Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Effects of different Bacillus licheniformis and Bacillus subtilis ratios on nutrient digestibility, fecal microflora, and gas emissions of growing pigs

  • Kim, Yong Ju (Division of Food and Animal Science, Chungbuk National University) ;
  • Cho, Sung Bo (Traditional Mongolian Medicine Research Institute, Inner Mongolia University for Nationalities) ;
  • Song, Min Ho (Department of Animal Science and Biotechnology, Chungnam National University) ;
  • Lee, Sung Il (Division of Food and Animal Science, Chungbuk National University) ;
  • Hong, Seok Man (Woosung Feed Co., Ltd.) ;
  • Yun, Won (Woosung Feed Co., Ltd.) ;
  • Lee, Ji Hwan (Division of Food and Animal Science, Chungbuk National University) ;
  • Oh, Han Jin (Division of Food and Animal Science, Chungbuk National University) ;
  • Chang, Se Yeon (Division of Food and Animal Science, Chungbuk National University) ;
  • An, Jae Woo (Division of Food and Animal Science, Chungbuk National University) ;
  • Go, Young Bin (Division of Food and Animal Science, Chungbuk National University) ;
  • Song, Dong Cheol (Division of Food and Animal Science, Chungbuk National University) ;
  • Cho, Hyun Ah (Division of Food and Animal Science, Chungbuk National University) ;
  • Kim, Hyeun Bum (Department of Animal Resource and Science, Dankook University) ;
  • Cho, Jin Ho (Division of Food and Animal Science, Chungbuk National University)
  • Received : 2022.01.20
  • Accepted : 2022.02.14
  • Published : 2022.03.31
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Abstract

The objective of this study was to evaluate the effects of different mixing ratios of Bacillus licheniformis and Bacillus subtilis in diets on nutrient digestibility, fecal microflora, and odor gas emissions of growing pigs. A total of four crossbred ([Landrace × Yorkshire] × Duroc) barrows with average body weight (BW) of 41.2 ± 0.7 kg were randomly allotted four diets over four periods in a 4 × 4 Latin square design. Treatments were as follows: Control (CON, basal diet), CON + 0.2% probiotic complex (L4S6, B. licheniformis and B. subtilis at a 4:6 ratio), CON + 0.2% probiotic complex (L5S5, B. licheniformis and B. subtilis at a 5:5 ratio), CON + 0.2% probiotic complex (L6S4, B. licheniformis and B. subtilis at a 6:4 ratio). Dietary probiotic supplementation showed higher crude protein (CP) digestibility values and lower Escherichia coli counts in fecal samples than the CON group (p < 0.05). There was no significant difference in NH3 or H2S emission until day 3. The positive effect of H2S and NH3 emissions was detected earlier with the L4S6 and L5S5 compared to the L6S4, which had a lower ratio of B. subtilis. Both the L4S6 and L5S5 probiotic complexes significantly decreased the fecal H2S and NH3 emission in days 4 and 6 (p < 0.05). On day 7, all probiotic complexes decreased (p < 0.05) H2S and NH3 emissions than the CON group. Our results agreed that the dietary supplementation of Bacillus licheniformis and Bacillus subtilis complexes in growing pigs can significantly improve CP digestibility and reduce fecal E. coli counts, NH3 and H2S emissions. Notably, the higher mixing ratio of Bacillus subtilis in probiotic supplementation is more effective in reducing the odor of manure.

Keywords

INTRODUCTION

Antibiotics and chemical therapeutics as feed additives have been used constantly in the pig industry for economic benefits, improving feed efficiency [1]. Nevertheless, due to the increases of antibiotic-resistant pathogens, many countries such as Europe, China, Korea, and the United States have banned the use of antibiotics [2–5]. In addition, as a result of high nutrient values in feed for high growth performance, the pig industry has faced minimizing acute environmental problems such as harmful gas emissions [6–8]. Furthermore, fermentation of undigested dietary proteins and endogenous proteins in the large intestine produces toxic metabolites. The metabolites contribute to compromise epithelial integrity and promote intestinal disorders such as postweaning diarrhea [9–11].

