Introduction

The main sources to meet the poultry’s dietary protein requirement are plant originated protein feeds. Soybean meal is the ideal protein source in poultry feed formulation because of its well-balanced essential amino acid profile (Ravindran 2013). A plant protein usually needs a supplementary source of amino acids or other protein sources to balance the amino acids profile (Baghban-Kanani et al. 2020) and the anti-nutritional factors content could limit their usage (Adeyemo and Longe 2007).

As a crop of fiber source for textile globally, cotton is produced in more than 80 countries and the Land under cotton cultivation in Iran has been 80,000 hectares in 2019. Although cotton is mainly planted for its fiber, for every 100 kg of lint fiber ginned from cotton, 150 kg of cottonseed is produced (Bolek et al. 2016). The cottonseed mainly contains lipids, proteins, carbohydrate, and minerals (Bellaloui and Turley 2013). The lipid fraction (oil) is dominantly used in the food industry (Bolek et al. 2016). The whole cottonseed and defatted cottonseed meal have been frequently used in animal feeds and also as garden fertilizers (Swiatkiewicz et al. 2016).

Recently, the industrial applications of the functional components of proteins and peptides derived from cottonseed meal and its protein isolates have received increased attention (Zhang et al. 2010). Enzymatic digestion of protein meals such as soybean meal or cottonseed meal has been reported to be an efficient and reliable method to produce small peptides with targeted functionalities, such as antioxidant activity. A broad range of antioxidant peptides and peptide mixtures (hydrolysates) have been produced from soy, corn, potato, peanut, milk, whey, egg, and meat proteins (Mine et al. 2011). The antioxidant efficacy of protein hydrolysates and peptides depends on the source of proteins, the protein substrate pretreatment, the type of the proteases used, and the hydrolysis conditions applied (Hou et al. 2017). Both pure and crude enzymes can be used to produce antioxidative peptides. However, to reduce the production cost, crude protein mixtures are preferred (Zarei et al. 2012).

Small peptides can be actively absorbed by the enterocytes via specific transporters. The peptides in the enterocyte are converted to single amino acids, which are released to the portal system or utilized by the gut itself. Another possibility is that the peptides are directly released into the portal system. In a study with healthy young pigs, the gut absorption kinetics of intact proteins, hydrolysates, and free amino acid mixes were found to be comparable (Deutz et al. 1996).

Bioactive peptides (BP) (Shahidi and Zhong 2008; Sharma et al. 2011) have been defined as specific protein fragments that have a positive impact on body functions or conditions and may favorably influence health (Kitts and Weiler 2005). More than 1500 different BP have been reported in a database named ‘Biopep’ (Sharma et al. 2011). Bioactive peptides could more comprehensively be defined as organic substances formed by amino acids joined by covalent bonds -amide or peptide bonds- with low molecular weight (Hou et al. 2017). Both BP and proteins play important roles in the metabolic functions of living organisms and, consequently, in human and animals health. Hormone or drug-like activities have been attributed to BP and as such they can be classified based on their mode of action as antimicrobial, antithrombotic, antihypertensive, opioid, immunomodulatory, mineral binding, and antioxidative peptides (Dhillon 2016).

Although the correlation between BP’s structure and functional properties is not well established, many BP share some structural features that include a peptide residue length between 2 and 20 amino acids (Manikkam et al. 2016), and the presence of hydrophobic amino acids in addition to proline, lysine or arginine groups. Some BPs have also shown to be resistant to the action of digestion by peptidases (Kadam et al. 2015). Wallace et al. (2010) have comprehensively reviewed the effect of plant-based BPs on productive traits and health in poultry. Bioactive peptides produced from CSM (Tang et al. 2012, 2018; Sun et al. 2013; Nie et al. 2015a), SBM (Feng et al. 2007b; Abdollahi et al. 2017, 2018), canola meal (Karimzadeh 2016; Karimzadeh et al. 2017), and sesame meal (Salavati et al. 2019) have been studied in meat chicken. However, most of these studies have utilized PBs produced through fermentation. Enzymatic hydrolysis is another technique applied to breakdown high molecular weight protein, destroy antinutritional factors, in order to improve the quality of vegetable protein meals (Rolle 1998; Dust et al. 2005; Sathe et al. 2005).

