Effect of feeding inulin oligosaccharides on cecum bacteria, egg quality and egg production in laying hens

Inulin is reported to improve the egg quality and production of laying hens. In the present study, we investigated the dietary effects of microcapsulated inulin oligosaccharide (INO) which is manufactured from Jerusalem artichoke (Helianthus tuberosus L.) on the cecum bacteria, egg quality and production of laying hens. 400 laying hens were randomly allocated to one of the following four treatment groups for 10 weeks: T1 (control without INO or inulin), T2 (200 mg INO/kg diet), T3 (250 mg INO/kg diet) and T4 (300 mg INO/kg diet). Egg production, Haugh unit, egg shell thickness and breaking strength were significantly higher in the T3 and T4 groups than in the T1 and T2 groups (P<0.05). The level of egg cholesterol was highest in the T1 group and decreased in the INO addition groups from 5.68 to 8.46% (P<0.05). When compared with the T1, triglycerides in the blood and total cholesterol decreased significantly in the T2, T3 and T4 groups by 11.75 to 13.45% and 9.41 to 9.85%, respectively (P<0.05). The growth of cecum Bifidobacterium and Lactobacillus was stimulated in the T2, T3 and T4 when groups compared with the T1 group, while the growth of Escheria coli and Salmonella was clearly inhibited (P<0.05). The results of this study demonstrate that the addition of microcapsulated inulin oligosaccharide (250 mg/kg) into a laying hen’s diet can promote the multiplication of beneficial cecum bacteria and simultaneously improve egg production and quality.


INTRODUCTION
A recent review demonstrated that indissoluble oligosaccharide, inulin extracted from the Jerusalem artichoke can be used as a prebiotic, which acts as an antibacterial promoter for the poultry diet (Rehman et al., 2008). Prebiotics can reach the lower gastrointestinal tract such as the colon and cecum without being hydrolyzed in the upper gastrointestinal tract of humans and animals, inhibiting the growth of pathogenic bacteria such as Salmonella, and simultaneously providing a carbohydrate substrate to promote the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus strains (Zsolt et al., 2011;Gibson and Rastall, 2006;Patterson and Burkholder, 2003). Inulin is a linear fructose polymer in which the fructose units are joined by a β (2→1) glycosidic linkage. More than 80% of *Corresponding author. E-mail: bspark@kangwon.ac.kr.  inulin reaches the large intestine without being hydrolyzed by animal gastric fluid and digestive enzymes, and they are used as a growth substrate for intestinal bacteria. Inulins are known as prebiotics because they selectively stimulate the growth of beneficial Lactobacillus and Bifidobacterium and have bifidogenic effects (Rehman et al., 2008). They have also been identified to promote gastrointestinal mineral absorption and modulate lipid metabolism (Azorin-Ortuno et al., 2009).
It has been established that inulin alone is partially broken down by acid in the upper gut. Inulin is also vulnerable to degeneration by air during commercial distribution. To address these problems, the production of inulin oligosaccharide (INO) using microcapsules has been developed (Park, 2008;Dorotea and Maris, 2005). These INO are reported to improve the immune capability of broilers and the growth capability of intestinal beneficial bacteria (Park and Park, 2011a), as well as to promote increased quality of chicken meat and longer storage life (Park and Park, 2011b). Frank (1999) reported that dietary inulin in layers improved the egg shell intensity by reducing the egg cholesterol through increased mineral absorption from the gut. However, there are virtually no reports regarding the effects of INO's addition and feeding on laying hens. Therefore, this study was conducted to examine the effects of adding varying levels of INO to the layer diet on egg production, egg quality, blood lipid and changes in cecum bacteria.

MATERIALS AND METHODS
All experimental procedures were approved by the IACUC (Institutional Animal Care and Use Committee of Kangwon National University, Republic of Korea).

Preparation of inulin oligosaccharides
Inulin with a mean degree of polymerization of 26 was extracted from Jerusalem artichokes (Helianthus tuberosus L.) using the hot water and cooling extraction method suggested by French (1989), and was subsequently freeze dried. 15 ppm of vitamin E and inulin were mixed with 70°C warm water, and a high pressure homogenate was obtained with a high pressure homogenizer (T25 Basic, IKA, German). Ultrafine powder coating materials (high pressure homogenate 9: Sureteric 1) were manufactured by shooting Sureteric (Colorcon, UK) in the form of an intestine-soluble film coated tablet, into the high-pressure homogenate with Park and Park 9517 compressed air. INO containing more than 90% inulin with a particle size of 100 to 200 μm was manufactured by drying with a spray dryer Buchi,Sweiss) (Park and Park, 2011a, b (Park and Park, 2011a,b;Park, 2008) and preliminary experiments.

