Effects of dietary supplementation of Trichoderma pseudokoningii fermented enzyme powder on growth performance, intestinal morphology, microflora and serum antioxidantive status in broiler chickens

Abstract

This study was conducted to evaluate the dietary supplementation of Trichoderma pseudokoningii enzyme powder (EP) and its effect on the growth performance, intestinal morphology and antioxidative status of broilers. In vitro experiments showed that xylanase and cellulase activities of EP were 487.96 ± 14.14 U/g and 18.55 ± 0.54 U/g, respectively. Total phenolic and ferulic acid content was 4.79 ± 0.35 mg GAE/g DW and 4.38 ± 0.32 μM/g DW, respectively. A total of 240 d-old Ross 308 male broiler chicks were randomly subjected to one of four treatments, namely diets supplemented with dry EP at 0% (control), 0.1%, 0.2% or 0.4% for 35d. Results showed that in the starter phase (1–21 d), the EP supplemented groups had improved body weight, weight gain and FCR compared to the control group. The villus:crypt ratio of both jejunum and ileum in all EP-treated groups was significantly higher than that of the control group. When testing serum antioxidant activity, results showed that 0.2% and 0.4% EP supplementation significantly increased superoxide dismutase activity in both the finisher phase and the overall experimental period. In addition, catalase activity was elevated in all EP supplemented groups in both the finisher phase and throughout the experimental period. 0.1% EP seemed to have no effect during the starter period. Serum malondialdehyde concentration was not affected by EP supplementation. These results suggest that EP is potential feed additive in terms of improving broiler performance and enhancing antioxidative status, while also providing an optimal intestinal environment.

Keywords:

• Trichoderma
• antioxidant
• intestinal morphology
• broilers

Introduction

Non-starch polysaccharides (NSPs) were general component in plant-derived feedstuffs. The major concern of NSPs is their resistance to the monogastric digestive enzymes, causing anti-nutritional effects and thus contributing to restricted utilisation of nutrition in animals. Therefore, NSP-degrading enzymes (NSPase) derived from microorganisms play an important role in the animal feed industry. They have been supplemented exogenously to animals to prevent adverse effects due to NSP-rich feedstuffs, such as a wheat-based diet. However, researchers recently found that the addition of NSPase to the corn-soybean based diet of broilers improved nutrient availability (Cowieson and Ravindran 2008), since broken down cell wall materials enhanced the access of endogenous enzymes and released nutritive contents. Xylanase has been reported to improve lactobacilli and bifidobacteria counts in the caecum of broilers fed a corn-soybean based diet (Nian et al. 2011). Passos et al. (2015) also proved that supplementation with xylanase improved nutrient digestion in pigs fed a corn-soybean based diet.

Filamentous fungi have been found to exert antioxidative properties by producing phenolic compounds during fermentation, suggesting they could be a promising source of natural antioxidants (Sugiharto et al. 2015). Exogenous antioxidants such as vitamin C or selenium powder were provided to synchronise the redox balance and endogenous antioxidants were replenished to avert negative factors caused by oxidative stress (Noori 2012), further preventing economic losses (Lara and Rostagno 2013).

Trichoderma spp. produce cell wall-degrading enzymes (xylanase and cellulase) that have been applied to soil borne pathogen-inhibiting biocontrol agents and industrial enzyme production (Harman et al. 2004). Goyal et al. (2008) produced xylanase with T. viride from xylan-rich lignocellulosic waste via submerged culture. Cacais et al. (2001) also produced xylan-degrading enzymes by submerged culture of T. harzianum. With regard to antioxidant production, Singh et al. (2010) discovered the ability of T. harzianum to modulate natural antioxidants in a soybean seed matrix by increasing the release of polyphenol. Due to this particularity, Trichoderma species are suitable for producing antioxidants and NSP enzymes. However, there are still limited studies utilising Trichoderma spp. in situ enzyme production on farm animals.

Our previous results showed that substituting 10% of a basal diet with Trichoderma pseudokoningii fermented wheat bran improved the growth performance and intestinal morphology of broilers (Chu et al. 2016), indicating the potential of T. pseudokoningii to mitigate high feed costs. However, before this fungal strain could be used as an exogenous enzyme-providing feed additive, the variety of substrate qualities and the cumbersome fermentation process would need to be further investigated, in order to confirm its consistent quality, convenience and efficiency.

This paper established the method of producing enzyme powder (EP) with antioxidant properties by T. pseudokoningii in situ enzyme production using solid-state fermentation (SSF). Furthermore, after dietary supplementation with EP, the growth performance and serum antioxidative parameters of broilers were tested in order to evaluate its potential as a functional feed additive.