De Oliveira Filho et al. (2020) studied different enzymatic and thermal treatments of cottonseed byproduct and found a higher antioxidant activity for the protein hydro-lysates produced through thermal pretreatment and enzymatic hydrolysis of cottonseed byproduct protein. They also reported an inhibitory effect for the heat-pretreated hydrolysates on the growth of C. gloeosporioides and S. aureus. Yuan et al (2020) studied the antioxidant and immunoenhancement effect of peptides derived from cottonseed meal protein hydrolysate in fish and found an improved hepatocyte metabolism and antioxidant properties and innate immunity.

Enzymatic hydrolysis is by far the most efficient and reliable method to produce peptides with targeted functionalities, such as antioxidative and antimicrobial activities. The hydrolysis of proteins can be achieved by a single enzyme (e.g., trypsin) or multiple enzymes (e.g., a mixture of proteases known as Pronase, pepsin and prolidase). The choice of the enzymes depends on the protein source and the degree of hydrolysis (Mine et al. 2011). Compared to acid, alkaline and microbial hydrolysis of proteins, the main advantages of enzyme hydrolysis of proteins are that: the hydrolysis conditions (e.g., like temperature and pH) are mild and do not result in any loss of amino acids; proteases are more specific and precise to control the degree of peptide-bond hydrolysis; and the small amounts of enzymes can be easily deactivated after the hydrolysis (e.g., 85 °C for 3 min). However, there might be some disadvantages to enzymatic hydrolysis of protein such as the relatively high cost and the potential presence of enzyme inhibitors in the raw protein materials which may result in an incomplete hydrolysis (Hou et al. 2017).

Considering the dearth of data on cotton seed bioactive peptides (CSBP) derived from enzymatic hydrolysis in poultry production, the current study was designed to evaluate the effect of different inclusion rate of CSBP, produced by enzymatic hydrolysis, on productive traits, serum lipid, ileal microflora and economical parameters of broiler chickens compared to an antibiotic growth promotor—herein zinc bacitracin. This research enriches the practical aspects on the knowledge on the cottonseed bioactive peptides, and would help in better understanding of the nutritional and functional properties of cottonseed protein for poultry feeding.

Materials and Methods

The experimental protocol was reviewed and approved by the animal care and use committee of the Isfahan Agricultural and Natural Resources Research and Education Center (protocol No. 2594-264-1). The present study was conducted at the research farm of the Isfahan Agricultural and Natural Resources Research and Education Center, Isfahan, Iran.

Preparation and Enzymatic Hydrolysis

The enzymatic hydrolysis of the CSM followed the method described by Alashi et al. (Alashi et al. 2014) with some modifications. Briefly, after preparation of the samples, CSM was hydrolysed using five food grade enzymes at an enzyme substrate ratio of 1:20 for all the enzymes, to obtain cottonseed protein hydrolysates following 4 h of incubation. The hydrolysis conditions for each enzyme used were as follow: Alcalase (pH 8.0 and 60 °C), chymotrypsin (pH 8.0 and 37 °C); pepsin (pH 3.0 and 37 °C); trypsin (pH 8.0 and 37 °C) and pancreatin (pH 8.0 and 40 °C). All the enzymes were obtained from Sigma Chemicals (St. Louis, MO). The pH was maintained for each hydrolysis process using either 1 M NaOH (Sigma Chemicals, St. Louis, MO) or 1 M HCl (Sigma Chemicals, St. Louis, MO) as appropriate, while the temperature was maintained using a thermostat. After the 4 h digestion period, the enzymes were inactivated by heating and holding at 85 °C for 15 min. The final product of the enzymatic hydrolysis was lyophilised and stored at − 18 °C until required for further analysis and use in the subsequent experiments.

The molecular weight distribution of SBM, CSM, and CSBP were determined using TSK gel 3000 PWXL columns (Tosoh, Japan) coupled with an HPLC system (Agilent 1100, Agilent Technologies Inc.). The acetonitrile in water (1:1, v/v) containing TFA (0.1%, v/v) was used as the mobile phase. The absorbance was monitored at 225 nm with a flow rate of 0.5 mL/min. Bovine serum albumin (BSA, MW: 66,000 Da), cytochrome C (MW: 12,384 Da), bacitracin (MW: 1423 Da), and reduced glutathione (GSH, MW: 307 Da) were used as the molecular weight standards.