Probiotic supplementation has been used to overcome the above-mentioned problems [12–14]. Probiotics can accelerate the breakdown of the carbohydrates which are resistant to digestion, thus it encourages the use of dietary fiber as a prebiotic substance, and promotes the substrate availability of colonic microbiota, and its population [15]. It has been extensively studied probiotics are able to support growth performance, gut micro-environment, feed utilization efficiency, immune system, and gastrointestinal tract (GIT) diseases for farm animals [16, 17]. Currently, the main groups of probiotics, commonly used in animal feeds are lactic acid bacteria, yeast, and Bacillus [18]. Among them, Bacillus-based probiotics, especially Bacillus subtilis and Bacillus licheniformis, are widely used as their spore-forming properties facilitate expanding the storage of feed and the resistance of low pH in the stomach [19–21].

The proliferating B. subtilis in the intestine help to maintain an anaerobic environment and lower pH in GIT, which in turn promotes lactic acid and regulates intestinal microflora. It can prevent and minimize GIT diseases [22]. Also, B. licheniformis can produce bacitracin, which is against pathogenic microorganisms [23, 24]. Bacitracin is a cyclic dodecyl-peptide antibiotic synthesized non-ribosomally by B. licheniformis [25]. Bacitracin is directed primarily against gram-positive bacteria via inhibition of the cell wall [26]. Both B. subtilis and B. licheniformis increased fecal Lactobacillus counts in finishing pigs without affecting fecal coliform counts [27]. Additionally, B. licheniformis and B. subtilis can survive in fecal after excretion and, they sustainedly degrade organic matters in the fecal [28], which could decrease fecal odor and reduce gas formation such as NH3 production. When growing pigs were given Bacillus direct-fed microbial (DFM), methane and NH3 emissions were reduced by 40% and 50%, respectively [29]. Thus, the Bacillus DFM has positive effects on fermentation and protein utilization in older animals.

Accordingly, it has been well-established that dietary Bacillus complex supplementation has beneficial effects on weaned to finishing pigs [30–33]. The 1:1 ratio of B. licheniformis and B. subtilis in growing-finishing pigs increased digestibility and fecal Lactobacillus and reduced fecal NH3 emission [34]. However, there is limited research on the mixing ratio of Bacillus complex in dietary supplementation for pigs. Therefore, this study evaluated the effects of different mixing ratios of B. licheniformis and B. subtilis on nutrient digestibility, fecal microflora, and odor gas emissions in growing pigs.

MATERIALS AND METHODS

The experimental protocol was approved (CBNUA-1619-21-02) by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea.

Source of probiotics

Probiotic product (Haedamun, Eumseong, Korea) was a mixture of spray-dried spores of B. licheniformis (CCTCC WL-04) and B. subtilis (CCTCC WL-22). It was guaranteed to contain at least 3 × 1010 CFU kg−1 of B. licheniformis and B. subtilis, respectively.

Experiment design and housing

A total of four crossbred ([Landrace × Yorkshire] × Duroc) barrows were randomly allotted to four diets over four periods in a 4 × 4 Latin square design. The pigs (average initial body weight [BW] of 41.2 ± 0.7 kg) were individually housed in 1.2 m × 0.7 m × 0.96 m stainless steel metabolism cages in an environmentally controlled room.

Diets and feeding

Diets were prepared to meet the NRC [35] nutritional requirements for pigs. Table 1 shows nutritional contents of the main ingredients used in this experiment. Treatments were as follows: Control (CON, basal diet), CON + 0.2% probiotic complex (L4S6, B. licheniformis and B. subtilis at a 4:6 ratio), CON + 0.2% probiotic complex (L5S5, B. licheniformis and B. subtilis at a 5:5 ratio), CON + 0.2% probiotic complex (L6S4, B. licheniformis and B. subtilis at a 6:4 ratio). The experiment was conducted for four weeks. The daily feed allowance was adjusted by 2.7 times the requirement to maintain digestible energy (DE, 2.7 × 110 kcal of DE/kg BW0.75) [35]. The daily feed was divided in half and mixed with water in a 1:1 ratio and fed at 8 and 17 o’clock. During the experiment, the pigs were allowed to drink water freely.

Table 1. Chemical composition of basal diets (%, as-fed basis)

1) Provided per kg of complete diet: vitamin A, 11, 025 IU; vitamin D3, 1, 103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; D-pantothenic, 29 mg; choline, 166 mg; and vitamin B12, 33 μg.

2) Provided per kg of complete diet: copper (as CuSO4 · 5H2O), 12 mg; zinc (as ZnSO4), 85 mg; manganese (as MnO2), 8 mg; iodine (as KI), 0.28 mg; and selenium (as Na2SeO3 · 5H2O), 0.15 mg.