Materials and methods

Micro-organisms

The T. pseudokonigii used for this study were kindly provided by the Department of Biotechnology, National Formosa University, Yunlin, Taiwan, and were capable of fermenting lignocellulose-rich materials. The micro-organisms were routinely maintained on a potato dextrose agar (PDA) plate at 25 °C with regular sub-cultivation (no longer than 1 week). An appropriate amount of sterilised 20% molasses solution was added to the plate before gently shaking it by hand to retrieve the spore suspension. The concentration of T. pseudokoningii spores in suspension was diluted to 1 * 10⁷–5 * 10⁷ spores per mL for subsequent usage as inoculum to prepare enzyme powder (EP). Wheat bran purchased from the Formosa Oilseed Processing Corporation, Taichung, Taiwan, was chosen as the substrate for SSF.

SSF

The techniques for large-scale production of EP are similar to SSF in the lab. About 50 g of wheat bran were weighed into a plastic bag and the moisture content was adjusted to 50% with Toyama’s solution. The contents of each bag were thoroughly mixed before being autoclaved at 121 °C ± 1 °C for 30 min. The autoclaved wheat bran was inoculated with 0.2 mL of T. pseudokoningii spore suspension per gram of wheat bran. The wheat bran was fermented aerobically under environmentally controlled conditions and maintained at 25 °C for 7 d. All samples were dried at 40 °C for 2 d then ground in a mill before being packed in plastic bags, sealed and stored at ambient temperature.

Enzyme extraction and assays

The 3 g of EP was weighted and diluted 10-fold by the buffer and stirred for 30 min at 4 °C to extract the extracellular enzyme solution. The solution was than centrifuged at 3000 rpm at 4 °C for 10 min and filtered through filter paper (Advantec No. 1, Tokyo, Japan), before detecting cellulase and xylanase activities. Both enzymes were assayed using the dinitrosalicylic acid (DNS) method (Miller 1959) to determine the reducing sugar content. Briefly, 0.1 mL of diluted enzyme solution was added to 0.9 mL of substrate and vortexed. Subsequently, the mixture was left to react for 10 min in a water bath. The reaction was stopped by adding 1 mL DNS and boiled a further 5 min. The colours that developed were measured spectrophotometrically at 540 nm. One international unit (IU) of cellulase and xylanase activity was defined as the quantity of enzyme required to liberate 1 μm of reducing sugar (glucose/xylose) of crude filtrate per minute under standard assay conditions (50 °C, pH 4.8 for cellulase and 55 °C, pH 5.3 for xylanase).

Bioactive compound extraction and determination

Air-dried EP powder was weighed and added to distilled water, which was then left to stand at 95 °C for 1 h. The solution was filtered (Advantec No. 1, Tokyo, Japan) to obtain the EP extract (EPE), which was stored at −20 °C for further analysis. The total phenolic contents were determined using a Folin–Ciocalteu reagent according to the method described by Kujala et al. (2000). The Folin–Ciocalteu reagent was mixed evenly with EPE before adding the sodium carbonate by using the equation obtained from the standard gallic acid graph, the phenolic compounds of the EPE (microgram of the gallic acid equivalent, mg GAE), was then determined.

Ferrous chelating capacity assay

The ferrous ion chelating activity of the EPE was determined as per the methods of Dinis et al. (1994). Briefly, 0.25 mL EPE was mixed with 0.025 mL 2 mM ferrous chloride solution and 0.925 mL methanol. After 30 min incubation at room temperature (RT), 0.05 mL 5 mM ferrozine was added to initiate the reaction and the mixture was then left to stand at RT for 10 min. When the reaction was complete, the absorbance of the mixture was measured spectrophotometrically at 562 nm. The inhibiting percentage of ferrozine–Fe²⁺ complex formation was calculated according to the following formula: where A0 is the absorbance of the control and A1 is the absorbance of the EPE and EDTA. The control contained ferrous chloride, ferrozine and complex formation molecules.

Determination of reducing power

The reducing power of the EPE was measured using the methods described by Oyaizu (1986). Briefly, the EPE was mixed with phosphate buffer (0.2 M, pH 6.6) and potassium ferricyanide (1%). The mixture was incubated at 50 °C for 20 min. before the reaction was terminated by adding 2.5 mL 10% trichloroacetic acid. About 5 mL of the solution was then mixed with the same volume of distilled water and 0.1% ferric trichloride. After 10 min of incubation, the absorbance was detected at 700 nm by spectrophotometer; ascorbic acid was used as a control. The increase in the reaction mixture absorbance indicated greater reducing power.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging capacity assay

The free radical scavenging ability of the EPE was investigated according to the methods of Blois (1958). Briefly, DPPH ethanol solution (0.1 mM) was prepared and added to the EPE at different concentrations. After 30 min incubation, the absorbance was measured at 517 nm against ascorbic acid. The lower absorbance of the reaction mixture indicated higher scavenging capacity. The scavenging effect was calculated according to the following equation: where A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of EPE.

