Effect of dietary mannan oligosaccharides and fructo-oligosaccharides on physico-chemical indices, antioxidant and oxidative stability of broiler chicken meat

The objective of this present study was to investigate the potentiality of prebiotics (mannan oligosaccharides-MOS and fructo-oligosaccharides-FOS) in replacement of antibiotic growth promoter and their relationship with physico-chemical indices, antioxidant and oxidative stability and carcass traits of broiler chickens meat. Accordingly, 240 day-old broiler chicks of uniform body weight divided in 6 treatment groups with 5 replicate each (5 × 6 = 30) having 8 birds in each replicate. Six corn based dietary treatments were formulated viz. T1 (control diet), T2 (T1 + Bacitracin methylene di-salicylate @ 0.002%), T3 (T1 + 0.1% MOS), T4 (T1 + 0.2% MOS), T5 (T1 + 0.1% FOS), and T6 (T1 + 0.2% FOS). Significant (p < 0.05) increase in cut up part yields (%) and reduction in cholesterol and fat content in T4 (0.2% MOS) group. The water holding capacity (WHC) and extract release volume (ERV) were increase (p < 0.05) in 0.1 or 0.2% MOS supplemented group. DPPH (1, 1-diphenyl-2-picrylhydrazy) was higher (p < 0.05) and lipid oxidation (free fatty acid and thio-barbituric acid reactive substances) was lower (p < 0.05) in T4 group. The standard plate count (SPC), staphylococcus and coliform counts were decreased (p < 0.05) in T3 or T4 group. Thus, it can be concluded that mannan oligosaccharides (MOS) may be incorporated at 0.2% level in diet for improved physico-chemical indices, antioxidant and oxidative stability and carcass characteristics of broiler chickens meat and it may be suitable replacer of antibiotic growth promoter.

. Effects of dietary inclusion of prebiotics on carcass characteristics and organ weight (% of live weight) in broiler chickens. T 1 (no MOS/FOS/BMD), T 2 (0.002% BMD), T 3 (T1 + 0.1% MOS), T 4 (T1 + 0.2% MOS), T 5 (T1 + 0.1% FOS), T 6 (T1 + 0.2% FOS). SEM = Standard error of mean; NS = Non-significant; n = 15. Mean values bearing the same superscript in a row did not differ significantly (p < 0.05).    www.nature.com/scientificreports/ Physico-chemical indices. The results of physico-chemical parameters as affected by feeding prebiotics to broiler chicken shown Table 3 indicated cholesterol and fat content of meat been significantly (p < 0.05) in birds fed 0.2% MOS (T4 group) which changed into statistically alike to MOS and FOS supplemented birds. The cholesterol and fat content of meat was higher in birds fed control diet (T 1 group) or BMD supplemented diet (T 2 group) which was statistically similar to T 3 , T 5 , and T 6 groups. The pH and drip loss (%) of chicken meat were not significantly (p > 0.05) influenced by dietary treatments. Significantly higher (p < 0.05) WHC and ERV of chicken meat was observed in birds fed 0.2% MOS (T 4 group) or 0.1% MOS (T 3 group) which were statistically similar to WHC and ERV of meat from FOS fed birds (T 5 and T 6 groups). The meat from birds fed control diet (T 1 group) or BMD supplemented diet (T 2 diet) revealed lower WHC and ERV values which did not differ significantly from FOS supplemented birds.
Lipid oxidation parameters. The lipid peroxidation parameters are given in Microbial load. The results of microbial load of chicken meat as influenced by prebiotic supplementation are given in Table 6. In case of fresh meat (0 d), the levels of standard plate count (SPC), coliform, and staphylococci were significantly (p < 0.05) reduced in meat of birds supplemented with 0.2% (T 4 ), 0.1% MOS (T 3 ) and 0.2% FOS compared to birds fed control diet (T 1 ) and antibiotics (T 2 ). Whereas, at 14 d of storage, SPC were significantly decreased in both the MOS supplemented group, coliform counts were reduced (p < 0.05) in both

