Impact of an indigenously produced multi-enzyme complex from Bacillus subtilis KT004404 on growth and blood parameters in broiler chicken

A 42-days experiment was conducted on a day old birds (n = 400) to evaluate the effect of enzyme supplements in feed on the growth, blood parameters, phosphorous content in bones, and nitrogen retention. Different treatments included: control (C) without enzyme supplement, while the other three groups included enzyme mixture T1 and T2 with two commercially available enzyme mix, and T3 with indigenously produced multi-enzyme complex from Bacillus subtilis KT004404. Birds that were fed with indigenously produced multi-enzyme complex showed significant weight gain as compared to other groups. The total feed intake of the birds fed with enzyme supplements was higher than the birds in the control group. The feed conversion ratio was significantly improved (p < 0.05) in treatment groups (T1, T2, T3) as compared to the control. The blood parameters which were analyzed included uric acid, triglycerides, total cholesterol, and serum proteins i.e. globulin and albumin. Birds fed with the enzyme in the group T1, T2 and T3 exhibited higher (p < 0.05) body weight gain. Tibia ash content was significantly higher (p < 0.05) in T1, T2, and T3 as compared to the control. The results of the current study indicate that supplementing poultry feed with the exogenous multi-enzyme produced from Bacillus subtilis KT004404 improved the growth of the birds, feed utilization, and exhibited beneficial effects on the blood parameters, phosphorous and nitrogen retention in broiler chicken.


Introduction
Food security is one of the most recurring challenge in most parts of the world due to which intensive production of meat, eggs, and milk is a necessity, however, this can only be achieved if the farm animals properly consume or digest the nutritious feed [1]. The high cost of conventional feed ingredients such as soybean meal and corn has posed major challenges for the poultry industry in the developing world [2]. Almost 75% of the total cost of animal production is comprised of feed and increasing costs have caused farmers and researchers to look into other less costly unconventional options [3]. Therefore, waste of fruits, vegetables, and other The birds were nearly equal in body weight and distributed randomly into 4 treatment groups of 100 birds each in 5 replicates (20 birds each replicate). Control group (C) was fed a commercial diet without an enzyme supplement. Other groups received the diets with enzyme supplements of 1000 g on ton diet and termed as T1, T2, T3 treatment group. The enzyme supplements mixed with the diet in T1 and T2 included commercially available enzyme mixture from WOOJIN, Co. Ltd (Phytezyme) and EWHA, Co.(Biomax) respectively. Phytezyme experimental diet contained xylanase (280 U/mL), phytase (130 U/mL), amylase (168 U/mL) and protease (102 U/mL) while Biomax contained phytase (57 U/mL), xylanase (322 U/mL), amylase (89 U/mL) and protease (258 U/mL).
Ltd enzymes preparation respectively while T3 feed was supplemented with an immobilized enzyme complex (IE) produced from indigenous Bacillus subtilis KT004404 and control group (C) contained no extracellular enzyme supplement. The study was terminated on the 42nd day. The mortality rate of the birds was recorded regularly during the experimental period. Mash Diet provided to the birds contained similar caloric value and same protein level across all the groups.

Feed compositional analysis
The chemical composition of the feed was determined through proximate analysis as shown in (Table 1) and ingredients were analyzed according to the proposed methods of (AOAC 2000). Dry Matter was determined by placing the samples in a hot air oven at 650˚C for 12 hours (method 930.15). Micro-Kjeldahl method was used for crude protein analysis and the ash

