the reduction of plasma triglycerides, total cholesterol and LDL- cholesterol (Wang et al. 2011; Saleh et al. 2018) as illustrated in Table 3.

Plasma creatinine concentration as an indicator of kidney functions was significantly decreased due to dietary protease supplementation. These results are in accordance with Dongare et al. (2013) who reported that blood creatinine level was decreased by adding protease. The elevating creatinine level in the blood is correlated with the breakdown of creatine and creatine phosphate in the muscles. Current findings may point out overall better amino acid utilization.

Increasing the supplemental protease levels were associated with decreasing the plasma levels of CHO, GLU, TG, and LDL-cholesterol and elevated HDL-cholesterol level in the current study. However, in another recent study (Ndazigaruye et al. 2019) it has been reported that serum total protein, albumin, TG, CHO, creatinine, HDL-cholesterol, GOT, and GPT levels did not change in broiler fed diets with protease. The disagreement between our data and their data may be due to some factors such as the nutrient composition of the diets (sufficient/insufficient protein), enzyme used (source of enzymes, doses), and raw protein sources used which could alter some conditions of the digestive tract such as the pH.

Hot carcass, cuts (breast and thigh muscles), and liver were increased with protease supplementation, while abdominal fat was decreased. It has been reported that enzymes improved amino acid utilization and decreased the quantity of nutrients excreted in faeces (Huo et al. 1993; Hajati et al. 2009; Tactacan et al. 2016). Proteases may cleave anti-nutrients such as trypsin inhibitors resulting in improve utilization and bio-availability of amino acids (Marsman et al. 1997; Hajati et al. 2009). In addition, it has been found that amino acids digestibility increased with protease inclusions by 5.4% for lysine, 7.8% for threonine, and 6.5% for methionine (Angel et al. 2011). Therefore, protease supplementation could increase the concentration of free amino acids in breast muscle. It has been reported that the levels of total amino acids in serum, liver, feather, and carcass can reflect the amino acids utilization (Kaczmarek et al. 2014; Azzam et al. 2015, 2017; Dong et al. 2016; Morales et al. 2017; Wecke et al. 2018; Middendorf et al. 2019). In addition, amino acids concentrations in carcass could be used to determine amino acid requirements accurately in broiler diets (Stilborn et al. 2010; Abd El-Moneim et al. 2019; Saleh et al. 2019b).

5. Conclusion

Based in the data presented above, it could be concluded that exogenous serine-protease (Ronozyme® ProAct) supplementation at levels 200∼300 mg/kg might be involved in enhancing protein utilization and resulted in positive effects on growth performance, nutrients utilization, lipid peroxidation and modified plasma lipids profile in broiler chickens.

Author contributions

A.A.S., M.M.D., M.M.A and K.A.A.; methodology, A.A.S., M.M.D and M.M.A; software, A.A.S. and M.M.A.; validation, A. A. S., M.M.A. and N.A.B.; formal analysis, A.A.S., M.M.A., T.A.E.; investigation, A.A.S.; resources, A.A.S. and M.M.A.; data curation, A.A.S., M.M.D. and T.A.E.; writing – original draft preparation, A.A.S., T.A.E. and M.M.A.; writing – review and editing, A.A.S., M.M.A. and T.A.E.; visualization, A.A.S.; supervision, A.A.S. and K.A.A and N.A.B.

Acknowledgement

The authors would like to extend their sincere appreciation to the staff members of the Poultry Production Department, Faculty of Agriculture, Kafrelsheikh University, Egypt; Also, The authors would like to thank Dr. Rashed Alhotan (King Saud University) for data analysis assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

ORCID

Ahmed A. Saleh http://orcid.org/0000-0002-2052-5248