Discussion
Carcass traits. Similar to the results of present study, Toghyani et al. 14 reported that carcass and cut-up parts yields were significantly higher in chicken fed prebiotic containing diet. However, in contrast to the present study Rehman et al. 15 reported no significant differences in breast, thigh, and carcass yields after dietary inclusion of prebiotics. Whereas, Ricke 12 observed no significant effect of prebiotics on the cut-up parts of chicken carcass. Therefore, based on the results of present study it can be assumed that the application of prebiotics has a positive effect on muscle weight. The principle effects of prebiotics have been reported by Cummings and Macfarlane 16 and include improvement of calcium and magnesium absorption, production of short-chain fatty acids, and selective increases in the population of lactate producing bacteria like Lactobacillus and Bifidobacterium. It has been shown that increased lactate concentration often decreases intestine pH and is a potent antimicrobial substance to several pathogenic species such as E.coli 17 . Thus, prebiotic helps to balance the intestinal microflora of poultry, consequently an improved utilization of diet nutrients i.e., protein and energy and higher feed intake leading to better cut up parts weight 14 .
Physico-chemical indices. The results of the present study are in line with the findings of Pilarski et al. 18 , who reported that prebiotics caused a decrease in meat cholesterol concentration in comparison to the control and antibiotic treated group. In contrast, Salma et al. 19 reported that no significant difference was observed in cholesterol concentration after dietary inclusion of prebiotics. The results of the present study were in accordance with the findings of Khaksefidi and Khaksefidi 20 , who observed that fat % of breast meat, was significantly lower in prebiotic supplemented chicken. Fat deposition in the abdominal area of broilers is considered as waste in the poultry production; subsequently it represents a loss in the market and consumer acceptability, and increases expenditure during the treatment of effluent produced when processing broilers. The obtained results of this study indicate that prebiotic supplementation of broilers diet has the potential to lessen this type of waste by reduction of the fat content in the abdominal area of birds 14 .
In the present study, the pH values were within the normal range and independent of dietary prebiotic supplementation. Similar to the results of the present study Tavaniello et al. 13 did not find any significant effect of dietary prebiotic supplementation on the pH values of chicken meat. However, Mir et al. 21 confirms that the  www.nature.com/scientificreports/ meat quality is influenced by pH changes which occur during rigor mortis. Generally meat with high pH has high WHC, although the present study does not support this correlation. The results of present study are in line with Habibi-Najafi et al. 22 , who reported that dietary supplementation of prebiotic increased the WHC of meat.
On the other hand, Harriet et al. 23 reported that dietary inclusion of prebiotic has no significant effect on WHC of meat during storage condition. It is remarkable to note that water loss reduces meat nutritional value because some nutrients may be lost in exudate resulting in meat becoming less tender and bad in flavour. Regarding ERV values in broiler chicken after the dietary inclusion of FOS and MOS, no such reports are available for comparing the results of this study.
Lipid oxidation parameters. The results of present study showed that prebiotic could inhibit both thigh and breast muscle lipid oxidation (MDA production) in broiler chicken, therefore protecting the peroxidation of labile PUFA enriched meat. The reduced shelf-life of meat occurs due to progressive oxidation and enzymatic hydrolysis of unsaturated fatty acid 24 FFA value is the measure of hydrolytic rancidity due to lipolytic enzyme activity of microbial and muscle origin resulting in accumulation of FFA which might impart undesirable flavour in foods 25 . The peroxide value test involves the measurement of peroxide and hydro peroxide formed during initial stage of lipid oxidation 26 . However, in contrast to the results of present study Konca et al. 27 reported that after the dietary inclusion of prebiotics, TBARS values were significantly increased. Furthermore, Ali 28 reported that dietary inclusion of prebiotics has no pivotal role in changing the TBARS activities in fresh as well as stored meat. No clear mechanisms have been reported responsible for the reduction of lipid synthesis by prebiotics. It might in part be due to increasing beneficial bacteria such as Lactobacillus that decrease the activity of acetyl-CoA carboxylase, which is the rate-limiting enzyme in fatty acids synthesis 14 .
Antioxidant parameters. The natural dietary antioxidant compounds of plant origin react with lipids and hydroxyl radicals and result into stable product. Simitzis et al. 29 reported that following absorption prebiotics have shown significant antioxidant activity in poultry meat after entering the systemic circulation. The lipid and cholesterol oxidation of broiler chicken meat was significantly reduced by dietary prebiotic supplementation in broiler chicken 30 . Inclusion of prebiotics in turkey diet increased the oxidation stability and retention of alpha tocopherol in the long term stored frozen turkey meat 31 . It is still unclear whether the dietary antioxidants consumed can be incorporated into fatty tissues in the same form as when the fat is stabilized in-vitro 32 . However in the present study, free radical inhibition percentage of thigh and breast meat of chicken fed 0.2% MOS was significantly greater than that of chicken fed control and antibiotic supplemented diet. These results indicate that antioxidant compounds from prebiotic prevented thigh and breast meat from oxidation.