The morphology and structure of the CSBP were further investigated by Field Emission Scanning Electron Microscopy (FE-SEM). FE-SEM was performed using a TESCAN, MIRA 3 field emission scanning electron microscope (Buali (Avicenna) Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran).

Ingredient and Dietary Chemical Analysis

All feed ingredients were analysed before the trial by Evonik Industries AG animal nutrition analytical lab for crude protein (AMINOProx®), amino acids (AMINONIR®), ether extract (AMINOProx®), dry matter (AMINOLab®) and total phosphorus as well as phytate phosphorous (AMINOProx®) contents. Furthermore, dietary samples were analysed for the profile of amino acids by AMINOLab® through high performance liquid chromatography (HPLC). The total nitrogen of the diet samples was determined by a kjeldahl method, then multiplied by a factor of 6.25 to calculate crude protein (AOAC 2000; method 990.03) and Ca and P by spectrophotometry (methods 968.08 and 965.17) as indicated by AOAC (2000). Free gossypol contents of CSM and CSBP were determined according to the method of the American Oil Chemists Society (AOCS 2009; method Ba 7b-96). The crude fiber content was determined by the filter bag technique after digesting with H2SO4 and NaOH (AOCS 2009; method Ba 6a-05). The Ash content of SBM, CSM, CSBP, and diets were analyzed by incineration at 550 °C for 24 h in a muffle furnace.

Experimental Diets and Animals’ Management

Male broiler chickens (Ross 308), 240 day-old, were purchased from a commercial hatchery and used in this experiment. Upon arrival, chicks were weighed, wing-banded and randomly assigned to the treatment groups so that the initial body weights were similar among different treatment groups (± 6.0 g). Five replicate cages of twelve chicks each were randomly allotted to four dietary treatments based on a completely randomized design across starter (1–15 days) and grower (16–35 days) periods. Each pen was equipped with a separate feeder and drinker. Experimental treatments included two basal diets without CSBP serving as the control diet or the control diet supplemented with zinc bacitracin as an antibiotic (40 mg/kg) and the other two treatments were supplemented with 15 or 20 g/kg CSBP by substituting an equal quantity of maize and soybean meal. Diets were formulated to be iso-energetic and iso-nitrogenous and to meet or marginally exceed nutrient requirements provided by Ross 308 manual (Aviagen, 2014; Table 1) across different periods. Birds had free access to feed and water throughout the experiment. The birds were reared in a power-ventilated broiler house equipped with battery cages (length 124 cm × width 65 cm). The illumination and ventilation programs followed the guidelines recommend by the breeder. The light was provided by incandescent bulbs, and the light intensity at bird’s level was 30 lx. Ambient temperature was kept at 32 °C for the first 3 days and gradually decreased thereafter to 24 °C by the end of the 3rd week. Relative humidity was maintained between 45 and 65%.

Table 1 Ingredients and nutrient composition of experimental diets1 during different growth periods (as-fed basis)
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Performance and Carcass Components

Feed intake and live body weight (BW) of chicks in each cage (replicate) were recorded individually from 1 to 35 days, and daily feed intake (DFI; g), daily weight gain (DWG; g), and feed conversion ratio (FCR; feed intake/weight gain) were calculated accordingly. Mortality was recorded daily. On day 35 of the experiment, three birds close to the mean BW of cage were individually weighed and slaughtered and the weight of abdominal fat and excised carcass was measured and expressed as g/100 g BW.

Blood Parameters

At 35 d of age, 5 ml of blood was collected from wing vein of 3 birds (randomly selected) in each replicate. The blood was centrifuging at 3000×g for 20 min (SIGMA 4–15 Lab Centrifuge, Germany), and serum was harvested and then stored at − 20 °C until assayed. The serum biochemical metabolites including triglyceride, total cholesterol, high density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured using the kit package (Pars Azmoon Co; Tehran, Iran). All sample analyses were performed in duplicate.

