Antibiotics and chemical therapeutics as feed additives have been used constantly in the pig industry for economic benefits, improving feed efficiency . 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 . 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 . 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 . 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 . Bacitracin is directed primarily against gram-positive bacteria via inhibition of the cell wall . Both B. subtilis and B. licheniformis increased fecal Lactobacillus counts in finishing pigs without affecting fecal coliform counts . Additionally, B. licheniformis and B. subtilis can survive in fecal after excretion and, they sustainedly degrade organic matters in the fecal , 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 . 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 . 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.
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.
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 were prepared to meet the NRC  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) . 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.
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.
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°C 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°C. 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 . 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°C for 48 h. MacConkey agar plates were incubated at 37°C 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°C 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.
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  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.
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.
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.
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.
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 . 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 . Similar to our results, the study of Mun et al.  showed a tendency to improve digestibility of CP with supplementation of B. licheniformis and B. subtilis complex in weanling pigs. Lee et al.  also reported the positive effects on ATTD of DM and CP in weanling pigs. Nevertheless, Conversely, Chen et al.  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 . 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.
Bacillus-based probiotics have been used steadily due to their positive effects on gut health, such as kinetics of nutrient transport through enterocytes , intestinal cell proliferation , 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 . During the weaning period, without affecting Lactobacillus counts dietary B. subtilis can result in fewer coliform counts  and the mixture of B. subtilis and B. licheniformis reduced E. coli counts . 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 . This is probably due to the higher resistance to intestinal pathogens in growing-finishing pigs.
NH3 and sulfur-containing compounds are the two most important toxic gases that cause odors and pollute the environment . 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 . The malodorous gases can be reduced by the improvement of nutrient digestibility and gut microbiota composition . Previous studies reported Bacillus-based probiotics decrease NH3 emissions in growing pigs [15,54] and NH3 and H2S emissions in sows . 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 . Particularly, B. subtilis consumes oxygen in the digestive tract and additionally produces certain enzymes such as subtilisin and catalase that can improve nutrient digestibility . Besides, B. subtilis can produce a glycosyl hydrolase, which aids in the hydrolysis of glycosidic bonds in complex sugars . Subsequently, higher B. subtilis in the mixtures could accelerate the protein and carbohydrate degradation and reduced the malodorous gases.
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.