Sampling and analysis

Each week, the experiment consisted of six days of adaptation period and one day of collecting urine and feces. Total feces were immediately collected in metabolic cages and packaged in plastic bags and stored at −20℃ for the duration of the experiment. Urine was collected once a day into buckets filled with 50 mL of 6 mol/L HCl under metabolic cages. The total urine collected was weighed and stored at −20℃. Fecal samples were dried in a forced air oven, then crushed on 1 mm screens and completely thawed prior to subsample collection for chemical analysis. The procedures used for the determination of dry matter (DM), and crude protein (CP) digestibility values were in accordance with the methods established by the AOAC [36]. 1 g of fecal samples from each cage were diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and homogenized. The viability of bacteria in fecal samples was obtained by plating a series of 10-fold dilutions (1% peptone solution) on MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and Lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany). Lactobacilli medium III agar plates were incubated anaerobically at 39℃ for 48 h. MacConkey agar plates were incubated at 37℃ for 24 h. The number of Escherichia coli or Lactobacillus colonies was measured immediately after removing the plate from the incubator.

For the odor gas estimation, 150 g of fresh fecal, 100 g of sawdust, and 50 g of urine were mixed. Mixtures of fecal, urine and sawdust were fermented at room temperature of 35℃ for 72 hours. The odor-causing materials (NH3 and H2S) were analyzed every 24 hours for 7 days with a gas detector (GV-110S, Gastec, Ayase, Japan) and tube namely, NH3 detector tube No. 3L, H2S detector tube No. 4LL.

Statistical analysis

Data generated in the present experiment were analyzed as a randomized design in a 4 × 4 Latin square arrangement of treatments. Data collected during the study were subjected to analysis of variance (ANOVA) for Completely Randomized Design [37] using General Linear Model Procedure (SAS, SAS Institute, Cary, NC, USA). The statistical model used to test the effects of treatment on nutrient digestibility, fecal microflora, and odor gas emissions are presented as follows: Yij = μ + Pi + Eij. Where: Yij = Observed value of a dependent variable; μ = Overall mean; Pi = Effect of different mixing ratios of B. licheniformis and B. subtilis; and Eij = Residual error. The differences between means were tested for significance (p < 0.05) using the least significant difference (LSD) range test.

RESULTS

Nutrient digestibility

Effects of dietary supplementation with B. licheniformis and B. subtilis probiotics in different mixing ratios on nutrient digestibility in growing pigs are shown in Table 2. CP digestibility values were greater (p < 0.05) for probiotic supplementation treatments than for the CON diet. However, there was no significant difference in CP digestibility and DM digestibility among all treatments.

Table 2. Effects of supplementation with Bacillus licheniformis and Bacillus subtilis probiotics at different mixing ratios on nutrient digestibility in growing pigs

Each value is presented as the mean value of four replicates (1 pig / cage; 4 × 4 latin square).

a,bMeans in the same row with different superscripts differ significantly (p < 0.05).

CON, basal diet; L4S6, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 4:6 ratio); L5S5, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 5:5 ratio); L6S4, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 6:4 ratio)

Fecal microflora

Effects of dietary supplementation with B. licheniformis and B. subtilis probiotics at different mixing ratios on fecal microflora in growing pigs are shown in Table 3. E. coli counts in fecal samples were lower (p < 0.05) at the probiotic supplementation treatments than the CON diet. However, there was no significant difference in E. coli counts between the different mixing ratios of B. licheniformis and B. subtilis. All treatments did not show a significant difference in Lactobacillus counts.

Table 3. Effects of supplementation with Bacillus licheniformis and Bacillus subtilis probiotics at different mixing ratios on fecal microflora in growing pigs

Each value is presented as the mean value of four replicates (1 pig / cage; 4 × 4 latin square).

a,bMeans in the same row with different superscripts differ significantly (p < 0.05).