Experimental birds and housing

The animal experiment was approved by the Animal Care and Use Committee of the National Chung Hsing University, Taiwan. A total of 240 1-d-old commercial Ross 308 male broiler chicks were randomly allocated to one of four treatments: 0% (control), 0.1%, 0.2% or 0.4% EP dietary supplementation, with four replicates and 15 birds per pen (a total of 60 birds/treatment). Room temperature was maintained at 34 ± 1 °C for the first 7 d, then gradually decreased to 26 °C ± 1 °C until the birds reached 21 d of age. A temperature of 26 °C was maintained until the end of the 35 d. The experimental period included two phases: starter phase (1–21 d) and finisher phase (22–35 d). At the beginning of the feeding trial, the average body weight of the birds was similar for all pens (approximately 43.4 ± 0.17 g/bird). All chicks were raised in a temperature-controlled house. The birds were kept in floor pens (2.5 × 4.0 m) with wire floors and rice bran litter material. In the starter phase (1–21 d), two fountain drinkers and one feed tray were installed per pen. In the finisher phase (22–35 d), these were replaced with nipple drinkers and a feeder. Vaccines for Marek’s disease, Newcastle disease and infectious bronchitis were provided to chicks immediately after birth. Moreover, vaccines for Newcastle disease and infectious bursal disease were administered on day 14. Water and feed were supplied ad libitum to the broilers. The diets (Table 1) were formulated to meet or exceed the nutrient requirements of broilers (NRC 1994). The proximate composition was analysed according to the AOAC (1980). Starter and finisher diets were offered to the birds from 1 to 21 d and from 22 to 35 d of age, respectively. Neither anti-coccidial nor anti-bacterial supplements were added to the feed mixtures.

Table 1. Ingredients and chemical composition of the basal diets of broilers.

Ingredient	Starter diet, g/kg	Finisher diet, g/kg
Yellow corn	472.6	518.0
Soybean meal (CP 44.0%)	345.2	295.9
Full fat soybean meal	100.0	100.0
Soybean oil	35.0	45.0
Calcium carbonate	16.2	13.4
Monocalcium phosphate	18.6	16.6
DL-Methionine	2.0	1.3
L-Lysine-HCl	3.7	3.2
NaCl	3.9	3.8
Choline-Cl	0.8	0.8
Vitamin premix^a	1.0	1.0
Mineral premix^b	1.0	1.0
Total	1000	1000
Calculated nutrient value
ME, kcal/kg	3050.1	3175.1
Crude protein, %	23.0	21.0
Calcium, %	1.05	0.90
Total phosphorus, %	0.75	0.70
Available phosphorus, %	0.50	0.45
Lysine, %	1.43	1.25
Methionine + cystein, %	1.07	0.96
Analyzed nutrition value
Crude protein, %	23.8	21.6
Dry matter, %	88.0	87.1

a Supplied per kg of diet: Vit A 15000 U; Vit. D3 3000 U; Vit. E 30 mg; Vit. K₃ 4 mg; Riboflavin 8 mg; Pyridoxine 5 mg; Vit. B₁₂ 25 μg; Ca-pantothenate 19 mg; niacin 50 mg; folic acid 1.5 mg; Biotin 60 μg.

b Supplied per kg of diet: Co (CoCO₃) 0.255 mg; Cu (CuSO4·5H₂O) 10.8 mg; Fe (FeSO4·H₂O) 90 mg; Zn (ZnO) 68.4 mg; Mn (MnSO4·H₂O) 90 mg; Se (Na₂SeO₃) 0.18 mg.

Performance, serum and intestinal content collection

The body weight of each broiler was measured at 1, 21 and 35 d of age. Feed consumption was recorded at the end of starter phase (21 d) and finisher phase (35 d). Overall feed cost and meat profit of each group were counted at the termination of the experimental period. Body weight gains and feed conversion ratios (FCR) and income over feed cost (IOFC) were calculated from these data. At 35 d, four birds were randomly selected from each pen for sampling. The birds were bled via the brachial vein and 5 mL blood samples were collected from the wing veins. Samples were centrifuged at 2000 × g for 15 min and the serum was stored at −20 °C until lipid profile evaluation. Catalase (CAT) activity, superoxide dismutase (SOD) activity and serum malondialdehyde (MDA) levels were measured calorimetrically using a spectrophotometer. The procedures were conducted using an assay kit purchased from Cayman Chemical Co. (Ann Arbor, MI). Antioxidant enzyme activities were expressed as units (U) per millilitre of serum. After the blood samples were collected, the chickens were euthanized by exsanguination and their abdominal cavities were opened. For each treatment group, the livers, hearts, bursae, leg muscles and contents of the ilea and caeca of three birds were collected for study.

