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. 2026 Mar 11;16:13158. doi: 10.1038/s41598-026-41542-y

Comparative evaluation of Bacillus subtilis delivery forms reveals their effects on biogenic element excretion in pigs

Anna Czech 1,✉, Łukasz Wlazło 2, Marcin Łukaszewicz 3, Agnieszka Lewińska 4, Bożena Nowakowicz-Dębek 2
PMCID: PMC13103316  PMID: 41813752

Abstract

Excess nitrogen, phosphorus and trace element losses from pig manure accelerate eutrophication and soil degradation. Probiotic Bacillus subtilis produces phytase, protease and other enzymes and postbiotics that may enhance nutrient retention, yet the influence of delivery matrix on environmental outcomes is unclear. Three hundred and eighty-four weaned pigs (Yorkshire × Landrace; 28 d) were assigned to four diets until 30 kg body weight: positive control (PC, complete diet), negative control (NC, no additives), BS (NC + 0.1% B. subtilis 87Y spores) and FR (NC + 8% rapeseed meal fermented with Bacillus subtilis 87Y). Apparent total-tract and ileal digestibility (AID), daily excretion and retention of macro- and micro-elements were determined over 72 d. FR pigs showed the greatest improvements: faecal nitrogen fell by 32% and urinary nitrogen by 29% versus NC, lowering total nitrogen output from 6.6 to 4.5 g pig/day. Total phosphorus excretion declined 39% (from 3.05 to 1.85 g pig/day) and calcium retention rose 187%. Faecal zinc and copper were reduced by 63% and 39%, respectively. FR increased AID of phosphorus (from 47 to 70%) and magnesium (from 22 to 43%) by > 20%-points; BS achieved intermediate gains. Predicted farm-gate savings equal 26 kg N and 3.6 kg phosphorus per 1 000 weaners. Embedding Bacillus subtilis in fermented rapeseed meal immobilizes enzymes and metabolites, protects spores and supplies prebiotic levan, delivering larger environmental benefits than free spores alone. Integrating this synbiotic into nursery diets simultaneously valorizes a regional oil-seed by-product, displaces imported soy, and helps swine operations meet nutrient-loss reduction targets.

Keywords: Synbiotic feed additive, Nutrient excretion, Eenvironmental mitigation, Biogenic element balance, Solid-state fermentation, Sustainable pig production

Subject terms: Biotechnology, Environmental sciences, Microbiology, Zoology

Introduction

In recent years, there has been a marked increase in interest toward natural feed additives that not only improve animal production performance but also help mitigate the negative environmental impacts of intensive farming. Optimizing feed composition and introducing innovative nutritional solutions may play a key role in reducing the excretion of mineral components, including biogenic elements, as well as lowering gas emissions to the atmosphere1. Among promising research directions, the use of probiotics such as Bacillus subtilis occupies a special place. This Gram-positive, aerobic, spore-forming bacterium is highly resistant to adverse environmental conditions. Due to its ability to produce various digestive enzymes, including amylase, glucoamylase, protease, pectinase, cellulase, and phytase, Bacillus spp. has been widely used as a functional feed additive2,3.

Additionally, Bacillus spp. exhibits antagonistic properties against pathogenic microorganisms such as Escherichia coli and Salmonella spp. through bacteriocin production and competition for space and nutrients within the host’s gastrointestinal tract2,4. The stabilization of the intestinal microbiota and the production of digestive enzymes by Bacillus spp. support improved digestion and nutrient absorption, thereby reducing metabolic losses associated with excessive excretion of mineral components, including biogenic elements5–7.

A key aspect determining the effectiveness of Bacillus spp. in enhancing the utilization of mineral components is the form of administration, that is, the applied matrix. In practice, various encapsulation technologies and carriers (lipid microcapsules, polysaccharide hydrogels, mineral-based preparations) are assessed, as they may affect the survival of bacteria in the gastrointestinal tract and their metabolic activity8,9. Another approach is fermenting feed components with strains such as Bacillus spp10. The involvement of these bacteria in the fermentation process increases enzymatic activity, leading to improved availability and digestibility of nutritional and mineral components, aids in the breakdown of antinutritional factors, and may improve the quality of the animals’ gut microbiota, contributing to better health and performance11.