Animal feeding and management
All scientific procedures involving experimental animals complied with the scientific and ethical procedures suggested by Swanson (2008). Experimental diets for treatment groups either satisfied or slightly exceeded the nutrient requirements recommended by the NRC (1994). Experimental diets mainly consisted of corn and soybean meal, and the different addition levels of INO were replaced by the same amount of corn (Table 1). Lighting during the laying period was adjusted to 17 h, and the experimental diet was provided ad libitum and they had access to water freely.

Egg production and quality
With egg production and weight recorded daily and dietary intake examined weekly, all the data were expressed as the mean values of the entire experimental period. Evaluation of egg quality measured from 20 eggs was selected through the average weight per treatment every week. Haugh unit (HU), egg shell thickness, egg shell breaking strength and egg yolk color was measured immediately in collected eggs using the egg multi tester EMT-5200 (Robotmation Co. Ltd. Tokyo. Japan).

Cholesterol and fatty acid of eggs
Lipids from the egg yolk were extracted according to the method developed by Folch et al. (1957). Egg cholesterol and fatty acid composition measured from 20 eggs were selected through the average weight per treatment every week. The gas chromatographic system (model GC-15A, Shimadzu Corp., Kyoto, Japan), which is equipped with a flame ionization detector, auto feeder (

Blood lipid
Eight chickens were randomly selected from each treatment group  once a week. 1 ml of blood was collected from the carotid artery of each laying hen using a syringe with a Hamilton 25 gauge needle into heparinized tubes (Becton Dickinson, Franklin Lakes, NJ USA), and the plasma was separated. Triglycerides, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol were analyzed by a commercial enzyme kit (Sigma Co. Ltd., USA) using the hematology Autoanalyzer (7150, Hitachi, Tokyo, Japan).

Cecum bacteria
20 laying hens were randomly selected from each treatment group at the end of the experiment and sacrificed by cervical dislocation, in accordance with recommendations for the euthanasia of experimental animals (Close et al., 1997). Cultures for cecum bacteria were obtained using sterilized selective plate media; Salmonella and E. coli were cultured at 37°C for 24 h under aerobic conditions, and Lactobacillus and Bifidobacterium were stationary cultured at 37°C for 48 h under anaerobic conditions in sealed anaerobic jars equipped with AnaeroGen sachets. All the numbers of bacterial colonies were presented in the common log as CFU (colony forming unit/g of wet of cecum content).

Statistical analysis
The statistical treatment of all analyzed data involved calculating the mean and standard error of each treatment group using the GLM procedure from SAS (2005), conducting ANOVA, and then verifying the statistical significance between treatment mean values using the Duncan's multiple range test at the 95% level (P<0.05). The statistical analysis indicated the degrees of freedom with four repeated measurements.

RESULTS
The effects of addition of different levels of INO on egg production, egg weight and dietary intake are presented in Table 2. Egg production, egg weight and dietary intake were significantly higher in the T3 and T4 groups than in the T1 and T2 groups (P<0.05), and there were no statistically significant differences between the T3 and T4 and between T1 and T2 groups. The results of this study confirm that supplementing the diet for laying hens with INO (250 mg INO kg/diet) can greatly increase egg production and weight.
The effects of adding different INO levels to the laying hen diet with regards to plasma lipids are presented in Table 3. Plasma triglycerides, total cholesterol and low density lipoprotein cholesterol content were significantly lower in T2, T3 and T4 groups than in the T1 group, and the ranges of decrease in the plasma triglycerides, total cholesterol and low density lipoprotein cholesterol in the INO addition group were 11.75 to 13.45, 9.41 to 9.85 and 4.46 to 4.89%, respectively (P<0.05).
The effects of the INO supplemental diet on HU, egg shell thickness, egg shell breaking strength and egg yolk color are shown in Table 4. HU, egg shell thickness and egg shell breaking strength were significantly higher in   the T3 and T4 groups than in the T1 and T2 groups (P<0.05), and there are no statistically significant differences between the T3 and T4 groups and between T1 and T2. Also, there was no statistically significant difference in the egg yolk color among treatment groups.
The composition of cholesterol and fatty acid in the eggs of laying hens fed INO are presented in Tables 5 and 6. The cholesterol content in eggs was highest in the T1 group followed in decreasing order of T4, T3 and T2 groups. There were statistically significant differences between treatment groups (P<0.05). As compared to the control group, the decreased rate of cholesterol for eggs in the INO addition group ranged from 5.68 to 8.46%. The composition of saturated fatty acid in eggs was lower in T3 and T4 groups as compared to T2 and T1 groups, while unsaturated fatty acid levels were significantly higher (P<0.05). There were no statistically significant differences between T3 and T4 and between T2 and T1. The effects of different levels of INO addition on the changes of bacteria in the cecum content of laying hens are presented in Table 7. The growth of cecum beneficial bacteria Bifidobacterium and Lactobacillus was higher in the T2, T3 and T4 groups as compared to T1, while that of harmful Escherichia coli and Salmonella was clearly inhibited (P<0.05).