Microbial populations of intestinal content

Ileum and caecum digesta were collected and diluted serially in PBS solution for enumeration of microbial populations. Escherichia coli was cultured with chromogenic medium agar (CHROMagar^TM ECC) under aerobic condition at 37 °C for 24 h and lactic acid bacteria was cultured with Difco™ Lactobacilli MRS Agar under anaerobic condition at 37 °C for 48 h. The microflora numbers were calculated and the bacterial populations were expressed as log₁₀ CFU per gram of intestinal contents.

Intestinal morphology

On days 21 and 35 of the experiment, one bird per replicate cage from each treatment group (four birds/treatment in total) was randomly selected and sacrificed. The jejunum (from the pancreatic loop to Meckel’s diverticulum) and ileum (from Meckel’s diverticulum to the ileo-caeco-colic junction) from each bird were sectioned. Approximately 3 cm of the segments were fixed in 10% formalin for the morphometrical assays. The formalin-fixed tissue was washed in PBS and embedded in paraffin wax. Three micrometre-sectioned tissue was stained using haematoxylin and eosin methods. Samples were analysed by light microscopy and computer software (Motic image plus 2.0, Motic Inc., Wetzlar, Germany) was used to measure the villus height and crypt depth in fifteen favourably oriented and representative samples per treatment. The ratio of villus height to crypt depth was also calculated.

Determination of lactic acid concentration in the intestinal digesta

The concentrations of lactic acid in the ileal and caecal digesta were analysed using a high-performance liquid chromatography (HPLC) system (Hitachi, Tokyo, Japan) according to the methods of Grigsby et al. (1992).

Statistical analysis

Data were subjected to ANOVAs as a completely randomised design using the GLM function of the SAS software (SAS 2004). Determination of the significant statistical differences among the mean values of the four treatment groups (0%, 0.1%, 0.2% or 0.4% EP dietary supplementation) used Tukey’s honestly significant difference test, with a significance level of p < .05.

Results

Bioactive compounds and enzyme activities

The bioactive compounds and enzyme activities of wheat bran (WB) solid state fermented with T. pseudokoningii are summarised in Table 2. Total phenolic content of EP was 4.79 ± 0.35 mg QE/g DW, which increased approximately 1.6- to 1.7-fold after fermentation compared to the untreated WB. The xylanase and cellulase activities detected in EP were 487.96 ± 1 4.14 and 18.55 ± 0.54 U/g DW, respectively.

Table 2. The content of bioactive compounds and enzyme activities of Trichoderma pseudokoningii fermented enzyme powder^a.

	WB	EP
Total phenolic content,mg GAE^b/g DW^c	2.92 ± 0.05	4.79 ± 0.35
Xylanase, U/g	ND	487.96 ± 14.14
Cellulase, U/g	ND	18.55 ± 0.54

a The value is expressed as the mean ± standard deviation (n = 4).

b GAE: Gallic acid equivalent.

c DW: Dry weight.

WB: wheat bran; EP: enzyme powder; ND: not detected.

DPPH radical scavenging capacity

Figure 1(A) illustrates the DPPH radical scavenging capacity of EPE. Compared with 0.1 mg/mL BHT, adding 10 mg/mL EPE exerted the equivalent of a 35% scavenging effect, while WBE only had a 25% chelating effect. The scavenging capacity of both EPE and WBE decreased when the concentration exceeded 10 mg/mL and only increased slightly as concentration increased.

Figure 1. DPPH scavenging capacity (A), reducing power (B) and chelating capacity (C) of WB and EP aqueous extracts. Values are mean ± SD.

Reducing power

The reducing power of EPE is shown in Figure 1(B). Ascorbic acid reached the maximum reducing power (A700 value) at 0.5 mg/mL and did not increase further with increasing concentration. The reducing powers exhibited when 50 mg/mL EPE and WBE were added were equivalent to 60% and 40% of the value associated with 0.5 mg/mL ascorbic acid, respectively.

Ferrous iron chelating capacity

Figure 1(C) shows the ferrous iron chelating capacity of the EPE. At 12.5 mg/mL, the chelating ability of EPE was 80%, while WBE exerted 50% compared with 0.1 mg/mL EDTA.

Growth performance

Table 3 shows the growth performance of 1- to 35-d-old broiler chickens supplemented with EP. The body weight, feed consumption and weight gain of all broilers supplemented with EP were significantly higher than the control group from 1 to 21 d of age (p < .05), leading to significantly better FCR overall during this period (p < .05). However, from 22 to 35 d of age, there were no significant differences in the growing traits. The overall growth performance showed the same tendency.

Table 3. Effect of Trichoderma pseudokoningii fermented enzyme powder supplemented in diet on growth performance of 1–35 d-old broilers.