Microbial Population of Ileum Content

At the end of the experiment (35 days), in order to determine the ileal microbial counts, 3 broiler chickens were selected and killed by cervical dislocation. The ileum content from the 3 killed birds were aseptically collected and transferred to peptone buffer in test tubes and sterile Whirl–Pak plastic bags for bacteriological culture. The samples were placed on ice and transported to the microbiology laboratory for bacteriological analysis that was carried out the same day. Sample weights were estimated by subtracting the weight of the container without sample from the weight with the samples and then, one gram of each sample was tenfold serially diluted (10–1 to 10–9) in 0.9% sterile bacteriological peptone diluents. Bacteriological analyses were performed on a total of 60 samples (pooled samples from 3 birds of the same pen constituting 1 sample). Lactobacillus spp. populations were quantified using Lactobacilli MRS Agar (Merck, Dusseldorf, Germany, Cat No. 146717) according to the manufacturer’s methods (ISO 152141998). Enterococcus spp. populations were determined on KF Streptococcal Agar (Merck, Dusseldorf, Germany, Cat No. 110707) and incubating at 37 °C for 48 h (Hayes et al. 2003). Clostridium perfringens was enumerated according to Knarreborg et al. (2002). Briefly, samples were spread on TSC (Tryptose Sulfite Cycloserine) agar (Merck, Dusseldorf, Germany, Cat No. 111972) and incubated anaerobically for 24 h at 37 °C. The generic Escherichia coli (E. coli) and other coliforms population was counted by EMB agar (Eosin methylene blue) (Merck, Dusseldorf, Germany, Cat No. 101347) and Mac Conkey agar (Merck, Dusseldorf, Germany, Cat No. 105465) as described previously (Diarra et al. 2007).

Economic Parameters

After calculation of livability percentage, FCR and cost of feed, the European Production Efficiency Factor (EPEF), European Broiler Index (EBI), Cost per kg of Live BW (CLBW), and Cost per kg of Carcass (CC) were used to evaluate the economic performance of broilers as suggested by and Marcu et al. (2013). EPEF, EBI, CLBW, and CC were calculated according to the following equations (Marcu et al. 2013).

BW = Body weight (kg); BWG = Body weight (g) at the end—Body weight (g) at start; ADG (g/chick/d) = BWG/days of growth period; FCR = Cumulative feed intake (kg)/Body weight gain (kg);

  1. (1)

    \({\text{Livability }}\left( \% \right) \, = \, 100 \, {-}{\text{ Mortality }}\left( \% \right)\)

  2. (2)

    \( \mathrm{E}\mathrm{P}\mathrm{E}\mathrm{F}=\frac{\mathrm{L}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} \left(\mathrm{\%}\right) \times \mathrm{B}\mathrm{W} (\mathrm{k}\mathrm{g})}{\mathrm{A}\mathrm{g}\mathrm{e} \left(\mathrm{d}\right) \times \mathrm{F}\mathrm{C}\mathrm{R} (\mathrm{k}\mathrm{g} \mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}/ \mathrm{k}\mathrm{g} \mathrm{G}\mathrm{a}\mathrm{i}\mathrm{n}) }\times100\)

  3. (3)

    \(\mathrm{E}\mathrm{B}\mathrm{I}=\frac{\mathrm{L}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} \left(\mathrm{\%}\right) \times \mathrm{A}\mathrm{D}\mathrm{G} (\mathrm{g}/\mathrm{C}\mathrm{h}\mathrm{i}\mathrm{c}\mathrm{k}/\mathrm{d})}{ \mathrm{F}\mathrm{C}\mathrm{R} (\mathrm{k}\mathrm{g} \mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}/ \mathrm{k}\mathrm{g} \mathrm{G}\mathrm{a}\mathrm{i}\mathrm{n}) \times10}\times100\)

  4. (4)

    Cost per kg of Live BW (% of the control group) = [(FCR × Cost of feed)/(FCR × Cost of feed) of the control group)] *100

  5. (5)

    Cost per kg of Carcass (% of the control group) = [(Cost per kg of Live BW/Carcass yields (%))/(Cost per kg of Live BW/Carcass yields (%) of the control group] × 100