Microbial load.
According to the hypothesis proposed by Kim et al. 33 the reduction in microbial load was due to production of different antimicrobial components by prebiotic which result in exclusion of common entero-pathogens and food spoilage organisms of broiler chicken. Though, the exact mechanism by which prebiotics might exert anti-microbial effects in broiler chicken meat remains unclear. Some of the proposed modes of actions are; maintaining a healthy balance of gut microflora, competitive exclusion and inhibition of microbial growth by lactic acid producing bacteria favoured by dietary prebiotics, enhancing gut immunity and integrity, improving digestive enzyme activities, digestion and neutralizing enterotoxins, etc. 34 . It is general hypothesis that prebiotics have been shown to alter gastrointestinal microflora, modify the immune system, reduce pathogen annexation including pathogens such as Salmonella Entritidis and E.coli 16 . Prebiotics supplementation of broilers diet also result in an increase of the pH of the gastro intestinal tract (GIT) and beneficial bacteria population such as lactobacillus and bifidobacterium, due to increasing production of volatile fatty acids 35 .

Conclusions
The results reported in this work indicate that 0.2% mannan oligosaccharides (MOS) could be used as natural growth promoter (NGP) to replace the antibiotic growth promoter (AGP) in improving the physico-chemical, oxidative stability, and microbiological quality of broiler chicken meat. Subsequent the appropriate guidelines and protocols will ensure eventually limited the use of feed antibiotic for poultry production and the induction of NGP in animal derived food products i.e., meat which will reduce the risk to the public. This NGP could be popularized among the farmers as a feed additive in poultry diets for production of safe, clean, and green poultry meat for human consumption.  (Table 7) dietary treatments were formulated viz. T 1 (control diet), T 2 (T 1 + bacitracin methylene di-salicylate @ 0.002%), T 3 (T 1 + 0.1% MOS), T 4 (T 1 + 0.2% MOS), T 5 (T 1 + 0.1% FOS), and T 6 (T 1 + 0.2% FOS). The birds were provided ad libitum respective feed and fresh water throughout the feeding trial of 42 days.