Enzyme and treatments
The enzyme activities in the enzyme mixture was determined by using xylan, sodium phytate starch and casein as substrate for xylanase, phytase, amylase and protease assays respectively [15][16][17] One unit of enzyme activity (xylanase, phytase, amylase, protease) was defined as the amount of enzyme which can liberate 1 μmol of product (xylose, inorganic phosphate, reducing sugar and tyrosine) from catalyzed substrate under assay conditions in one minute. The extracellular enzyme supernatant obtained from the Bacillus subtilis KT004404 contained xylanase (113 U/mL), phytase (132 U/mL) and amylase (110 U/mL) and protease (137 U/mL). The thermal stability of adsorbed and free enzymes was studied by carrying out the reaction in the range of incubating temperature. The results showed that the optimal temperature for immobilized enzymes and few enzymes is between 35-40˚C. The stability of the enzyme was enhanced after adsorption as immobilized enzymes showed better relative activities 90% at higher temperature (50-80˚C). The free enzymes lost the activity after 7 hours under the heat treatment of 80˚C while immobilized enzyme on the medium retained 80 percent of their initial activities at the similar temperature. Thermal stability enhancement of the immobilized enzyme was due to the reason that the molecules are stabilized due to hydrogen bonding or hydrophobic interactions between the functional groups of an amino chain of xylanase, amylase and phytase and the hydroxyl group on the adsorbed medium surface [18]. This improved thermal stability of immobilized xylanase benefits the application of adsorbed enzyme at hightemperature industrial and pelleting processes during feed preparation [19]. T1 experimental diet contained xylanase (280 U/mL), phytase (130 U/mL), amylase (168 U/mL) and protease (102 U/mL) while T2 contained phytase (57 U/mL), xylanase (322 U/mL), amylase (89 U/mL) and protease (258 U/mL).

Sample collection and analysis of growth performance parameters in broiler
Different parameters such as body weight gain [BWG (grams on period) = BW (g) at the end period-BW (g) in first day], feed intake (FI), and feed conversion ratio [FCR (g feed/g gain) = cumulative feed intake (g)/total weight gain (g)] were recorded for 42 days. At the beginning of the experiment, birds were weighed individually. Mean initial body weights of day-old chicks were almost similar (41.2 ±0.6 g per bird) among all treatment groups. Later in the experiment, at 6 day intervals, randomly selected 5 birds from each treatment group were weighed using a precision weighing balance (Mettler Toledo, USA) for 42 days. On day 35 th , 3 birds from each group were weighed and the blood sample was collected in non-heparinized tubes. These samples were centrifuged at 3500 rpm for 15 minutes and serum was collected and stored at -20˚C. Serum total protein was determined by the biuret method (Cannon), albumin, uric acid, triglycerides, and cholesterol concentrations were determined by commercially available kits (Labtest Diagnostica, Brazil). Globulin levels were obtained by the difference between serum total protein concentration and serum albumin.

Analysis of enzyme in intestinal homogenate and pancreas
Three birds from each replicate per treatment were selected for current studies. International standards to conduct the animal research were followed to avoid the pain and suffering of the birds. Bird's pancreas and intestinal segments were collected at 4˚C in Tris-HCL buffer (pH 8).
Tissues were homogenized using Dounce homogenizer (Bio Vision, USA), centrifuged at 6000 rpm for 20 minutes at 4˚C and the supernatant was stored at -80˚C for analysis of enzymes activities i.e. trypsin, amylases, alkaline phosphatase, and lipase. Amylase activity was determined by following an already reported method [20] while trypsin was measured following the method by [21]. Alkaline phosphatase activity was determined by the method proposed by [22] and lipase activity was determined following) [23] method. Enzymatic units were defined as the ability of the enzyme to hydrolyze one micromole substrate in one minute at 25˚C.

Analysis of phosphorous and nitrogen content in excreta
After the 35 th day, three birds per replicate were randomly selected for total excreta collection for five consecutive days to determine the content of excreted phosphorous and nitrogen. After collection, the excreta were processed according to the method proposed by [24] and frozen at -20˚C. Phosphorous content was determined by the vanadate-molybdate calorimetric method [25] while nitrogen content was calculated using the Kjeldahl method [26].