Statistical Analysis

Data obtained were first check for normality and then subjected to analysis of variance procedures appropriate for a completely randomized design using the General Linear Model procedure of SAS 9.4 (SAS Institute 2016), according to the following model:

$${\text{Yij}} = \mu \, + {\text{ Ti}} + {\text{ eij,}}$$

where μ is the overall mean, Ti is the effect of experimental treatments and eij is the random residual error. Cage was the experimental unit for growth performance and economical parameters and the mean of 3 broiler chicken per cage was the statistical unit for all blood parameters, ileal microbial counts, and carcass characteristics. When a significant F-test was detected (P < 0·05), corresponding means were separated by Tukey test (HSD). Values in the tables are presented as means and pooled standard error of means (SEM). Also, differences among treatments were separated using polynomial orthogonal contrasts to determine linear and quadratic responses.

Results

Hydrolysis of CSM by enzymatic method produced BP of different molecular weight mostly ranging from 180 to 500 Da including 40.8% di and tri-peptides, 39.4% oligopeptides and polypeptides (500 to > 3000 Da), and 6.3% free amino acids (< 180 Da). The enzymatic hydrolysis increased all the amino acid content of CSBP compared to CSM (Table 2). The biggest increase was observed for Arg and Ile by nearly 28% followed by Val at 20%. The FE-SEM images for the CSBP are shown in Fig. 1. The FE-SEM images confirm that the CSBP are uneven and granulated, which compared to the albumin structure, it appears to be a loose and unstable peptide structure. The results are consistent with the concept that the enzymatic hydrolyze is a desirable solution to form small digestible peptides.

Table 2 Chemical composition of soybean meal, cottonseed meal, and cottonseed meal bioactive peptides (dry matter basis) (analytical values, means of triplicate analyses)
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Fig. 1
figure 1

The surface morphology of cottonseed bioactive peptide visualized by Field emission scanning electron microscopy to determine the peptides structure. This image confirms that the CSBP are uneven granulated one. The results are consistent with the concept that the enzymatic hydrolyze is a desirable solution to form small digestible peptides

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The highest and lowest weight gain was recorded in the antibiotic and control groups, respectively (P < 0.05), and the birds fed the CSBP diets had no significant difference with the other treatments, although their weight gain was numerically higher than the control group. Antibiotic and the 20 g/kg CSBP group had higher feed intake than the control group (P < 0.05). Antibiotic or CSBP supplementation increased the feed conversion ratio, but only the difference between the control group and the group fed 20 g/kg CSBP, was significant (P < 0.05; Fig. 2).

Fig. 2
figure 2

Performance parameters of broiler chickens in response to dietary treatment over the entire production period (1–35 days)

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The highest and the lowest livability rates were observed in the antibiotic and control groups, respectively. The use of CSBP improved livability compared to the control group, but the differences with the control and antibiotic groups were not significant. Antibiotic supplementation improved the EBEF and EBI of broiler chicks compared to the other groups, and the improvement in the 15 g/kg CSBP group was also significant compared to the control group. Inclusion of antibiotic, 15 and 20 g/kg CSBP increased the feed cost per kilogram body weight gain by 9, 11 and 16 percent, respectively, compared to the control group. Feeding the antibiotic and CSBP (15 and 20 g/kg) diets also resulted in approximately 4, 3 and 11 percent increase in feed cost per kilogram of carcass compared to the control group, respectively (Table 3).

Table 3 Effect of dietary treatments on economic parameters from broilers
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As summarized in Table 4, serum total cholesterol and HDL concentrations were not affected by the dietary treatments (P > 0.05). Serum triglyceride level was higher in the CSBP-fed groups than in the antibiotic-treated and the control groups, and the difference with the 20 g/kg CSBP group was statistically significant (P < 0.05). Antibiotic and CSBP supplementation decreased serum LDL compared to the control group and the lowest LDL level was observed in the group fed 20 g/kg CSBP (P < 0.05). The LDL to HDL ratio was lower in the antibiotic and CSBP fed groups than in the control group (P < 0.05). Antibiotic or CSBP administration significantly reduced relative weight of abdominal fat compared with the control group (P < 0.05).