Experimental period (d) and parameter	Experimental diets^1				SEM^3	p Values
	Control	0.1% EP	0.2% EP	0.4% EP
1–21 d
Body weight, g/bird^1	646.8^b	712.0^a	699.4^a	705.1^a	6.11	.009
Feed consumption, g/bird^2	805.0^b	873.7^a	874.3^a	867.7^a	5.60	.002
Weight gain, g/bird	603.1^b	668.5^a	655.9^a	661.7^a	6.09	.009
FCR	1.36^a	1.31^b	1.32^a^,^b	1.29^b	0.007	.050
22–35 d
Body weight, g/bird	1821.1	1932.7	1935.6	1948.3	29.96	.313
Feed consumption, g/bird	1816.8	1914.4	1896.4	1875.1	52.46	.616
Weight gain, g/bird	1174.3	1220.6	1236.2	1243.1	65.45	.796
FCR	1.63	1.59	1.55	1.51	0.014	.369
1–35 d
Feed consumption, g/bird	1310.9	1394.1	1385.3	1371.4	105.13	.953
Weight gain, g/bird	888.7	944.6	946.0	952.4	59.66	.975
FCR	1.48	1.45	1.43	1.40	0.027	.956

1 Results are provided as the means of four replicates (15 birds/replicate) in each control and treatment group (n = 4).

2 Results are provided as the means of 60 birds in each control and treatment group (n = 60).

3 SEM: standard error of the mean.

EP: enzyme powder.

a,b Means within the same rows without the same superscript letter are significantly different (p < .05).

Lactate concentration of intestinal contents

The effect of EP supplementation on the lactate concentration in the intestinal contents of the broiler chickens is shown in Table 4. The lactate concentration in the caecal content of all EP supplemented groups was significantly higher from 35 d of age compared with the control group. Overall, 0.2% EP supplementation caused the greatest elevation in caecal lactate concentration while EP seemed to have no effect on ileal lactate concentration.

Table 4. Effect of Trichoderma pseudokoningii fermented enzyme powder (EP) supplemented in diet on lactate concentration of intestinal content of 1–35 d-old broilers.

Lactate concentration^2, μM	Experimental diets^1				SEM^3	p Values
	Control	0.1% EP	0.2% EP	0.4% EP
Ileum
Day 21	64.3	68.7	64.0	67.2	1.84	.485
Day 35	44.0	43.8	47.5	45.3	1.24	.718
Overall	55.6	56.3	54.1	56.3	2.88	.991
Caecum
Day 21	110.8	113.9	119.8	118.9	2.62	.640
Day 35	95.1^c	113.4^a	115.4^a	105.6^b	6.52	.001
Overall	102.9^b	113.7^a^,^b	117.9^a	110.9^a^,^b	1.92	.013

1 Control: basal diet; 0.1% EP: 0.1% enzyme powder; 0.2% EP: 0.2% enzyme powder; 0.4% EP: 0.4% enzyme powder.

2 The results are provided as the means of four replicates (four birds/replicate) in each control and treatment group (n = 4). EP: enzyme powder.

3 SEM: standard error of the mean.

a–c Means within the same rows without the same superscript letter are significantly different (p < .05).

Microbial population in ileum and caecum

The effects of EP dietary supplementation on the microbial population in broiler ilea and caeca after 35 d are presented in Table 5. The lactic acid bacteria population was increased in the caeca of the 21 d old chickens that received EP supplementation, in which 0.2% supplementation produced the best results. EP supplementation significantly lowered the coliform population throughout the entire experimental period; however, no difference in the lactic acid bacteria population was observed.

Table 5. Effect of Trichoderma pseudokoningii fermented enzyme powder (EP) supplemented in diet on microbial parameter in intestinal content of 1–35 d-old broilers.

	Experimental diets^1
Microbial parameter, log cfu/g	Control	0.1% EP	0.2% EP	0.4% EP	SEM^2	p Value
Day 21
Lactic acid bacteria
Ileum	8.15	8.05	8.08	8.42	0.10	.529
Caecum	10.79^b	11.17^a^,^b	11.88^a	11.29^a^,^b	0.11	.017
Coliform
Ileum	7.16	7.09	6.58	6.90	0.11	.334
Caecum	10.48	10.63	9.54	9.67	0.24	.079
Day 35
Lactic acid bacteria
Ileum	9.26	9.87	10.19	10.07	0.11	.071
Caecum	13.53	13.93	13.36	13.49	0.14	.602
Coliform
Ileum	9.67	9.06	8.62	8.89	0.22	.415
Caecum	11.57	10.37	9.79	9.94	0.25	.092
Overall
Lactic acid bacteria
Ileum	8.67	8.94	9.14	9.12	0.847	.200
Caecum	12.44	12.55	12.62	12.39	0.995	.340
Coliform
Ileum	8.41	8.08	7.81	7.90	0.873	.280
Caecum	11.01^a	10.20^a^,^b	9.46^b	9.43^b	0.013	.018

1 The results are provided as the means of four replicates (four birds/replicate) in each control and treatment group (n = 4).