Bone mineral content analysis
At the end of the experiment, 3 birds per treatment were selected for collection of the right tibia for bone mineral content quantification. The tibia of the birds was autoclaved for 30 minutes at 121˚C and adhered material was cleaned. Endcaps of the tibia were removed, and each tibia was cut into two pieces [27]. Fat extraction from the tibia was done using ice-cold ethyl ether. The tibia was then dried for 24 hours at 100˚C and then heated at 650˚C for 24 hours. Ashed samples were dissolved in 25% hydrochloric acid by following the method 968.08 (AOAC 1998.) and then total phosphorous content was calculated using the calorimetric method by the molybdate-vendate method [25].

Statistical analysis
Statistical analysis of data was done using IBM SPSS Statistics 11.0 software. One-way analysis of variance (ANOVA) univariate and repeated measure models indicated the effect of enzymes supplement on broiler performance, blood parameters, phosphorus content in bone, and nitrogen retention. Group comparisons were made by using Tukey post hoc test. Probabilities (p) values less than 0.05 (p<0.05) were considered significant.

Broiler growth performance
The results of the current study revealed that the bodyweight of birds in the experimental group (T3) increased by +2.3% in the birds from 31 to 36 days and +2.8% from 37 to 42 days as compared to groups T1and T2. From 31 to 36 days the average weight gain per bird in T3 group was 486.78 g per bird while from 37 to 42 days the average weight gain was observed to be 488.1 g per bird. This shows that our supplementary enzyme mixture in the feed has a significant impact on increasing the weight in the birds. The effects of various enzyme treatments on body weight between 37 to 42 days in different treatment groups is represented in Fig 1. The results show that in the birds aged 13 to 18 days, experimental group (T3) feed intake significantly (p<0.05) decreased as compared to control (-11.9%), T1 (-12.26%), and T2 groups (-12.9%). From 19 to 24 days, feed intake in the birds in treatment groups was observed to be significantly reduced by -18% in T1, -8.6% in T2 and -4.4% in T3 as compared to the control. From 25 to 30 days, T1, T2 and T3 groups' feed intake was significantly higher for T1 (+9.3%), T2 (+10.15%) and T3 (+11.4%) than the control group. However, the feed intake of 31 to 36 days old birds in T1, T2 groups was observed to be significantly reduced as compared to the control group. From 37 to 42 day, birds in the treatment groups showed a significant increase in feed intake (+2.8% for T1, +3.8% for T2, and +3.1% for T3) as compared to the control group (Fig 2).
When the bird's FCR was analyzed during the study, results revealed that enzyme treatments have diverse impacts on FCR in the birds depending on the age of the birds. The post hoc test showed a significant (p<0.01) decline in FCR (-23%) of the T3 group as compared to the T1 group from 13 to 18 days, but increase in FCR (+ 20%) in 19 to 24 days old birds as compared to the same group. Whereas, the FCR of T1 from 19 to 24 days was significantly (p<0.05) lower than the control group (Fig 3).
When the serum uric acid concentration was analyzed, it was found that that enzymes' treatment has significantly decreased uric acid concentration in the treatment group T1, T2, T3 as compared to control group (Fig 4). Enzyme's treatment exhibited no significant effect on triglycerides and total cholesterol levels as shown in Fig 5. When the impacts of mixture of enzyme supplementation on serum protein, globulin and albumin were analyzed it was observed that serum protein (p<0.01) and globulin levels were effected in the treatment groups however, no significant effect was observed on albumin levels. Post-hoc testing for groups' comparisons and differences revealed that the protein level of T1, T2, and T3 groups were significantly (p<0.01) higher (+ 20% for T1, + 18% for T2 and +23% for T3) than the control group. Globulins level in the treatment groups T1, T2, and T3 were observed to be significantly increased as compared to the control group (Fig 6)

Enzyme activities in the intestinal homogenate and pancreas
During current studies the impact of enzyme supplements on the protease, α-amylase, trypsin, and lipase activity in the pancreatic and intestinal homogenate was determined. The activity of pancreatic protease in T3 increased (+50%) as compared to control group while trypsin activity declined by -24% for T1, -18% for T2 and -19% for T3 as compared to the control group (Table 2). Similarly, in the intestinal homogenate, the enzyme supplement significantly increased the protease, α-amylase, trypsin, and lipase activity are significantly higher (p<0.05) in the treatment groups as compared to the control group as shown in Table 2.

