Effect of Bacillus subtilis

Effect of Bacillus subtilis C-3102 spores as a probiotic feed supplement on growth performance, noxious gas emission, and intestinal microflora in broilers J. S. Jeong and I. H. Kim1 Department of Animal Resource and Science, Dankook University, Cheonan, Choongnam, 330-714, Republic of Korea ABSTRACT Bacillus subtilis C-3102 has been used as a direct-fed microbial or probiotic product since 1986 to improve production performance in broilers worldwide. This study was conducted to determine and confirm the effect of B. subtilis C-3102 spore supplementation to feed on growth performance, nutrient digestibility, carcass quality, blood profiles, noxious gas emission, and intestinal and excreta microflora in broilers. A total of 816 one-day-old male Ross 308 broilers (46.06 ± 0.67 g) were used in a 5-wk study with Calsporin, B. subtilis final product (1.0 10 9 cfu/g of B. subtilis). Broilers were randomly allotted to 1 of 3 dietary treatments consisting of 16 replicate cages with 17 broilers each: I) CON (control, basal diet), II) BS300 (CON + 300 mg of B. subtilis/kg of feed), and III) BS600 (CON + 600 mg of B. subtilis/kg of feed). Regarding probiotic effect, B. subtilis significantly increased Lactobacillus counts in the cecum, ileal, and excreta, and reduced Escherichia coli counts in the cecum and excreta, compared with CON. In addition, supplementation also tended to reduce Clostridium perfringens counts in the large intestine and excreta, while linearly reducing Salmonella counts in the cecum, ileal, large intestine, and excreta, compared with CON. Regarding growth performance, B. subtilis enhanced ADG in the starter and overall experimental periods, without any effects on feed intake compared with CON. Consequently, feed conversion ratio in the grower-finisher and overall experimental periods decreased significantly. The inclusion of B. subtilis improved the digestibility of DM and gross energy, as well as reducing ammonia emission, compared with CON. No significant difference in breast muscle color, water holding capacity, and drip loss, and relative organ weights, as well as in white blood cells, red blood cells, lymphocyte counts, and IgG amount, were observed. Overall, B. subtilis C-1302 is capable of providing a probiotic effect leading to improved growth performance and feed efficiency, due to the manipulation of intestinal microflora, with minimal side effects in broilers. Key words: Bacillus subtilis, broiler, excreta microflora, growth performance, intestinal microflora 2014 Poultry Science 93 :3097 3103 http://dx.doi.org/10.3382/ps.2014-04086 INTRODUCTION Restrictions or total bans on the use of growth promoting antibiotics in poultry feed are currently in place, to limit and prevent negative effects associated with overusage, such as the induction of microbial antibiotic resistance (Hooge et al., 2004). As such, alternatives are currently being proposed and sought out, of which probiotics have been specifically targeted for use in the poultry industry (Patterson and Burkholder, 2003; zhang et al., 2012). As a general category, probiotics tend to refer to bacterial cultures capable of 2014 Poultry Science Association Inc. Received April 6, 2014. Accepted August 19, 2014. 1 Corresponding author: inhokim@dankook.ac.kr stimulating intestinal microflora, which in turn are capable of modifying the gastrointestinal environment in a positive manner, benefitting beneficial bacteria and improving the growth performance and feed efficiency of broilers (Tabidi et al., 2013). Contrary to most known probiotics, which are sensitive and unable to survive high temperatures, during feed processing, spore-forming probiotics are metabolically dormant, and very resilient to external conditions, such as low and high ph and extreme high and low temperatures (Nicholson, 2002). Various Bacillus species, including Bacillus subtilis, Bacillus cereus, and Bacillus clausii, have been extensively used as directfed microbials for animals and humans (Spinosa et al., 2000; zhang et al., 2013; Park and Kim, 2014). Moreover, B. subtilis C-3102 spores are widely used as probiotics and their use has reportedly led to modulation of the intestinal microflora to inhibit and prevent prolif- 3097