2 SEM: standard error of the mean. EP: enzyme powder.

a,b Means within the same rows without the same superscript letter are significantly different (p < .05).

Intestinal morphology

Table 6 demonstrates the effects of dietary EP supplementation on the intestinal morphology of the broiler chickens. Jejunal villus height was increased by EP supplementation during both the starter and finisher periods. The villus:crypt ratio in the jejunum and ileum were improved in all EP supplemented groups compared to the control group in both 21- and 35-d-old chicken (Figure 2).

Figure 2. Photomicrography of jejunum and ileum of 21 and 35 d old broiler fed with control and fermented wheat brans. (A)–(D) Jejunum of 21 d broiler, control; 0.1% EP; 0.2%; 0.4% EP. (E)–(H) Jejunum of 35 d broiler, control; 0.1% EP; 0.2%; 0.4% EP. (I)–(L) Ileum of 21 d broiler, control; 0.1% EP; 0.2%; 0.4% EP. (M)–(P) Ileum of 35 d broiler, control; 0.1% EP; 0.2%; 0.4% EP haematoxylin and eosin stain. 40×.

Table 6. Effect of Trichoderma pseudokoningii fermented enzyme powder (EP) supplemented in diet on intestinal morphology of 1–35 d-old broilers.

Item	Experimental diets^1				SEM	p Values
	Control	0.1% EP	0.2% EP	0.4% EP
Jejunum
Day 21
Villus height, μm	1090.4^b	1140.6^a^,^b	1223.5^a	1215.7^a	16.48	.021
Crypt depth, μm	218.9	211.3	235.7	228.3	4.70	.270
Villus:crypt	4.85^b	5.49^a	5.31^a	5.39^a	0.09	.050
Day 35
Villus height, μm	1096.3^b	1331.2^a	1450.3^a	1317.6^a	24.30	<.0001
Crypt depth, μm	206.2	215.0	245.3	223.6	4.95	.084
Villus:crypt	5.43^b	6.20^a	6.17^a	5.99^a	0.10	.012
Ileum
Day 21
Villus height, μm	857.6	889.3	860.4	856.9	16.47	.282
Crypt depth, μm	147.5^b	155.2^a	142.4^a^,^b	141.6^a^,^b	2.43	.035
Villi:crypt	5.93^b	6.52^a	6.48^a	6.10^a	0.08	<.0001
Day 35
Villus height, μm	1166.4^a	1075.5^b	1081.2^b	1119.5^a^,^b	13.59	.039
Crypt depth, μm	214.8^a	173.5^b	180.1^b	185.8^b	3.76	.007
Villus:crypt	5.58^b	6.28^a	6.15^a	6.17^a	0.09	.016

1 The results are provided as the means of twenty spots corresponding to four birds for the control group and treatment groups, control: basal diet; 0.1% EP: 0.1% enzyme powder; 0.2% EP: 0.2% enzyme powder; 0.4% EP: 0.4% enzyme powder. EP: enzyme powder. SEM: standard error of the mean.

a,b Means within the same rows without the same superscript letter are significantly different (p < .05).

Serum antioxidant enzyme activities

Table 7 shows the effects of dietary EP supplementation on serum antioxidant enzyme activity in broiler chickens at different periods. CAT activity was significantly elevated in the 0.2% and 0.4% EP supplemented groups from 1 to 21 d of age. From 22 to 35 d of age, 0.4% and 0.2% EP supplementation significantly raised SOD and CAT activity, respectively. Overall, SOD and CAT activities were increased by EP supplementation, with 0.4% supplementation producing the best effect.

Table 7. Effects of Trichoderma pseudokoningii fermented enzyme powder (EP) supplemented in diets on the serum antioxidant enzymes activities and MDA levels of 1–35 d-old broilers.

Items	Experimental diets^1				SEM^2	p Values
	Control	0.1% EP	0.2% EP	0.4% EP
Day 21
SOD^3, U/mL	9.06	9.48	9.41	9.13	0.14	.071
CAT^3, U/mL	11.08^b	14.21^b	27.24^a	32.47^a	1.09	<.0001
MDA^3, μM	24.55	22.73	23.30	23.47	0.61	.761
Day 35
SOD, U/mL	8.54^c	9.03^b^,^c	9.39^b	10.26^a	0.10	<.0001
CAT, U/mL	13.48^b	15.84^a^,^b	24.20^a	19.71^a^,^b	1.23	.008
MDA, μM	28.01	27.61	25.91	29.12	0.70	.455
Overall
SOD, U/mL	8.86^c	9.22^b^,^c	9.40^a^,^b	9.83^a	0.08	.001
CAT, U/mL	11.73^b	19.05^a^,^b	25.42^a	23.27^a	1.48	.027
MDA, μM	26.28	25.17	24.60	26.11	0.53	.637

1 Control: basal diet; 0.1% EP: 0.1% enzyme powder; 0.2% EP: 0.2% enzyme powder; 0.4% EP: 0.4% enzyme powder. Results are given as the means of 16 birds’ serum samples for each treatment at day 21 and 35.