Table 4 Lipid parameters of broiler chickens in response to dietary treatment
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Diet supplementation with antibiotic decreased the population of Clostridium perfringens and Enterococcus spp. in the ileum compared to other groups (P < 0.05; Table 5). The ileal Escherichia coli counts were significantly lower in birds fed the AGP and both CSBP supplemented diets compared to the control group (P < 0.05). The highest Lactobacillus spp. population was observed in the ileum contents of the birds fed 15 g/kg CSBP supplemented diets and the differences with the antibiotic group was significant (P < 0.05).

Table 5 Effect of dietary treatments on counts of bacteria (CFU) in the ileal content from broilers
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Discussion

Enzymatic hydrolysis of cottonseed meal has been shown to effectively reduce the anti-nutritional substances in the meal (Kamnerdpetch et al. 2007). In the present study, feeding broilers with hydrolyzed peptides of cottonseed meal was able to improve the growth performance of the birds compared to the control group and lead to comparable results with the antibiotic group, although the feed conversion ratio did not improve in line with body weight gain as the birds fed the CSBP supplemented diets had a higher feed intake compared to the control birds. Nie et al. (2015a) reported improved daily weight gain and also feed conversion ratio by using fermented cottonseed meal in the broiler chickens diet. However contrary to the current findings, Wang et al (2017) showed that two different levels (8.9% and 17.9%) of fermented cottonseed meal did not influence the broiler chickens productive traits. In another experiment using 6% fermented cottonseed meal, contrary to the results of the present study, growth rate was not affected but feed conversion ratio was significantly improved (Niu et al. 2019). Similarly, hydrolyzed soybean meal has also been reported to positively affect weight gain (Mathivanan et al. 2006; Wang et al. 2011) and feed conversion ratio (Abdollahi et al. 2017, 2018). The improved performance traits by using hydrolyzed products has been attributed to the increased nutrient digestibility (Nie et al. 2015a) as a result of an enhanced activity of digestive enzymes such as amylase, trypsin, lipase, and protease (Feng et al. 2007a, b; Karimzadeh 2016).

Triglyceride in the blood serum can reflect the status of lipid metabolism in the body. In this study, serum triglyceride content of chickens increased with increasing the amount of CSBP in the diet. In similar experiments using fermented cottonseed meal and canola meal, contrary to our results, adding fermented products significantly reduced serum triglycerides concentration (Karimzadeh et al. 2017; Niu et al. 2019). Triglycerides are made from glycerol and fatty acids, so that their fatty acids are almost from the source of dietary fatty acids (Lee et al. 2009).

The different effects of hydrolyzed or fermented products on fat metabolism have been attributed to the presence of secondary metabolic products such as the concentration of essential amino acids, the size and type of the peptides produced, the concentration of some vitamins and even the presence of some sources of probiotics in the fermented products (Nie et al. 2012; Tang et al. 2012). However, by studying the expression of genes involved in fat metabolism, it has been suggested that the use of fermented cottonseed meal can greatly upregulate the expression of the genes involved in fat metabolism in male birds and ultimately decrease the fat storage in the abdominal area of male birds (Nie et al. 2015b; Niu et al. 2019).

Niu et al. (2019), in an experiment examined the fat profile of fermented cottonseed meal, and stated that the unsaturated fatty acids in the fat content of cottonseed meal were significantly higher than soybean meal, due to the presence of these fatty acids in cotton seed meal. Azelaic acid, a 9-carbon linear saturated dicarboxylic acid, is typically measured as an indicator in urine to assess the fatty acid beta-oxidation status (Amer et al. 2017). It has been shown that azelaic acid increases in the long-chain unsaturated fatty acid beta-oxidation process (Mastrofrancesco et al. 2010). It has also been shown that the amount of azelaic acid in the urine increases when fermented cottonseed replaces part of the dietary soybean meal (Niu et al. 2019).

The presence of poly-unsaturated fatty acids in the diet can alter fat metabolism, thereby significantly reducing fat storage in the abdominal fat content (Gaíva et al. 2003). In this regard, the effect of poly-unsaturated fatty acid docosahexaenoic acid on the inhibition of enzyme activity (Gaíva et al. 2003) as well as the expression of fatty acid synthase genes (Sampath and Ntambi 2004) was noted, which can reduce the synthesis of De Novo fatty acids synthesis and ultimately decreasing the body’s fat storage.