Therefore, both probiotics and fermented components possess similar potential to improve health and production outcomes12. Thus, it is of interest to determine which of these forms, free spores or a fermented component serving as a probiotic matrix, will prove more effective in minimizing the excretion of biogenic elements into the environment, a critical factor for the sustainable development of animal production. The research hypothesis assumes that supplementation of pigs with Bacillus spp. 87Y in the form of free spores and in a matrix of fermented rapeseed meal differs in terms of its effect on the absorption and retention of mineral components and the reduction of their losses to the environment. It is expected that the use of fermented rapeseed meal as a probiotic carrier will increase the efficiency of the utilization of these components compared to administering free spores of Bacillus spp. 87Y.

The aim of this study was to evaluate the effect of adding Bacillus spp. 87Y in the form of free spores and in a matrix of fermented rapeseed meal (FRSMb) on the mechanism of action of the fermented component as a probiotic carrier and its potential to reduce mineral component losses. This aspect is important for both production economics and environmental protection. The findings provide information relevant for developing effective nutritional strategies to enhance mineral utilization while simultaneously reducing environmental burdens associated with these elements, which aligns with the assumptions of sustainable intensification in pig production.

Results

Nutrient intake

Pigs in the FR and BS groups consumed significantly (p ≤ 0.001) more feed, resulting in increased intake of nitrogen and mineral elements (Na, K, Mg, Cu, Zn, Fe, Mn, and Se) compared to pigs in the PC and NC groups (Tables 1, 2, 3 and 4).

Table 1.

Effects of fermented rapeseed meal (FRSMb) or Bacillus subtills without feed additives on apparent total tract digestibility (ATTD) and apparent ileal digestibility (AID) of sodium (Na), potassium (K) and magnesium (Mg) in weaner pigs (n = 6/group).

Item1 PC ± SD NC ± SD FR ± SD BS ± SD P value SEM
Sodium
Intake of Na, g d−1 0.498b ± 0.054 0.504b ± 0.020 0.521a ± 0.011 0.539a ± 0.030  < 0.001 0.004
Faecal excretion of Na, g d−1 0.040bc ± 0.001 0.052a ± 0.002 0.039c ± 0.003 0.046ab ± 0.005  < 0.001 0.002
Urinary excretion of Na, g d−1 0.238b ± 0.038 0.342a ± 0.018 0.223b ± 0.020 0.229b ± 0.044 0.001 0.015
Absorption of Na, g d−1 0.459b ± 0.012 0.452b ± 0.002 0.482a ± 0.014 0.493a ± 0.006  < 0.001 0.004
Retention of Na, g d−1 0.221a ± 0.039 0.109b ± 0.018 0.259a ± 0.018 0.264a ± 0.039  < 0.001 0.017
ATTD of Na, % 91.93a ± 0.165 89.64b ± 0.295 92.51a ± 0.621 91.42a ± 0.877  < 0.001 0.266