2 SEM: standard error of the mean.

3 CAT, SOD and MDA represent catalase, superoxide dismutase and malondialdehyde, respectively.

a–c Means within the same rows without the same superscript letter are significantly different (p < .05).

Economic benefits

An evaluation of the economic benefits of adding EP to the broiler diet is summarised in Table 8. The income over feed cost (IOFC) of the control, 0.1%, 0.2% and 0.4% EP groups were 55.6, 58.7, 58.4 and 58.1 NT$/bird, respectively.

Table 8. Evaluation of the economic benefit of Trichoderma pseudokoningii-fermented enzyme powder (EP) supplemented in diet.

Item	Experimental diets
	Control	0.1% EP	0.2% EP	0.4% EP
Feed cost, NT$/bird
1–35 d	26.3	28.3	28.7	29.6
Meat income, NT$/bird
1–35 d	81.9	87.0	87.1	87.7
Income over feed cost, NT$/bird
1–35 d	55.6	58.7	58.4	58.1

Feed cost: based on the costs (NT$/kg) of the ingredients as follows: yellow corn 10.48, soybean meal 13.63, full fat soybean meal 18.3, soybean oil 38.00, monocalcium phosphate 16.95, calcium carbonate 1.94, l-Lysine-HCl 300, dl-Methionine 230, salt (NaCl) 2.70, choline chloride, 50% 33.8, vitamin premix 109.5, and mineral premix 43.0. The fees for processing of basal ration per kg were 9.00 for grain mixture of control group, 10.10 for 0.1% EP group, 10.03 for 0.2% EP group and 10.36 for 0.4% EP group during 1–21 d, respectively. The fees for processing of basal ration per kg were 17.33 for grain mixture of control group, 18.24 for 0.1% EP group, 18.70 for 0.2% EP group and 19.26 for 0.4% EP group during 22–35 d, respectively.

Discussion

Trichoderma spp. are known to produce NSPase by fermenting various lignocellulosic substrates with different features. Pang et al. (2006) produced xylanase from local isolated Trichoderma spp. FETL c3-2 via SSF with sugar cane bagasse and palm kernel cake, and retrieved 75.0 U/mg glucosamine xylanase activity after 4 d fermentation. Azin et al. (2007) recorded xylanase activity of 592.7 U/g substrate after fermenting wheat bran and wheat straw with Trichoderma longibrachiatum. Our previous study also found that T. pseudokoningii produced 179.7 ± 6.62 U/g and 15.19 ± 0.15U/g of xylanase and cellulase activity, respectively, by fermenting wheat bran (Chu et al. 2016). However, it was widely recognised that microbial enzyme activity during fermentation was affected by the form and property of the substrates. Based on our previous study, this indicated the potential of T. pseudokoningii fermented WB producing higher enzyme activity. Kalogeris et al. (2003) studied the optimal incubation environment of Thermoascus aurantiacus to produce xylanase and determined that enhanced extracellular enzyme activity was produced from carbon sources with a finer particle size (< 0.5 mm). In this study, the higher xylanase activity (487.96 ± 14.14 U/g), compared with our previous study (179.7 ± 6.62 U/g), was due to the modification of particle size (< 0.5 mm), day of incubation and the addition of Toyama’s mineral solution (1% (NH₄)₂SO₄, 0.3% KH₂PO₄, 0.05% MgSO₄·7H₂O and 0.05% CaCl₂·2H₂O, pH 5.5).

According to our results, EP supplementation caused a significant improvement in body weight, feed consumption, weight gain and FCR in broilers during the starter period, compared with the control group. However, there were no significance differences during the finisher period or in the overall period. Xylanase supplementation has been shown to be beneficial to the performance of feed-restricted broilers (Pinheiro et al. 2004). Nian et al. (2011) also demonstrated that the addition of xylanase to a corn–soybean-based diet in broilers significantly improved feed energy as well as FCR. This improvement could be due to the ability of NSPase to break down the encapsulation of starch and proteins in botanical resources. The lack of significant improvements in the finisher period and the overall experiment can be explained by the complementary development of digestion enzymes and the ability of older chickens to utilise feed nutrients (Noy and Sklan 1995).

In this study, we observed an improvement in gut microflora by supplementation of broiler diet with EP, which was in agreement with Nian et al. (2011), who found that lactobacilli and bifidobacteria counts increased with the supplementation of xylanase in broilers. Bedford (2000) also showed that exogenous enzymes could alter microbial populations indirectly within the GI tract through their utilisation of the substrates that bacteria use as a carbon source. Furthermore, lactic acid concentration in the caecum was significantly increased by the addition of EP in the finisher period. This was in consistent with the findings of Pluske et al. (2001), who reported carbohydrase supplementation increased the proportion of lactic and organic acids and promoted gut health by suppressing the growth of pathogens. The increasing lactic acid concentration, caused by the increasing population of lactic acid bacteria, contributed to an acidic environment in the GI tract that partially influenced the growth of pathogenic microorganisms (Yang et al. 2015). These studies supported the ability of dietary EP supplementation to enhance gut microflora and inhibit the growth of E. coli as well.