CON, basal diet; L4S6, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 4:6 ratio); L5S5, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 5:5 ratio); L6S4, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 6:4 ratio)

Odor gas emissions

Effects of dietary supplementation with B. licheniformis and B. subtilis probiotics at different mixing ratios on odor gas emissions in growing pigs are shown in Table 4. There was no significant difference in NH3 or H2S emission until day 3. On days 4 and 5, the L4S6 and L5S5 showed lower (p < 0.05) fecal H2S emissions than the CON diet. On day 6, the L6S4 also showed lower H2S emission (p < 0.05). NH3 emission in the L4S6 and L5S5 was significantly decreased (p < 0.05) compared to that in the CON diet. On day 7, all of probiotics supplementation decreased (p < 0.05) both NH3 and H2S emissions than the CON treatment.

Table 4. Effects of supplementation with Bacillus licheniformis and Bacillus subtilis probiotics at different mixing ratios on odor gas emissions in growing pigs

Each value is presented as the mean value of four replicates (1 pig / cage; 4 × 4 latin square).

a,bMeans in the same row with different superscripts differ significantly (p < 0.05).

CON, basal diet; L4S6, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 4:6 ratio); L5S5, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 5:5 ratio); L6S4, CON + 0.2% probiotic complex (B. licheniformis and B. subtilis at a 6:4 ratio).

DISCUSSION

Nutrient digestibility

Dietary supplementation with B. subtilis and B. licheniformis can increase nutrient digestibility by producing extracellular enzymes including proteases and α-amylase [38, 39] to improve feed conversion in pigs [40]. In our study, all ratios of B. licheniformis and B. subtilis complex increased the apparent total tract digestibility (ATTD) of CP compared to the CON diet, but there was no different ATTD of CP among the groups. And all treatments did not improve DM. Many studies argue the advantage of B. licheniformis and B. subtilis complex in growing and/or finishing pig. The dietary supplementations with B. subtilis and B. licheniformis were expected more effective in weanling pigs than in growing pigs where they are under the development or impairment of gut microbiota [41]. Similar to our results, the study of Mun et al. [42] showed a tendency to improve digestibility of CP with supplementation of B. licheniformis and B. subtilis complex in weanling pigs. Lee et al. [30] also reported the positive effects on ATTD of DM and CP in weanling pigs. Nevertheless, Conversely, Chen et al. [43] have reported that B. subtilis based multi probiotics show no effect on ATTD of DM or CP in growing-finishing pigs. On the other hand, a previous study reported dietary supplementation of B. subtilis and B. licheniformis complex (1:1 ratio) can improve ATTD of DM and CP in growing pigs [33]. Currently, it is difficult to confirm the benefits of B. subtilis and B. licheniformis complex due to various environmental conditions such as gender, health status, environment, composition of ingredients, and strain of probiotic. Therefore, more research is needed to clarify this.

Fecal microflora

Bacillus-based probiotics have been used steadily due to their positive effects on gut health, such as kinetics of nutrient transport through enterocytes [44], intestinal cell proliferation [45], and modulation of the gut immune system [46, 47]. In our study, dietary supplementation with B. licheniformis and B. subtilis complex in all ratios decreased fecal E. coli count, but no difference in fecal Lactobacillus count. In general, Bacillus supplementation provides more positive and consistent results in weanling pigs than in growing-finishing pigs [48]. During the weaning period, without affecting Lactobacillus counts dietary B. subtilis can result in fewer coliform counts [30] and the mixture of B. subtilis and B. licheniformis reduced E. coli counts [49]. In contrast, dietary Bacillus based probiotic supplementation in growing-finishing pigs did not indicate the positive effects on the reduction of fecal Lactobacillus or E. coli counts [50]. This is probably due to the higher resistance to intestinal pathogens in growing-finishing pigs.

Odor gas emissions

NH3 and sulfur-containing compounds are the two most important toxic gases that cause odors and pollute the environment [51]. Exposure to high levels of malodorous gases such as NH3, volatile sulfur, and volatile organic compounds is not only negatively affecting animal health and performance, but also affects human health and causes environmental problems [52]. The malodorous gases can be reduced by the improvement of nutrient digestibility and gut microbiota composition [53]. Previous studies reported Bacillus-based probiotics decrease NH3 emissions in growing pigs [15, 54] and NH3 and H2S emissions in sows [55]. As expected, at the end of experiment, NH3 and H2S emissions were significantly reduced in all ratios but, the treatments L4S6 and L5S5 showed a significant decrease in H2S emissions from day 4 and the lower NH3 from day 6. In our digestibility study, all ratios of B. licheniformis and B. subtilis complex indicated the increase of ATTD of CP and, the higher digestibility of CP could decrease the fecal NH3 and H2S. Improvements in ATTD in CP may reflect decreased NH3 excretion due to increased protein absorption in the upper GIT and decreased protein fermentation in the lower GIT. The low nitrogen concentration in fecal samples can reduce fecal NH3 and H2S emissions [56]. Particularly, B. subtilis consumes oxygen in the digestive tract and additionally produces certain enzymes such as subtilisin and catalase that can improve nutrient digestibility [57]. Besides, B. subtilis can produce a glycosyl hydrolase, which aids in the hydrolysis of glycosidic bonds in complex sugars [58]. Subsequently, higher B. subtilis in the mixtures could accelerate the protein and carbohydrate degradation and reduced the malodorous gases.