3098 Jeong and Kim eration of pathogens, thereby rendering improvements to the health, immune status, and performance of broilers (Fritts et al., 2000; Hooge et al., 2004). The main mode of action of B. subtilis C-3102 spores appears to be in their ability to create an anaerobic environment within the intestine after germination, which has been hypothesized to favor growth and proliferation of native microfloral lactobacilli, which can lead to competitive colonization exclusion of pathogenic bacteria and production of lactic acid to control and limit pathogenic bacteria in the intestine. Therefore, the primary objective of this preliminary study is to determine and confirm the effect of feed supplementation of B. subtilis C-3102 spores as a probiotic on growth performance, nutrient digestibility, carcass quality, blood profiles, noxious gas emission, and intestinal and excreta microflora in broilers. Bacterial Strain MATERIALS AND METHODS The B. subtilis C-3102 final product, Calsporin, was provided by a commercial company (Calpis Co. Ltd., Tokyo, Japan) and is composed of spray-dried sporeforming B. subtilis C-3102 endospores. The product was determined to contain at least 1.0 10 9 cfu/g of B. subtilis and was kept in a sterilized container before use. Experimental Design, Birds, Housing, and Diets A total of 816 conventional healthy 1-d-old male Ross 308 broilers (BW of 46.06 ± 0.67 g) were obtained from a local commercial hatchery (Yang Ji Company, Cheonan, Choongnam, South Korea). All birds were raised in stainless-steel pens of identical size (1.75 1.55 m) and provided with continuous light. Room temperature was maintained at 33 ± 1 C for the first 3 d, and then gradually reduced by 3 C a week until reaching 24 C and maintained for the remainder of the experiment. Broilers were randomly allotted into 3 treatments, and each dietary treatment consisted of 272 animals with 16 replicate cages, with 17 broilers in each cage. Dietary treatments included I) basal diet (CON); II) basal diet + 300 mg of B. subtilis/kg of feed (BS300), and III) basal diet + 600 mg of B. subtilis/kg of feed (BS600). The CON diet was formulated to meet or exceed the nutritional requirements of broilers during starter (d 1 to 21) and grower (d 22 to 35) phases, according to the NRC (1994) recommendations for Ross 308 broiler chickens. All broilers were allowed ad libitum access to water and feed and were handled in accordance with the guidelines set forth by the Animal Care and Use Committee of Dankook University (Cheonan, Choongnam, Korea). The ingredient and chemical compositions of the experimental diets used in this study are shown in Table 1. The CP, ME, Ca, P, lysine, and methionine levels in the 3 diets were adjusted to the same level. Sampling and Measurements Broilers were weighed and feed intake was recorded at d 1, 21, and 35. Body weight gain and feed intake were measured, and feed conversion ratio (FCR) was calculated. At d 35, 16 broilers per treatment (1 per cage) were randomly selected, and blood samples were collected from the brachial vein into a sterile syringe. After collection, sample was aliquoted into both a vacuum (clot activator with gel) and K 3 EDTA vacuum tube, separately (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ), and stored at 4 C. Samples for serum analysis were then centrifuged at 3,000 g for 15 min at 4 C, separated, and stored at 4 C. The white blood cells, red blood cells, lymphocyte percentage, and IgG concentrations were analyzed using an automatic blood analyzer (ADVIA 120, Bayer, New York, NY). After blood collection, the same broilers were individually weighed, killed by cervical dislocation, and exsanguinated. The liver, spleen, bursa of Fabricius, left breast muscle, gizzard, and abdominal fat were then removed by trained personnel and weighed. The breast muscle was stored at 20 C for the following analysis. All organ weights were expressed as a percentage of BW. The diets were mixed with 0.2% Cr 2 O 3 during d 28 to 35. At d 35, the ileal digesta from the same cage were collected from 2 broilers per cage during d 34 to 35, pooled and frozen, until being lyophilized and ground. Ileal and feed samples were ground through a 1-mm screen and then analyzed for apparent total tract digestibility (ATTD) of DM (method 934.01, AOAC