Material and methods
Carcass characteristics. Toward the finish of 42 days trial period, 15 birds from every treatment (three birds for each replicate) were electrically stunned (200 V applied for 3 s) and slaughtered by exsanguination after 12 h of fasting with ad libitum drinking water. The carcass characteristics (dressing and eviscerated yield), cut up parts (thigh, breast, back, wings and drumstick) and relative weight of organs (heart, liver, and gizard) were determined.
Collection of sample. The breast and thigh meat samples were collected separately from every slaughtered bird for the study of physico-chemical, oxidative stability, and microbial characteristics.
Physico-chemical indices. Fat content (percentage, dry basis) of meat was determined by refluxing 2 g dried meat sample in 150 mL petroleum ether in Soxhlet extraction equipment for 6 h at 60°C 36 . For cholesterol estimation about 1 g meat sample was extracted in 15 mL chloroform methanol mixture (2:1) and the concentration of cholesterol within the extract was determined by spectrophotometer at wavelength of 560 nm 26 .
The pH of meat sample was measured with the assistance of digital pH scale meter by mixing 5 g meat sample with 25 mL distilled water for 2 min 21 . For the estimation of purge loss/drip loss, the frozen meat samples were weighed and recorded as the initial weight (W1). The weighed samples were placed into polyethylene bags, labelled, and keep hanging at 4 °C for 24 h. The meat samples were weighed once more and final weight (W2) was recorded. Drip loss was calculated as shown in the equation below: To determine the extract release volume (ERV) of meat samples, 15 g samples were blended with 60 mL phosphate buffer solution (0.05 M; pH 5.8) for two minutes and the homogenate was filtered through Whatman filter paper No. 1 for a fixed time period of 15 min to the filtrate measured as ERV 37 . Water holding capacity (WHC) of meat samples was determined by mixing 10 g minced meat sample in 15 mL of 0.6 M NaCl for 2 min followed  Lipid peroxidation parameters. The lipid peroxidation was determined by estimating the thio-barbituric acid reactive substance (TBARS) in the selected meat sample. About 5 g meat sample was extracted in 12.5 mL 20% TCA (made in 2 M orthophosphoric acid) solution for 2 min and the slurry was mixed with 12.5 mL cold distilled water followed by filtration through Whatman paper No. 1. Then 3 mL of filtrate was mixed with 3 mL of TBA reagent (0.005 M), mixture was kept in dark cabinet for 16 h and absorbance (O.D) was measured by a spectrophotometer (UV/VIS, Varian, make up of spectrophotometer) at fixed wavelength of 532 nm against the blank made by mixing of 3 mL of 10% TCA and 3 mL of TBA reagent 39 . TBARS value was calculated as mg malonaldehyde (MDA) per Kg of sample by multiplying O.D value with K-factor of 5.2. The free fatty acid value and peroxide value was determined in the selected meat sample. About 5 g meat sample was blended with 30 mL chloroform for 2 min in presence of anhydrous sodium sulphate powder followed by filtration into conical flask through No. 1 Whatman paper 40 . For free fatty acid value about 2-3 drops of 0.2% phenolphthalein indicator was added to the chloroform extract followed by titration with 0.1 N alcoholic potassium hydroxide to get the pink colour end point. For peroxide value 30 mL of glacial acetic was added to 25 mL of chloroform extract, then 2 mL potassium iodide solution was added, and the mixture was allowed to stand for 2 min with occasional shaking. Then, 100 mL distilled water and 2 mL fresh 1% starch solution were added to the mixture following titration with 0.1 N sodium thiosulphate till the end point was reached (non-aqueous layer turned colourless). The calculations were made as follows: Antioxidant parameters. About 5 g meat sample was triturated in 20 mL ethanol for 2 min followed by filteration through Whatman paper No. 42. For ABTS + (2, 2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) assay 2 mL of ABTS working solution (7 mM) was added to 1 mL filtrate and absorbency was measured by spectrophotometer (UV/VIS, Varian, make up of spectrophotometer) at fixed wavelength of 734 nm after 20 min (At 20 ) 41 . For DDPH (1, 1-diphenyl-2-picrylhydrazyl) assay 1 mL filtrate was mixed with 1 mL 0.1 M Tris-HCl buffer (pH 7.4) and 1 mL DPPH reagent (250 µM). The absorbency was measured immediately (At 0 ) and after 20 min (At 20 ) by spectrophotometer (UV/VIS, Varian) at fixed wavelength of 517 nm 42 . The calculations were made as follows: Microbial load. The microbial load of the meat samples were estimated in terms of specific plate count (SPC), coliform count, and staphylococcus count. About 1 g sample was homogenized with 10 mL of 0.1% peptone water (Hi-media, make up of all agars used in this study) with the aid of sterile pestle and mortar under aseptic condition to give a 10:1 initial dilution. The homogenate was used for the preparation of tenfold serial dilution up to 10 6 :1 with 0.1% peptone water in sterile test tubes. One mL aliquot of each dilution was placed in identified sterile petri dishes aseptically. About 12-15 mL of sterile molten and cooled (45 °C) specified agar (Himedia) was poured on each petri dish and mixed gently. After setting, the plates were incubated at 37 °C for 48 h and colonies were counted using a Quebec colony counter. The counts were multiplied by the respective dilution and calculated per gram of sample as log 10 cfu. Statistical analysis. The experimental unit for the data analysis was the sampled bird. Prior to the analysis, all the data were tested for normality and homogeneity of variances with the Shapiro-Wilk test and Levene's test, respectively. The data were analysed by one way ANOVA by using the General Linear Model procedure (IBM SPSS software-20). However, data of the measurements repeated after 14 days were subjected to mixed model procedure for repeated measure analysis. The Tukey post-hoc analysis was done to test the significant mean differences between the groups with significance level defined at p < 0.05.
Ethical approval. All applicable institutional guidelines for the care and use of animals were followed. The experimental procedures carried out in this study were approved by the Institutional Animal Ethics Committee www.nature.com/scientificreports/

Data availability
The datasets analysed during the current study are available from the corresponding author on reasonable request.