CONCLUSION

This study suggested that dietary probiotic supplementation with B. licheniformis and B. subtilis in growing pigs significantly improved crude protein digestibility. It also significantly reduced E. coli counts and gas emission. Although there were no significant differences among different mixing ratios of B. licheniformis and B. subtilis in digestibility and fecal microflora, odor gas emissions showed significantly different. Particularly, H2S and NH3 emissions were decreased in L4S6 and L5S5 treatments. Therefore, the 5:5 or 4:6 ratio of probiotics mixture with B. licheniformis and B. subtilis has the potential advantage of odor gas reduction.

References

  1. Chu GM, Lee SJ, Jeong HS, Lee SS. Efficacy of probiotics from anaerobic microflora with prebiotics on growth performance and noxious gas emission in growing pigs. Anim Sci J. 2011;82:282-90. https://doi.org/10.1111/j.1740-0929.2010.00828.x
  2. Flynn D. South Korea bans antibiotics in animal feed [Internet]. Food Safety News. 2011 [cited 2022 Jan 7]. https://www.foodsafetynews.com/2011/06/south-korea-bans-antibiotics-in-animal-feed/
  3. Chu GM, Jung CK, Kim HY, Ha JH, Kim JH, Jung MS, et al. Effects of bamboo charcoal and bamboo vinegar as antibiotic alternatives on growth performance, immune responses and fecal microflora population in fattening pigs. Anim Sci J. 2013;84:113-20. https://doi.org/10.1111/j.1740-0929.2012.01045.x
  4. Martin MJ, Thottathil SE, Newman TB. Antibiotics overuse in animal agriculture: a call to action for health care providers. Am J Public Health. 2015;105:2409-10. https://doi.org/10.2105/AJPH.2015.302870
  5. Walsh TR, Wu Y. China bans colistin as a feed additive for animals. Lancet Infect Dis. 2016;16:1102-3. https://doi.org/10.1016/S1473-3099(16)30329-2
  6. Jongbloed AW, Lenis NP. Alteration of nutrition as a means to reduce environmental pollution by pigs. Livest Prod Sci. 1992;31:75-94. https://doi.org/10.1016/0301-6226(92)90057-B
  7. Paik IK, Blair R, Jacob J. Strategies to reduce environmental pollution from animal manure: principles and nutritional management - a review. Asian-Australas J Anim Sci. 1996;9:615-36. https://doi.org/10.5713/ajas.1996.615
  8. Jeong TS, Heo PS, Lee GY, Kim DH, Ju WS, Kim YY. The influence of phase feeding methods on growth performance, meat quality, and production cost in growing-finishing pigs. J Anim Sci Technol. 2010;52:29-36. https://doi.org/10.5187/JAST.2010.52.1.029
  9. Nousiainen J. Comparative observations on selected probiotics and olaquindox as feed additives for piglets around weaning: 2. effect on villus length and crypt depth in the jejunum, ileum, caecum and colon. J Anim Physiol Anim Nutr. 1991;66:224-30. https://doi.org/10.1111/j.1439-0396.1991.tb00290.x
  10. Davila AM, Blachier F, Gotteland M, Andriamihaja M, Benetti PH, Sanz Y, et al. Intestinal luminal nitrogen metabolism: role of the gut microbiota and consequences for the host. Pharmacol Res. 2012;68:95-107. https://doi.org/10.1016/j.phrs.2012.11.005
  11. Jha R, Berrocoso JFD. Dietary fiber and protein fermentation in the intestine of swine and their interactive effects on gut health and on the environment: a review. Anim Feed Sci Technol. 2016;212:18-26. https://doi.org/10.1016/j.anifeedsci.2015.12.002
  12. Simon O. Micro-organisms as feed additives-probiotics. Adv Pork Prod. 2005;16:161-7.
  13. Le Bon M, Davies HE, Glynn C, Thompson C, Madden M, Wiseman J, et al. Influence of probiotics on gut health in the weaned pig. Livest Sci. 2010;133:179-81. https://doi.org/10.1016/j.livsci.2010.06.058