International, 2000) and nitrogen (method 968.06, AOAC International, 2000). Nitrogen was determined by Kjeltec 2300 Nitrogen Analyzer (Foss Tecator AB, Höganäs, Sweden). Gross energy (GE) was determined by measuring the heat of combustion in the samples, using a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL). Chromium was determined by UV absorption spectrophotometry (Shimadzu, UV-1201, Kyoto, Japan). Meat Quality Analysis Breast muscle samples were collected for meat quality at the end of the experiment. Lightness (L*), redness (a*), and yellowness (b*) of meat color was determined by a Chroma meter (model CR-410, Minolta Co., Tokyo, Japan). Drip loss was measured using approximately 2 g of meat sample, according to the plastic bag method described by Honikel (1998). A 2-cm thick piece of meat was kept in the refrigerator and weighed on 0, 1, 3, 5, and 7 d to calculate drip loss. The waterholding capacity (WHC) was measured according to the methods of Kauffman et al. (1986). In brief, 0.2-g sample was pressed at 3,000 g for 3 min, on 125-mm diameter filter paper. The areas of the pressed sample

RESEARCH NOTE 3099 Table 1. Ingredient composition of experimental diets (as-fed basis) Item Prestarter; crumble Starter; crumble Grower-finisher; pellet Ingredient, 1 % Corn 37.645 37.375 30.035 Wheat 15.00 10.00 25.00 Lupin, dehulled 5.00 6.00 5.00 Tapioca 1.00 1.66 2.00 Wheat bran 0.30 0.30 0.30 SBM, 45% 20.50 19.50 15.10 Rapeseed meal, 38% 3.00 6.00 Corn gluten 2.51 0.58 4.00 DDGS 6.00 8.00 8.00 Fish meal 1.00 Tallow 0.83 5.00 5.00 Soy oil 1.00 Limestone 1.42 1.15 1.41 DCP 1.52 1.31 1.19 Salt 0.15 0.18 0.18 NaHCO 3 0.22 0.10 0.10 Met, 99% 0.35 0.37 0.37 Lys, 24% 1.79 1.78 1.66 Thr, 98.5% 0.19 0.19 0.19 Vitamin premix 2 0.06 0.06 0.045 Vitamin E, 10% 0.03 Choline, 50% 0.13 0.10 0.10 Mineral premix 3 0.10 0.10 0.10 CuSO 4 0.04 0.04 Phytase, 5000FT 0.01 0.01 0.01 NSP enzyme 0.04 0.04 0.04 Antioxidant 0.025 Emulsifier 0.08 0.08 0.08 Organic acid 0.10 0.075 0.05 Total 100.00 100.00 100.00 Chemical composition DM, % 87.16 87.35 87.90 ME, kcal/kg 3,000.48 3,139.87 3,249.92 CP, % 21.98 20.99 19.87 Crude fat, % 4.32 7.38 7.24 Crude fiber, % 2.77 3.14 2.66 Crude ash, % 6.07 5.72 5.17 Ca, % 1.05 0.90 0.90 Total P, % 0.67 0.63 0.56 Lys, % 1.43 1.40 1.18 Met, % 0.68 0.68 0.67 Cys, % 0.37 0.36 0.34 Thr, % 0.99 0.96 0.87 Trp, % 0.23 0.23 0.20 Met + Cys, % 1.07 1.05 1.02 1 SBM = soybean meal; DDGS = dried distillers grains with solubles; DCP = dicalcium phosphate; NS = nonstarch polysaccharides. 2 Provided per kilogram of premix: retinyl palmitate, 4.95 mg; cholecalciferol, 0.09 mg; dl-α-tocopheryl acetate, 37.5 mg; menadione sodium bisulfite, 2.55 mg; thiamine mononitrate, 3 mg; riboflavin, 7.5 mg; cyanocobalamin, 24 mg; niacin, 51 mg; folic acid, 1.5 mg; biotin, 126 mg; pantothenic acid, 13.5 mg. 3 Provided per kilogram of complete diet: Fe (as FeSO 4 7H 2 O), 37.5 mg; Cu (as CuSO 4 5H 2 O), 3.75 mg; Zn (as ZnSO 4 ), 37.5 mg; Mn (as MnO 2 ), 37.5 mg; I (as KI), 0.83 mg; Se (as Na 2 SeO 3 5H 2 O), 0.23 mg. and expressed moisture were delineated and then determined with a digitizing area-line sensor (MT-10S, M. T. Precision Co. Ltd., Tokyo, Japan). The ratio of water:meat areas was calculated, giving a measure of WHC (smaller ratio indicates higher WHC). Excreta and Intestinal Microflora The excreta and content samples of jejunum, colon, and large intestine were used for subsequent analysis of the bacterial populations by serial dilution. To accomplish this, serial dilution (10 2 to 10 7 ) of the contents of the intestine was made using anaerobic diluents and then plated onto MacConkey agar plates (Difco Laboratories, Detroit, MI), lactobacilli medium III agar plates (Difco Laboratories), sheep blood agar base plates (Oxoid, Basingstoke, UK), and Salmonella-Shigella agar plate, to isolate Escherichia coli, Lactobacillus, Clostridium perfringens, and Salmonella, respectively. The lactobacilli medium III agar plates were incubated for 48 h at 39 C, under anaerobic conditions. The Mac- Conkey agar plates, sheep blood agar base plates, and Salmonella-Shigella agar plates were incubated for 24 h at 37 C. Escherichia coli, Lactobacillus, C. perfringens, and Salmonella colonies were counted immediately upon removal from the incubator.