AID of Na, % 91.40a ± 0.165 88.80b ± 0.309 91.87a ± 0.616 90.71a ± 0.826  < 0.001 0.285
Retention:intake, % 44.30a ± 7.59 21.74b ± 3.43 49.78a ± 3.51 48.98a ± 7.51  < 0.001 3.23
Retention:absorption, % 48.20b ± 8.32 24.27c ± 3.89 53.82a ± 3.96 53.61a ± 8.52  < 0.001 3.47
Potasium
Intake of K, g d−1 3.39b ± 0.017 3.36b ± 0.014 3.51a ± 0.007 3.61a ± 0.030  < 0.001 0.025
Faecal excretion of K, g d−1 0.482b ± 0.006 0.620a ± 0.017 0.433b ± 0.030 0.468b ± 0.043  < 0.001 0.019
Urinary excretion of K, g d−1 2.58a ± 0.052 2.19b ± 0.067 1.32c ± 0.172 1.50c ± 0.259  < 0.001 0.136
Absorption of K, g d−1 2.92ab ± 0.018 2.75b ± 0.017 3.07a ± 0.034 3.14a ± 0.043  < 0.001 0.040
Retention of K, g d−1 0.337b ± 0.055 0.557b ± 0.057 1.75a ± 0.188 1.64a ± 0.226  < 0.001 0.166
ATTD of K, % 85.80b ± 1.19 81.59c ± 0.484 87.65a ± 0.878 87.03ab ± 1.17  < 0.001 0.559
AID of K, % 82.27b ± 0.184 78.41c ± 1.04 86.49a ± 0.867 85.49a ± 2.01  < 0.001 0.756
Retention:intake, % 9.92b ± 1.62 16.53b ± 1.64 50.00a ± 5.27 45.40a ± 6.14  < 0.001 4.61
Retention:absorption, % 11.56b ± 1.86 20.27b ± 2.13 57.04a ± 5.79 52.24a ± 7.67  < 0.001 5.20
Magnesium
Intake of Mg, g d−1 0.889b ± 0.004 0.892b ± 0.004 0.973a ± 0.005 0.977a ± 0.006  < 0.001 0.011
Faecal excretion of Mg, g d−1 0.558b ± 0.017 0.695a ± 0.020 0.540b ± 0.023 0.517b ± 0.026  < 0.001 0.019
Urinary excretion of Mg, g d−1 0.160a ± 0.011 0.178a ± 0.021 0.081b ± 0.003 0.089b ± 0.010  < 0.001 0.011
Absorption of Mg, g d−1 0.331b ± 0.014 0.198c ± 0.017 0.434a ± 0.027 0.461a ± 0.026  < 0.001 0.027
Retention of Mg, g d−1 0.171b ± 0.020 0.020c ± 0.011 0.353a ± 0.027 0.372a ± 0.021  < 0.001 0.037
ATTD of Mg, % 37.23b ± 1.68 22.17c ± 1.92 44.57a ± 2.58 47.14a ± 2.66  < 0.001 2.26
AID of Mg, % 35.28b ± 1.70 21.84c ± 1.86 43.14a ± 2.50 45.62a ± 2.57  < 0.001 2.16
Retention:intake, % 19.24b ± 2.33 2.23c ± 1.25 36.27a ± 2.59 38.04a ± 2.15  < 0.001 3.78
Retention:absorption, % 51.55b ± 4.08 9.74c ± 5.12 81.32a ± 1.25 80.72a ± 1.543  < 0.001 7.58
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a–c Values within a row lacking without a common superscript letter are significantly different (P < 0.05).Data are least squares means for 6 replicate pens per diet1PC, positive control group – weaned piglets receiving a standard diet with feed additives and ZnO, in accordance with NRC13 recommendations; NC, negative control group – weaned piglets receiving the same diet as in the PC group but without feed additives and ZnO; FR, weaners which received a diet with 8% FRSMb containing Bacillus subtilis 87Y without feed additives and ZnO; BS, weaners which received a diet with 0.1% Bacillus subtilis 87Y without feed additives and ZnO; SD, standard deviation; SEM, Standard Error of the Mean.