  14. Zimmermann JA, Fusari ML, Rossler E, Blajman JE, Romero-Scharpen A, Astesana DM, et al. Effects of probiotics in swines growth performance: a meta-analysis of randomised controlled trials. Anim Feed Sci Technol. 2016;219:280-93. https://doi.org/10.1016/j.anifeedsci.2016.06.021
  15. Payling L, Kim IH, Walsh MC, Kiarie E. Effects of a multi-strain Bacillus spp. directfed microbial and a protease enzyme on growth performance, nutrient digestibility, blood characteristics, fecal microbiota, and noxious gas emissions of grower pigs fed corn-soybean-meal-based diets-a meta-analysis. J Anim Sci. 2017;95:4018-29. https://doi.org/10.2527/jas.2017.1522
  16. Gaggia F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. Int J Food Microbiol. 2010;141:S15-28. https://doi.org/10.1016/j.ijfoodmicro.2010.02.031
  17. Kang J, Lee JJ, Cho JH, Choe J, Kyoung H, Kim SH, et al. Effects of dietary inactivated probiotics on growth performance and immune responses of weaned pigs. J Anim Sci Technol. 2021;63:520-30. https://doi.org/10.5187/jast.2021.e44
  18. Liu Y, Espinosa CD, Abelilla JJ, Casas GA, Lagos LV, Lee SA, et al. Non-antibiotic feed additives in diets for pigs: a review. Anim Nutr. 2018;4:113-25. https://doi.org/10.1016/j.aninu.2018.01.007
  19. Feliciano MAR, Saad FMOB, Logato PVR, Aquino AA, Jose VA, Roque NC. Effects of probiotics on digestibility, faecal score, and hematologic characteristics in dogs. Arq Bras Med Vet Zootec. 2009;61:1268-74. https://doi.org/10.1590/S0102-09352009000600003
  20. Felix AP, Netto MVT, Murakami FY, Marcon de Brito C, Gisele de Oliveira S, Maiorka A. Digestibilidade e caracteristicas das fezes de caes suplementados com Bacillus subtilis na dieta. Cienc Rural. 2010;40:2169-73. https://doi.org/10.1590/S0103-84782010005000166
  21. Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28:214-20. https://doi.org/10.1016/j.fm.2010.03.007
  22. Hu Y, Dun Y, Li S, Zhao S, Peng N, Liang Y. Effects of Bacillus subtilis KN-42 on growth performance, diarrhea and faecal bacterial flora of weaned piglets. Asian-Australas J Anim Sci. 2014;27:1131-40. https://doi.org/10.5713/ajas.2013.13737
  23. Kayalvizhi N, Gunasekaran P. Production and characterization of a low-molecular-weight bacteriocin from Bacillus licheniformis MKU3. Lett Appl Microbiol. 2008;47:600-7. https://doi.org/10.1111/j.1472-765X.2008.02473.x
  24. Anthony T, Rajesh T, Kayalvizhi N, Gunasekaran P. Influence of medium components and fermentation conditions on the production of bacteriocin(s) by Bacillus licheniformis AnBa9. Bioresour Technol. 2009;100:872-7. https://doi.org/10.1016/j.biortech.2008.07.027
  25. Ishihara H, Takoh M, Nishibayashi R, Sato A. Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis. Curr Microbiol. 2002;45:18-23. https://doi.org/10.1007/s00284-001-0041-5
  26. MacIver RH, Stewart R, Frederiksen JW, Fullerton DA, Horvath KA. Topical application of bacitracin ointment is associated with decreased risk of mediastinitis after median sternotomy. Heart Surg Forum. 2006;9:E750-3. https://doi.org/10.1532/hsf98.20051187
  27. Kornegay ET, Risley CR. Nutrient digestibilities of a corn-soybean meal diet as influenced by Bacillus products fed to finishing swine. J Anim Sci. 1996;74:799-805. https://doi.org/10.2527/1996.744799x
  28. Vainshtein M. Probiotics for environmental sanitation: goals and examples. In: Cao G, Orru R, editors. Current environmental issues and challenges. Dordrecht: Springer; 2014. p. 127-35.