3100 Jeong and Kim Noxious Gas Emission During the last 3 d of the experiment, excreta samples were collected from each replicate (6 hens per replicate), for the analysis of noxious gas emission. The excreta samples were stored in 2.6-L plastic boxes, in duplicates. Each box had a small hole in the middle of one side wall, which was sealed with adhesive plaster. The samples were permitted to ferment for a period of 7 d at room temperature (25 C). After the fermentation period, the Gastec (model GV-100) gas sampling pump was used for gas detection (Gastec Corp., Kanagawa, Japan). In these measurements, the adhesive plaster was punctured and 100 ml of headspace air was sampled approximately 2 cm above the feces. After air sampling, each box was again covered with adhesive plaster. Headspace measurements were again performed every 48 h. The gas contents were averaged by 2 measurements from the same box. Statistical Analysis All data were analyzed with SAS 2003 (v. 9.1, SAS Institute Inc., Cary, NC) using the MIXED procedure, with the following statistical model: Y ijk = μ + t i + r k + e ijk, where Y ijk was an observation on the dependent variable ij, μ was the overall population mean, t i was the fixed effect of B. subtilis C-3102 treatments, r k was the cage as a random effect, and e ijk was the random error associated with the observation ijk. A significant difference level of 0.05 was used to determine statistical significance, and a level of 0.10 was considered a trend. In addition, orthogonal comparisons were conducted, using polynomial regression, to measure the linear and quadratic effects of increasing the dietary concentration of B. subtilis C-3102. RESULTS AND DISCUSSION With antibiotics being eliminated from poultry diets, intestinal pathogen counts need to be reduced to safe levels for the prevention of necrotic enteritis and associated mortality, and for consumer safety. Although some strains of Bacillus are capable of producing antibiotics, enzymes, or both, B. subtilis C-3102 has a different main mode of action to improve intestinal health in broilers (Hooge et al., 2004). Spores are popular as direct-fed microbials, though little is known about their mode of action. It has been hypothesized that B. subtilis C-3102 spores can favor beneficial intestinal microfloral lactic acid-producing bacteria, by creating a positive anaerobic environment due to rapid oxygen consumption resulting from germination (Hoa et al., 2000). Lactobacilli are facultative anaerobes, which proliferate under anaerobic conditions, resulting in enzyme production and competitive exclusion of colonization of pathogenic bacteria. In addition, Lactobacilli can produce lactic acid, which can function as a natural antimicrobial, disrupting the outer membrane of gramnegative bacteria (Alakomi et al., 2000), and reduces the intestinal ph, causing inhibition of the growth of pathogenic bacteria (Spring et al., 2000). Our results demonstrated that B. subtilis supplementation significantly increased Lactobacillus counts in the cecum (linear, P = 0.002), ileum (linear, P = 0.024), and excreta (linear, P = 0.022), whereas it significantly reduced E. coli counts in the cecum (linear, P < 0.001) and excreta Table 2. Effect of Bacillus subtilis C-3102 supplementation on intestinal and excreta microflora in broilers Item, log 10 cfu/g Cecum Lactobacillus 6.25 6.59 7.16 0.122 0.002 0.63 Escherichia coli 6.12 5.74 5.43 0.073 <0.001 0.77 Clostridium perfringens 3.00 2.92 2.78 0.123 0.19 0.82 Salmonella 2.66 2.23 2.13 0.077 0.004 0.27 Ileal Lactobacillus 7.03 7.32 7.63 0.109 0.025 0.95 E. coli 5.91 5.71 5.38 0.238 0.37 0.91 C. perfringens 2.87 2.79 2.77 0.112 0.19 0.52 Salmonella 2.87 2.58 2.42 0.061 0.002 0.87 Large intestine Lactobacillus 6.50 6.71 7.07 0.213 0.28 0.87 E. coli 4.94 4.80 4.51 0.242 0.47 0.88 C. perfringens 3.14 2.97 2.86 0.052 0.058 0.93 Salmonella 3.04 2.59 2.44 0.064 <0.001 0.19 Excreta Lactobacillus 6.98 7.12 7.29 0.054 0.022 0.87 E. coli 6.26 5.81 5.77 0.102 0.048 0.33 C. perfringens 3.31 3.17 3.03 0.124 0.067 0.59 Salmonella 2.88 2.76 2.61 0.053 0.044 0.86