Table 2.

Effects of fermented rapeseed meal (FRSMb) or Bacillus subtills without feed additives on apparent total tract digestibility (ATTD) and apparent ileal digestibility (AID) of nitrogen (N), calcium (Ca) and phosphorus (P) in weaner pigs (n = 6/group).

Item1 PC ± SD NC ± SD FR ± SD BS ± SD P value SEM
Nitrogen
Intake of N, g d−1 14.52b ± 0.113 14.56b ± 0.225 15.88a ± 0.200 15.42a ± 0.323  < 0.001 0.157
Faecal excretion of N, g d−1 2.42c ± 0.113 3.78a ± 0.185 2.56bc ± 0.194 2.80b ± 0.144  < 0.001 0.142
Urinary excretion of N, g d−1 2.93a ± 0.143 2.78a ± 0.232 1.96b ± 0.015 2.18b ± 0.020  < 0.001 0.109
Absorption of N, g d−1 12.10b ± 0.180 10.78c ± 0.292 13.32a ± 0.167 12.62b ± 0.423  < 0.001 0.248
Retention of N, g d−1 9.17b ± 0.280 8.00c ± 0.398 11.36a ± 0.658 10.44a ± 0.432  < 0.001 0.337
ATTD of N, % 83.30a ± 0.825 74.03b ± 1.34 83.88a ± 1.10 81.85a ± 1.20  < 0.001 0.931
AID of N, % 82.82a ± 0.820 73.30b ± 1.33 83.30a ± 1.10 81.28a ± 1.19  < 0.001 0.947
Retention:intake, % 63.10c ± 1.68 54.91d ± 2.04 71.56a ± 0.949 67.70b ± 1.46  < 0.001 1.64
Retention:absorption, % 75.75b ± 1.39 74.18b ± 2.35 85.31a ± 0.204 82.71a ± 0.659  < 0.001 1.24
Calcium
Intake of Ca, g d−1 6.51b ± 0.038 6.34b ± 0.031 6.86a ± 0.038 6.94a ± 0.058  < 0.001 0.064
Faecal excretion of Ca, g d−1 3.18b ± 0.036 4.01a ± 0.275 2.59c ± 0.260 2.81bc ± 0.188  < 0.001 0.147
Urinary excretion of Ca, g d−1 0.958a ± 0.073 0.931a ± 0.045 0.250b ± 0.022 0.121c ± 0.016  < 0.001 0.099
Absorption of Ca, g d−1 3.33b ± 0.028 2.34c ± 0.252 4.27a ± 0.292 4.12a ± 0.190  < 0.001 0.204
Retention of Ca, g d−1 2.37b ± 0.051 1.40c ± 0.219 4.02a ± 0.281 4.00a ± 0.190  < 0.001 0.292
ATTD of Ca, % 51.10b ± 0.448 36.82c ± 4.11 62.27a ± 3.96 59.43a ± 2.70  < 0.001 2.34
AID of Ca, % 50.80b ± 0.446 36.47c ± 4.07 58.28a ± 3.71 58.26a ± 1.62  < 0.001 2.11
Retention:intake, % 36.39b ± 0.908 22.13c ± 3.53 58.63a ± 3.80 57.69a ± 2.67  < 0.001 4.01
Retention:absorption, % 71.22b ± 2.00 59.88c ± 3.06 94.13a ± 0.445 97.05a ± 0.411  < 0.001 4.04
Phosphorus
Intake of P, g d−1 5.88b ± 0.025 5.87b ± 0.029 6.36a ± 0.035 6.38a ± 0.054  < 0.001 0.064
Faecal excretion of P g d−1 2.21b ± 0.076 3.05a ± 0.169 1.85c ± 0.159 1.91bc ± 0.174  < 0.001 0.129
Urinary excretion of P, g d−1 2.59a ± 0.137 1.37b ± 0.078 0.456c ± 0.021 0.548c ± 0.081  < 0.001 0.222
Absorption of P, g d−1 3.68b ± 0.090 2.82c ± 0.145 4.51a ± 0.190 4.47a ± 0.171  < 0.001 0.182
Retention of P, g d−1 1.08c ± 0.125 1.45b ± 0.137 4.05a ± 0.211 3.93a ± 0.200  < 0.001 0.355
ATTD of P, % 62.41b ± 1.37 48.01c ± 2.67 70.91a ± 2.64 70.11a ± 2.69  < 0.001 2.15
AID of P, % 59.35b ± 1.30 47.30c ± 2.74 70.19a ± 2.69 69.09a ± 3.52  < 0.001 2.17
Retention:intake, % 18.42c ± 2.13 24.67b ± 2.41 63.74a ± 2.99 61.52a ± 3.10  < 0.001 5.37
Retention:absorption, % 29.51c ± 3.36 51.34b ± 2.94 89.85a ± 0.886 87.72a ± 1.99  < 0.001 6.58
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a–c Values within a row lacking without a common superscript letter are significantly different (P < 0.05).Data are least squares means for 6 replicate pens per diet.1 See Table 2.

Table 3.

Effects of fermented rapeseed meal (FRSMb) or Bacillus subtills without feed additives on apparent total tract digestibility (ATTD) and apparent ileal digestibility (AID) of copper(Cu), zinc (Zn) and ferrum (Fe) in weaner pigs (n = 6/group).