Many of the reports attributed the improvement of intestinal morphology to NSPase dampening the deleterious effects of NSP-rich feedstuffs on the intestinal villi (Wang et al. 2005; Luo et al. 2009). However, in our study, this effect should be regarded as a beneficial effect of EP addition as it caused pathogenic bacteria inhibition as well as beneficial bacteria promotion. The increase in villi height represents an improvement in the area available for nutrient absorption in the intestine (Xu et al. 2003). The crypt region is the origin of the villi epithelial cells and the depth of the crypts represents the cell turnover rate. With regard to farm animal feed efficiency, low depth was favoured in order to save energy for animal growth (Parsaie et al. 2007). Stressors presented in the digesta can cause dynamic changes in the intestinal mucosa, due to the close proximity of the mucosal surface and the intestinal contents (Giannenas et al. 2010). In our study, EP supplemented to the broilers diet was found to be phenolic-rich and capable of preventing oxidative stress, which may be one of the reasons why gut morphology improved. Similar results were showed in the study of Viveros et al. (2011), suggesting dietary polyphenol-rich grape pomace concentrate (60 g/kg) could improve gut morphology (villus height:crypt depth) and increase feed efficiency in broiler chicks. Lai et al. (2015) also found that fermented soybean hulls containing phenolic-rich Pleurotus eryngii stalk residues could increase ileum villus height:crypt depth.

Phenolic content had been proven to contribute to the antioxidant capacity of fungal mycelia (Wu and Hansen 2008). Antioxidants can be classified into four categories according to their operating mechanisms: chain breaker, peroxide decomposer, metal chelator and reactive oxygen scavenger (Krinsky 1992). The phenolic content of EP was first detected followed by several in vitro assays determining different aspects of antioxidant capacities in EP. DPPH is a stable free radical (Eklund et al. 2005); decolouring them represents the ability of a sample to directly remove free radicals. Hydrogen donating ability was indicated in the reducing power assay, which was reported to be one of the antioxidant abilities of mushrooms (Shimada et al. 1992). Ferrous iron (Fe²⁺) could initiate many radical reactions by transferring single electrons, indicating the important role of iron chelation agents as antioxidants. Trichoderma spp. had been shown to enhance the antioxidant defence of plants by secreting fungitoxic phenolic compounds (Mastouri et al. 2012; Bernal-Vicente et al. 2014). However, few studies have examined such filamentous fungi exerting antioxidant capacities on animals.

In animal cells, non-enzymatic and enzymatic antioxidant systems are developed to limit the potential toxicity of ROS. Enzymatic antioxidants are synthesised and regulated endogenously, which are also essential indicators of the oxidative status of animal tissues (Jiang et al. 2007; Giannenas et al. 2010). SOD and CAT are two of the endogenous antioxidant enzymes that make up the antioxidant cellular enzymatic system (Blokhina et al. 2003). SOD catalyses the dismutation of O₂- to H₂O₂ and O₂, and also functions in conjugation with CAT, which converts H₂O₂ to H₂O (Weiss 1986). Filamentous fungi have been shown to produce secondary metabolites possessing antioxidant abilities (Yadav et al. 2014; Wang et al. 2017), and are also capable of improving broiler oxidative status (Wang et al. 2017). In our study, the improved CAT and SOD activity of broilers supplemented with EP was due to the antioxidant properties of polyphenols produced by T. pseudokoningii during fermentation (Table 2). Wang et al. (2017) supplemented broilers with white rot fungi fermented wheat bran and claimed the phenolic content improved serum CAT activity. Increased broiler CAT and SOD activities mediated by the phenolic content of Pleurotus eryngii stalk residues and soybean hulls fermented by Aureobasidium pullulans were also found by Lai et al. (2015), suggesting that fungi phenolic content is able to improve animal oxidative status, which is in consistent with the current findings.

Conclusions

In conclusion, supplementing the corn-soybean based diet of broilers with T. pseudokoningii fermented enzyme powder improves their performance in the starter phase, as well as optimising the intestinal environment by enhancing intestinal microflora and gut morphology. The antioxidant properties exerted by T. pseudokoningii also contributed to the increased in SOD and CAT activities and improved broiler oxidative status. Based on our results, we recommend 0.4% as the optimal amount to add to broiler feed.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors thank the Ministry of Science and Technology (MOST 103-2628-B-005-001-MY3) for their financial support of this study.