  29. Prenafeta-Boldu FX, Fernandez B, Vinas M, Lizardo R, Brufau J, Owusu-Asiedu A, et al. Effect of Bacillus spp. direct-fed microbial on slurry characteristics and gaseous emissions in growing pigs fed with high fibre-based diets. Animal. 2017;11:209-18. https://doi.org/10.1017/S1751731116001415
  30. Lee SH, Ingale SL, Kim JS, Kim KH, Lokhande A, Kim EK, et al. Effects of dietary supplementation with Bacillus subtilis LS 1-2 fermentation biomass on growth performance, nutrient digestibility, cecal microbiota and intestinal morphology of weanling pig. Anim Feed Sci Technol. 2014;188:102-10. https://doi.org/10.1016/j.anifeedsci.2013.12.001
  31. FEEDAP [EFSA Panel on Additives and Products or Substances used inAnimal Feed]. Safety and efficacy of BioPlus 2B® (Bacillus subtilis DSM 5750 and Bacillus licheniformis DSM 5749) as a feed additive for sows, piglets, pigs for fattening, turkeys for fattening and calves. EFSA J. 2016;14:e04558. https://doi.org/10.2903/j.efsa.2016.4558
  32. Balasubramanian B, Li T, Kim IH. Effects of supplementing growing-finishing pig diets with Bacillus spp. probiotic on growth performance and meat-carcass grade quality traits. Rev Bras Zootec. 2016;45:93-100. https://doi.org/10.1590/S1806-92902016000300002
  33. Lewton JR, Woodward AD, Moser RL, Thelen KM, Moeser AJ, Trottier NL, et al. Effects of a multi-strain Bacillus subtilis-based direct-fed microbial on weanling pig growth performance and nutrient digestibility. Transl Anim Sci. 2021;5:txab058. https://doi.org/10.1093/tas/txab058
  34. Lan R, Kim IH. Effects of Bacillus licheniformis and Bacillus subtilis complex on growth performance and faecal noxious gas emissions in growing-finishing pigs. J Sci Food Agric. 2019;99:1554-60. https://doi.org/10.1002/jsfa.9333
  35. NRC [National Research Council]. Nutrient requirements of swine. 10th rev. ed. Washington, DC: National Academies Press; 1998.
  36. AOAC [Association of Official Analytical Chemists] International. Official methods of analysis of AOAC International. 16th ed. Washington, DC: AOAC International; 1995.
  37. Steel RGD, Torrie JH. Principles and procedures of statistics: a biometrical approach. 2nd ed. New York, NY: McGraw-Hill; 1980.
  38. Priest FG. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev. 1977;41:711-53. https://doi.org/10.1128/br.41.3.711-753.1977
  39. Carlisle GE, Falkinham JO 3rd. Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis. Appl Environ Microbiol. 1989;55:3026-8. https://doi.org/10.1128/aem.55.11.3026-3028.1989
  40. Davis ME, Parrott T, Brown DC, de Rodas BZ, Johnson ZB, Maxwell CV, et al. Effect of a Bacillus-based direct-fed microbial feed supplement on growth performance and pen cleaning characteristics of growing-finishing pigs. J Anim Sci. 2008;86:1459-67. https://doi.org/10.2527/jas.2007-0603
  41. Stavric S, Kornegay ET. Microbial probiotic for pigs and poultry. In: Wallace RJ, Chesson A, editors. Biotechnology in animal feeds and animal feeding. Weinheim: Wiley-VCH; 1995. p. 205-31.
  42. Mun D, Kyoung H, Kong M, Ryu S, Jang KB, Baek J, et al. Effects of Bacillus-based probiotics on growth performance, nutrient digestibility, and intestinal health of weaned pigs. J Anim Sci Technol. 2021;63:1314-27. https://doi.org/10.5187/jast.2021.e109
  43. Chen YJ, Min BJ, Cho JH, Kwon OS, Son KS, Kim HJ, et al. Effects of dietary Bacillus-based probiotic on growth performance, nutrients digestibility, blood characteristics and fecal noxious gas content in finishing pigs. Asian-Australas J Anim Sci. 2006;19:587-92. https://doi.org/10.5713/ajas.2006.587
  44. Walsh MC, Rostagno MH, Gardiner GE, Sutton AL, Richert BT, Radcliffe JS. Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids. Part I: effects on growth performance, microbial populations, and immune status. J Anim Sci. 2012;90:261-71. https://doi.org/10.2527/jas.2010-3598