Phosphorous and nitrogen content in excreta
The effects of various enzymes' treatment on dry matter intake (DMI), dry matter excreted (DME), nitrogen intake (NI), nitrogen in excreta (NE), phosphorous intake (PI) and phosphorous excreted (PE) is shown in Table 3. Results of the current studies showed that dry matter intake of T1 (609.093 ± 29 g), T2 (611.40 ±36 g) and T3 (621.02 ±19 g) groups were significantly (p<0.05) lower as compared to the control group (C). No significant difference within the treatment groups was observed. The data for DME showed that the treatment groups had a lower level of dry matter in excreta (for T1, T2, T3) as compared to the control. Similarly, the decline in the excreted nitrogen level was only observed in T2 as compared to the control group while phosphorous in excreta was found to be reduced in T1, T2 and T3 by -24%, -21.1% and -11.6% as compared to control. However, enzyme supplements showed no

Fig 2. Effects of enzymes' treatment on feed intake in birds.
Values represent mean ± (SD). Significant differences by Tukey's test: � p<0.01 from control group; ++p<0.01 and +p<0.05 from treatment T1 group and #p<0.01 from T2 group.

Bone mineral content
Tibia ash values and phosphorus content in bones for the birds fed with exogenous enzyme supplements in treatment group T1, T2, and T3 were found to be significantly higher (p < 0.05) as compared to control. Exogenous enzyme supplementation was found to have no effect on the length or width of the bones among the treated and control groups as shown in Table 4.