RESEARCH NOTE 3101 Table 3. Effect of Bacillus subtilis C-3102 supplementation on growth performance in broilers Item Prestarter (d 0 7) 2 ADG, g 114 119 119 1.1 0.11 0.33 ADFI, g 140 143 143 1.1 0.28 0.48 FCR 1.23 1.23 1.21 0.011 0.41 0.63 Starter (d 8 21) ADG, g 658 685 700 7.3 0.017 0.69 ADFI, g 980 979 1,003 6.4 0.14 0.34 FCR 1.49 1.43 1.45 0.016 0.227 0.26 Grower-finisher (d 22 35) ADG, g 800 826 843 11.6 0.14 0.87 ADFI, g 1,435 1,393 1,404 17.8 0.48 0.49 FCR 1.79 1.69 1.67 0.018 0.004 0.24 Overall (d 0 35) ADG, g 1,573 1,630 1,662 12.9 0.004 0.63 ADFI, g 2,555 2,515 2,550 20.1 0.92 0.39 FCR 1.63 1.54 1.54 0.011 <0.001 0.071 2 FCR = feed conversion ratio. (linear, P = 0.048) compared with CON (Table 2). Bacillus subtilis supplementation tended to reduce C. perfringens counts in the large intestine (linear, P = 0.058) and excreta (linear, P = 0.067), while significantly reducing Salmonella counts in the cecum (linear, P = 0.004), ileum (linear, P = 0.002), large intestine (linear, P < 0.001), and excreta (linear, P = 0.044), compared with CON (Table 2). Overall, these results corroborate with some recent broiler studies (Hooge, 2007; Baltzley et al., 2010; Knap et al., 2011), thus confirming that broiler intestinal microflora can be possibly manipulated by supplementation of B. subtilis C-3102, thereby supporting its potential use as a probiotic alternative for use with broilers. Broilers grown in a pathogen-free environment grow 15% faster than those grown under conventional conditions, where they are exposed to bacteria and viruses (Klasing et al., 1987). The inclusion of B. subtilis spores significantly enhanced the ADG in the starter period (linear, P = 0.017), and overall experimental period (linear, P = 0.004), but not in the prestarter and grower-finisher periods, compared with CON (P > 0.10; Table 3). However, B. subtilis treatment was ineffective on ADFI compared with CON. Consequently, FCR in the grower-finisher and overall experimental periods decreased significantly (linear, P = 0.004 and P < 0.001, respectively) and tended to decrease during the overall experimental period (quadratic, P = 0.071), compared with CON. Overall, the results corroborate studies by Fritts et al. (2000), Hooge et al. (2004), and Knarreborg et al. (2008), which all suggest that the effects of supplementing a broiler diet with Bacillus species can improve growth performance. However, there are some conflicting studies. For example, Knap et al. (2011) reported nonsignificant differences in ADG and FCR with B. subtilis DSM17299 supplementation. Similarly, Albino et al. (2000) reported nonsignificant changes in ADG and FCR after B. subtilis C-3102 supplementation. In addition, Santoso et al. (1999) reported that supplementation with B. subtilis does not affect ADG, ADFI, and FCR, compared with CON. We speculate that the variation in the results of the aforementioned studies can be ascribed to several factors, including the age of the animals, the dose of Bacillus species, diet composition, feed form, and interaction with other dietary feed additives (Chesson, 1994). Nonetheless, our results indicate that the supplementation of Bacillus species in broilers can improve growth performance, which significantly improved FCR in a consistent fashion. In addition, our results also demonstrated that the ATTD of DM (linear, P = 0.021) and GE (linear, P = 0.030) were improved by dietary supplementation with B. subtilis C-3102, compared with CON (Table 4), thereby indicating that the tendency for improved Table 4. Effect of Bacillus subtilis C-3102 supplementation on nutrient digestibility in broilers Item DM 73.4 76.5 77.2 0.68 0.021 0.37 Nitrogen 65.3 66.2 67.8 1.19 0.40 0.91 Gross energy 75.3 78.8 79.9 0.86 0.030 0.49