Our results regarding a significant decrease in low density lipoprotein (LDL) levels and significant decrease in abdominal fat pad suggest that, like most previous reports, enzymatic hydrolysis of cottonseed protein alike its fermented products, had no effect on the fatty acid content, but should have positively affected beta-oxidation of fatty acids leading to a decreased abdominal fat pad deposition. Optimal digestive function is essential for the production and health of birds and plays a major and undeniable contribution to the overall productive performance and also quality of poultry meat. The “gastrointestinal health” plays a vital role in overall health and productivity of broiler chickens (Kogut and Arsenault 2016). In this respect, the intestinal microflora has been shown to be the key player in the digestion and absorption of nutrients as well as the control of pathogens (Baurhoo et al. 2009). The results of our experiment showed that the use of hydrolyzed cottonseed meal in the diet has the potential to improve intestinal microflora by increasing the lactobacillus counts and decreasing E. coli population. While, the antibiotic reduced the growth of all bacteria in the ileum (pathogenic and beneficial bacteria). Similarly, other experiments have also reported positive effect of fermented cottonseed meal on intestinal microflora in ileum (Jazi et al. 2017) and cecal contents (Sun et al. 2013) of broiler chickens, indicating that peptides from CSBP can be particularly effective as antibacterial peptides to reduce the population of pathogenic bacteria.

The effect of fermented products on the population of beneficial intestinal bacteria could be due to the low acidity of fermented products. Acidic environments inhibit the production and proliferation of pathogenic bacteria and provide a suitable environment for the proliferation of beneficial anaerobic bacteria, including lactic acid producing bacteria, and improve the digestion and absorption process in mono-gastric animals (Van Winsen et al. 2001; Engberg et al. 2009; Naji et al. 2015; Jazi et al. 2017). In this regard, Niba et al. (2009), by comprehensively examining the fermented products on the gastrointestinal health, suggested the term fermbiotic to better describe the properties of these products. As the end product of both fermentative and enzymatic degradation processes are small peptides, it may be possible to attribute the improvement of the gastrointestinal microbial flora to the existence of antimicrobial peptides that are rich in cysteine molecules. Many peptides have antimicrobial and immune-system modulating effects, each of which can have independent functions in the immune system and cellular defense, but almost all of these peptides have cysteine molecules in their structure (Hiemstra and Zaat 2013). Liu et al. (2018) reported that the oligopeptide obtained from solid-state fermented cottonseed meal significantly enhanced the immune activity and showed a protective role on the immunosuppression effect of cyclophosphamide in mice.

Analysis of cottonseed meal peptides showed that vicillin-like antimicrobial peptides are highly abundant in this product (He et al. 2018). However, whether the improved microbial flora is due to the activities of these peptides or other metabolites with different mechanisms requires further complementary research. The results of this experiment showed that the use of hydrolyzed cottonseed meal could improve carcass yield and decrease relative weight of abdominal fat compared to the control group. In previous studies, improvement of carcass characteristics and significant reduction in abdominal fat by inclusion of hydrolyzed cottonseed meal, were attributed to an increase in the number of lactic acid producing bacteria, because fermentative bacteria were used to hydrolyze the cottonseed protein (Jazi et al. 2017). It has been reported that increasing lactic acid concentration can decrease acetyl-coenzyme-A carboxylase activity and thereby decrease body fat deposition (Santoso et al. 1995) and thereby improve carcass yield. Furthermore, it has also been hypothesized that BPs positively affect digestibility and absorption of proteins; and/or indirectly upregulate the activity of the enzymes involved in the fat or protein storage process by affecting the microbial population of the gastrointestinal tract (increasing the population of beneficial bacteria and reducing the activity of the enzymes involved in the fat storage), and also directly affecting the activity of the enzymes involved in the process of fat or protein metabolism in the bird body (Nie et al. 2015a).

Conclusions

This study evaluated the nutritional, antioxidant and antimicrobial properties of bioactive peptides derived from cottonseed meal. In conclusion, the bio-peptides derived from cottonseed meal by enzymatic hydrolysis had a positive effect on the growth rate, antioxidant status, abdominal fat deposition, serum LDL and intestinal microflora of broiler chickens and these compounds have the potential to be used as an alternative to antibiotic growth promoters in broiler diets.