Discussion
The study was performed to evaluate the efficacy of indigenously produced multi-enzyme by Bacillus subtilis KT004404 in comparison with the multi-enzyme complexes available in the market. Bodyweight gain (BWG) was not significantly increased up to 30 days as enzyme supplements have no significant impact on the broiler body weight from 0-30 days. However, enzyme supplements containing phytase, xylanase, protease, and amylase were observed to increase the body weight in T3 treatment from 31-42 days. As compared to a commercially available enzymes, the body weight of the birds in the treatment group (T3) significantly increased between 37 to 42 days. Similar results were reported previously in which the food conversion ratio increased due to the addition of exogenous enzymes (cellulase, amylase, and protease) in broiler diet [28]. Endogenous enzyme production is also improved by the addition of exogenous enzymes in diets which increases the nutrients utilization in the feed. Arabinoxylans and phytate are anti-nutritional factors hence, the inclusion of enzyme supplements containing xylanase and phytase improve the broiler performance [29,30]. An increase in body weight by the addition of enzymes is also in agreement with numerous previously conducted studies [31,32]. Increase in FCR after the addition of multiple enzymes in the broiler feed indicates improved nutrient utilization [33]. These studies concluded that exogenous enzymes improves the nutrient utilization which is responsible for the increase in the body weight in the groups fed with enzyme supplemented feed as compared to the group fed with a similar level of the nutrient without enzyme supplement [34]. However, the findings are in contradiction with some previous which didn't report any [35] significant effect of enzyme supplement on the growth performance of broilers. Similarly, no significant impact was observed on the body weight gain and FCR due to the inclusion of exogenous enzymes in diets from 1 to 35 days [36].
In the current studies, broiler feed intake decreased (p < 0.05) with enzyme supplements treatment as compared to the control group (C) from 13 to 24 days. However, in 37 to 42 days old birds the feedd intake (FI) increased significantly in treatments (T1, T2, T3) as compared to the control. The increase in body weight of 37 to 42 days old birds is also an indication of higher feed intake. These results are in agreement with previously conducted studies [2,31]. Feed intake decreases in the birds fed with exogenous enzymes because the nutrient requirement of birds is fulfilled due to enhanced digestibility in birds [37]. Some earlier studies have reported indigenously produced multi-enzyme to be as effective as a commercially available enzyme, improving the nutrient digestibility, safer to be utilized as a poultry feed supplement and poses no adverse effects on the vital organs in broilers [33][34][35][36][37][38][39][40][41].
The effect of enzyme supplements on the blood parameters was determined by analyzing uric acid as an indicator of renal function. It was found that uric acid in the serum obtained from treatment T 1, T2, and T3 birds were lower than the group without enzyme supplementation. It suggested that exogenous enzyme preparations increase protein anabolism. In birds, purines are formed from excessive amino nitrogen [42]. The purine bases are degraded into uric acid and excreted out of the body. Increased protein anabolism results in a decline in excessive amino acid hence, lower level of uric acid in the serum. Enzyme inclusion in the feed does not affect serum triglycerides and total cholesterol level [43,44].
An increase in blood cholesterol and triglycerides by the addition of multi-enzyme in the feed has been reported [30]. Similar results have been reported in numerous previous studies [45,46] in which increase in blood cholesterol, plasma total protein, and globulin increased by the addition of NSP enzymes in broiler feed. The increase in blood lipid profile after the addition of mixture of enzyme may have association with the improved bile salt functioning and emulsification properties of the chyme that enhances the total cholesterol in the blood [47]. Current studies found higher serum globulins levels in the birds from T1, T2, T3 as compared to the control group which contradicts some earlier reports where no significant effect on the blood serum protein concentration was observed [48][49][50]. Enhanced concentration of globulin in the serum indicated the enhanced immune response in the birds [51]. The current study found no significant effect on the pancreatic lipase and amylase activity in the groups fed with exogenous enzyme (T1, T2, T3) and the groups without any enzyme supplement (C). However, trypsin activity declined in the treatment groups T1 and T2 while protease script was slightly higher in the group T3. Pancreatic juice contains multiple enzymes such a lipase, trypsin, and amylase are involved in digestive function. Hence, activity of pancreatic enzyme is an effective index to determine the digestive capacity in the birds [52]. The decline in pancreatic trypsin activity by exogenous enzyme supplement is in agreement with some earlier reports [53,54]. Enzyme supplements impacted intestinal enzyme activity. Current studies showed a decline in lipase activity in the treatment group (T3) fed with indigenous enzyme from Bacillus subtilis KT004404 but amylase, trypsin, and protease were found to be higher in the treatment groups (T1, T2, T3) fed with exogenous enzymes. These results are not in agreement with some studies which report decline in amylase and trypsin in the intestinal content [55].
The qualitative and quantitative characteristics of the excreta of broiler showed that phosphate percentage excreted was lower in the groups fed with enzyme supplement in broiler diet as compared to control. This indicates high utilization of minerals and nutrients when the broiler feed was supplemented with exogenous enzyme. Phytase containing exogenous enzymes when added to the poultry diet also reduced in excreted phosphorous [2,[56][57][58]. The current studies found that Tibia ash percentage in groups fed with exogenous enzyme (T1, T2, T3) was higher than control which indicates improvement in bone mineralization and higher phosphorous deposition in the bone [59]. Percentage of tibia ash was increased when the feed was supplemented with phytase enzyme [58,60]. The increase in tibia ash and phosphorous content is attributed to higher availability of calcium from the ingredients and organic phosphorous which results in a higher phosphorous deposition in the bones.

Conclusion
The results of the current experiment demonstrate the efficacy of indigenously produced enzymes from Bacillus subtilis KT004404 to be used as feed supplement. The impact of enzyme on the performance of boiler and blood parameters suggests that the indigenous enzyme supplement is effective and safe to be utilized in the poultry feed and has equal efficacy as the commercially available mixture of enzyme supplements. The results provide a significant knowledge base for conducting future studies on the impact of multi-enzyme supplements on the intestinal morphology and microbiome of poultry.