DISCUSSION
In our results, we verified that a feeding diet with INO can further improve the egg production as compared to the control diet without INO or inulin. There are some reports that inulin increases the absorption rate of nutrition in the upper gut of animals and provides an excellent substrate for Bifidobacterium and Lactobacillus, leading to improved immune capabilities (Absolonne et al., 1995;Roberfroid, 1993). It is estimated that egg production is lower in the control group without INO or inluin than in the INO groups due to the reasons described above.
Inulin is known to decrease the level of blood lipid in humans and animals (Fiordaliso et al., 1998). Causey et al. (2000) reported similar results involving a chicory inulin supplement for humans with hypercholesterolemia that reduces blood triglycerides, total cholesterol and low density lipoprotein cholesterol. Inulin reduces blood triglycerides because short chain fatty acids generated from inulin decrease the synthesis of de novo fatty acid, inhibiting the entire lipogenic enzymes' gene expression (Delzenne and Kok, 1999). Inulin reduces blood cholesterol because the short chain fatty acids inhibit the biosynthesis of cholesterol in the liver, and propionic acids influence its colonic absorption path and the modulation of carbohydrate and lipid metabolism (Wolever et al., 1995). Moreover, blood cholesterol is also removed in part by the Bifidobacterium and Lactobacillus in fermentation products (Van and Schaafsma, 1996). Another way in which inulin reduces blood cholesterol is by inhibiting the liver's cholesterol synthesis, through both the increased excretion of bile acids and the inhibition of activity of HMG-CoA reductase, a restriction enzyme associated with cholesterol synthesis (Kim and Shin, 1998). The reduction of blood lipid in the INO addition group is considered a part of this particular mechanism.
It is estimated that the egg HU, eggshell thickness and eggshell breaking strength are higher in the INO addition group (250 mg INO kg/ diet) than in the control group because of the increased absorption rate of nutrients of inulin and minerals such as calcium, a main component of eggshells (Azorin-Ortuno et al., 2009;Lopez et al., 1998). In addition, inulin is reported to improve the mineral absorption rate through the synergistic action of multiple Bifidobacterium and short chain fatty acid in the human colon (Mitsuoka, 1990), which mirrors the results of this study. HU, eggshell thickness, eggshell breaking strength and egg yolk color are important factors in determining overall egg quality. As consumers are increasingly pursuing physical well-being, internal and external egg quality become increasingly important for enhancing the egg's marketing value, and HU in particular can be used as a standard for internal egg quality (Lesson and Summers, 1991).
In this study, it is estimated that a greater cholesterol decrease in eggs of the INO addition group reflects the lowest total cholesterol level in the blood of laying hens (Table 3). It is well known that cholesterol in animals is immediately transported through the blood to body tissues and eggs and then accumulated in vivo. Also, the results of this study are supported by the report that inulin has the effect of inhibiting lipid synthesis and promoting lipid degeneration (Causey et al., 2000). The lower saturated fatty acid and higher unsaturated fatty acid in the INO addition group (250 mg INO kg/diet) may be related to the biosynthesis and metabolism of short chain fatty acids generated in the lower gastrointestinal tract by inulin inflow into the liver through the hepatic portal vein (Scheppach, 1994).
The number of cecum beneficial bacteria is higher and the number of harmful bacteria is lower in the INO addition group because the microencapsulated INO bypasses the stomach and the small intestine of laying hens, reducing the decomposition rate and absorption rate of inulin. Meanwhile, most of the INO are simultaneously transported to the cecum, and inulin eventually dissolved in the cecum is utilized as a substrate for the growth of Lactobacillus and Bifidobacterium (Gong et al., 2002). Park (2008) and Rada et al. (2001) support this study's results by reporting that inulin supplements for broilers and laying hens significantly increase Bifidobacterium in the cecum. The reduction of E. coli and Salmonella in the INO group is due to the significant increase of Bifidobacterium and Lactobacillus in the cecum and the strong antibacterial activity of inulin (Ahn et al., 2007;Park, 2008).

Conclusion
This study demonstrates that supplementing the layer diets with microcapsulated inulin oligosaccharides resulted in selective stimulation of beneficial Bifidobacterium and Lactobacillus growth and inhibition of harmful bacteria in the cecum and also improved egg production and quality.