3102 Jeong and Kim Table 5. Effect of Bacillus subtilis C-3102 supplementation on noxious gas emission in broilers Item, ppm Ammonia 9.9 8.2 7.8 0.33 0.009 0.32 Mercaptan 11.8 11.4 10.7 0.41 0.30 0.81 Hydrogen sulfide 35.1 34.8 33.1 0.49 0.58 0.84 Acetic acid 16.5 16.1 15.3 0.88 0.59 0.94 ADG and FCR may occur as a result of a significant improvement in digestibility, but not in ADFI, which is partly in accordance with Abaza et al. (2008), who reported that the supplementation of B. subtilis and Saccharomyces cerevisae to broiler diets significantly improved the digestibility of DM. The effects of singular supplementation of B. subtilis C-3102 or Bacillus species on nutrient digestibility in broilers requires further investigation. Ammonia is a major pollutant contributing to manure malodor, which is an acute environmental problem associated with intensive animal agriculture (Kristensen and Wathes, 2000) that must be resolved because it can adversely affect the health of animals and workers (Wang et al., 2009). Ferket et al. (2002) have suggested that fecal odor and ammonia emission are related to nutrient utilization and the intestinal microflora ecosystem. Interestingly, because B. subtilis supplementation has been purported to influence the intestinal microflora ecosystem, there are reported studies of dietary B. subtilis supplementation being capable of reducing ammonia emission in poultry by improving the activity of enzymes and the utilization of nitrogen (Zhang and Kim, 2013). In our study, ammonia emission was significantly lower in BS 300 and BS600 than that of CON (linear, P = 0.009), whereas no significant differences were observed in mercaptan, hydrogen sulfide, or acetic acid, compared with CON (P > 0.10; Table 5). In addition, B. subtilis supplementation did not affect the ATTD of nitrogen (Table 4). Overall, our results demonstrated that B. subtilis supplementation resulted in a significant reduction in ammonia emission, without any accompanying improvement in nitrogen digestibility, in spite of the fact that some Lactobacillus strains are capable of nitrogen fixation. Further study is needed to elucidate the possible mechanism(s) of reducing ammonia emission from feces. Lastly, the potential use of B. subtilis C-1302 as a probiotic replacement for broilers appears to be very positive, with no negative side effects being observed. Broilers fed the B. subtilis-supplemented diets did not differ in breast muscle color (L*, a*, and b*), WHC, or drip loss, compared with those fed CON (P > 0.10). Moreover, no significant differences were observed in the relative organ weights for B. subtilis-supplemented diets compared with CON (P > 0.10; data not shown). In addition, when examining blood profiles, B. subtilis supplementation did not affect white blood cells, red blood cells, lymphocyte counts, or IgG amount, compared with CON (P > 0.10; data not shown). In conclusion, B. subtilis C-1302 supplementation at 300 and 600 mg/kg of feed produced beneficial changes to intestinal and excreta microflora (increased Lactobacillus counts and reduced E. coli, C. perfringens, and Salmonella counts), resulting in favorable effects on growth performance (increased ADG and decreased FCR), nutrient digestibility (higher ATTD of DM and GE), and noxious gas emission (reduced ammonia) in broilers. REFERENCES Abaza, I. M., M. A. 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