  45. Babinska I, Rotkiewicz T, Otrocka-Domagala I. The effect of Lactobacillus acidophilus and Bifidobacterium spp. administration on the morphology of the gastrointestinal tract, liver and pancreas in piglets. Pol J Vet Sci. 2005;8:29-35.
  46. Scharek L, Guth J, Filter M, Schmidt MFG. Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets. Arch Anim Nutr. 2007;61:223-34. https://doi.org/10.1080/17450390701431540
  47. Schierack P, Wieler LH, Taras D, Herwig V, Tachu B, Hlinak A, et al. Bacillus cereus var. toyoi enhanced systemic immune response in piglets. Vet Immunol Immunopathol. 2007;118:1-11. https://doi.org/10.1016/j.vetimm.2007.03.006
  48. Mingmongkolchai S, Panbangred W. Bacillus probiotics: an alternative to antibiotics for livestock production. J Appl Microbiol. 2018;124:1334-46. https://doi.org/10.1111/jam.13690
  49. Hu J, Kim YH, Kim IH. Effects of two Bacillus strains probiotic supplement on reproduction performance, nutrient digestibility, blood profile, fecal score, excreta odor contents and fecal microflora in lactation sows, and growth performance in sucking piglets. Livest Sci. 2021;244:104293. https://doi.org/10.1016/j.livsci.2020.104293
  50. Giang HH, Viet TQ, Ogle B, Lindberg JE. Effects of supplementation of probiotics on the performance, nutrient digestibility and faecal microflora in growing-finishing pigs. Asian-Australas J Anim Sci. 2011;24:655-61. https://doi.org/10.5713/ajas.2011.10238
  51. Jongbloed AW, Lenis MP. Excretion of nitrogen and some minerals by livestock. In: Nitrogen flow in pig production and environmental consequences: proceedings of the First International Symposium on Nitrogen Flow in Pig Production and Environmental Consequences; 1993; Wageningen, Netherlands. p. 22-36.
  52. Ushida K, Hashizume K, Miyazaki K, Kojima Y, Takakuwa S. Isolation of Bacillus sp. as a volatile sulfur-degrading bacterium and its application to reduce the fecal odor of pig. Asian-Australas J Anim Sci. 2003;16:1795-8. https://doi.org/10.5713/ajas.2003.1795
  53. Ferket PR, van Heugten E, van Kempen TATG, Angel R. Nutritional strategies to reduce environmental emissions from nonruminants. J Anim Sci. 2002;80:E168-82. https://doi.org/10.2527/animalsci2002.80E-Suppl_2E168x
  54. Upadhaya SD, Kim SC, Valientes RA, Kim IH. The effect of Bacillus-based feed additive on growth performance, nutrient digestibility, fecal gas emission, and pen cleanup characteristics of growing-finishing pigs. Asian-Australas J Anim Sci. 2015;28:999-1005. https://doi.org/10.5713/ajas.15.0066
  55. Jeong J, Kim J, Lee S, Kim I. Evaluation of Bacillus subtilis and Lactobacillus acidophilus probiotic supplementation on reproductive performance and noxious gas emission in sows. Ann Anim Sci. 2015;15:699-710. https://doi.org/10.1515/aoas-2015-0018
  56. Cho JH, Chen YJ, Min BJ, Yoo JS, Wang Y, Kim IH. Effects of reducing dietary crude protein on growth performance, odor gas emission from manure and blood urea nitrogen and IGF-1 concentrations of serum in nursery pigs. Anim Sci J. 2008;79:453-9. https://doi.org/10.1111/j.1740-0929.2008.00549.x
  57. Kampf D. Mode of action of Bacillus subtilis and efficiency in piglet feeding. Feed Compounder; 2012;2:36-7.
  58. Larsen N, Thorsen L, Kpikpi EN, Stuer-Lauridsen B, Cantor MD, Nielsen B, et al. Characterization of Bacillus spp. strains for use as probiotic additives in pig feed. Appl Microbiol Biotechnol. 2014;98:1105-18. https://doi.org/10.1007/s00253-013-5343-6