Item PC ± SD NC ± SD FR ± SD BS ± SD P value SEM
Copper
Intake of Cu, mg d−1 16.86b ± 0.072 16.88b ± 0.068 18.13a ± 0.037 18.14a ± 0.103  < 0.001 0.163
Faecal excretion of Cu, mg d−1 11.92b ± 0.258 15.80a ± 0.469 9.59c ± 0.567 8.98c ± 0.257  < 0.001 0.697
Urinary excretion of Cu, mg d−1 0.151b ± 0.012 0.182a ± 0.013 0.101c ± 0.026 0.129bc ± 0.012  < 0.001 0.009
Absorption of Cu, mg d−1 4.95b ± 0.235 1.09c ± 0.445 8.54a ± 0.592 9.16a ± 0.230  < 0.001 0.838
Retention of Cu, mg d−1 4.80b ± 0.243 0.905c ± 0.451 8.44a ± 0.609 9.03a ± 0.233  < 0.001 0.845
ATTD of Cu, % 29.36b ± 1.42 6.44c ± 2.65 47.08a ± 3.20 50.50a ± 1.32  < 0.001 4.03
AID of Cu, % 28.65b ± 0.941 7.32c ± 1.26 46.30a ± 2.40 49.17a ± 2.05  < 0.001 3.85
Retention:intake, % 28.46b ± 1.47 5.36c ± 2.68 46.52a3.29 49.80a ± 1.33  < 0.001 4.59
Retention:absorption, % 96.94a ± 0.368 79.52b ± 12.98 98.80a ± 0.371 98.59a ± 0.140 0.003 2.54
Zinc
Intake of Zn, mg d−1 159.5a ± 0.680 44.96c ± 1.18 47.44b ± 1.264 48.55b ± 0.941  < 0.001 12.59
Faecal excretion of Zn, mg d−1 120.6a ± 2.65 30.05b ± 0.809 11.06c ± 0.453 11.89c ± 0.607  < 0.001 11.68
Urinary excretion of Zn, mg d−1 3.88a ± 0.204 1.38b ± 0.181 0.608d ± 0.053 1.03c ± 0.103  < 0.001 0.331
Absorption of Zn, mg d−1 38.93a ± 2.75 14.92b ± 0.753 36.38a ± 0.692 36.66a ± 0.790  < 0.001 2.54
Retention of Zn, mg d−1 35.05a ± 2.93 13.53b ± 0.864 35.78a ± 0.673 35.63a ± 0.710  < 0.001 2.48
ATTD of Zn, % 24.39c ± 1.70 33.18b ± 1.71 76.68a ± 1.06 75.50a ± 1.31  < 0.001 5.48
AID of Zn, % 24.25c ± 1.69 32.85b ± 1.69 74.29a ± 0.682 74.54a ± 0.916  < 0.001 5.31
Retention:intake, % 21.96c ± 1.82 30.10b ± 1.97 75.40a1.05 73.38a1.17  < 0.001 6.30
Retention:absorption, % 89.95b ± 1.25 90.67b ± 1.49 98.32a0.137 97.19a ± 0.236  < 0.001 0.994
Ferrum
Intake of Fe, mg d−1 140.5c ± 0.715 145.5b ± 0.710 158.1a1.20 158.4a ± 1.33  < 0.001 2.03
Faecal excretion of Fe, mg d−1 90.72b ± 6.23 115.4a ± 1.36 88.68b6.20 92.27b ± 4.81  < 0.001 3.03
Urinary excretion of Fe, mg d−1 22.75a ± 0.359 22.43a ± 1.22 15.82b0.817 17.87b ± 0.958  < 0.001 0.770
Absorption of Fe, mg d−1 49.86b ± 6.76 30.04c ± 0.837 69.45a5.34 66.21a ± 5.85  < 0.001 4.20
Retention of Fe, mg d−1 27.11b ± 7.05 7.61c ± 0.823 53.62a5.47 48.34a ± 5.59  < 0.001 4.86
ATTD of Fe, % 35.45b ± 4.68 20.64c ± 0.637 43.93a3.63 41.76ab ± 3.42  < 0.001 2.17
AID of Fe, % 34.63b ± 4.32 19.44c ± 0.823 41.44a2.37 40.68a ± 2.76  < 0.001 2.09
Retention:intake, % 19.27b ± 4.94 5.23c ± 0.579 33.92a3.65 30.48a ± 3.35  < 0.001 3.00
Retention:absorption, % 53.67b ± 6.88 25.27c ± 2.10 77.09a1.92 72.86a ± 2.28  < 0.001 5.35
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a–c Values within a row lacking without a common superscript letter are significantly different (P < 0.05).Data are least squares means for 6 replicate pens per diet.1 See Table 2.

Table 4.

Effects of fermented rapeseed meal (FRSMb) or Bacillus subtills without feed additives on apparent total tract digestibility (ATTD) and apparent ileal digestibility (AID) of manganese (Mn) and selenium (Se) in weaner pigs (n = 6/group).

Item1 PC ± SD NC ± SD FR ± SD BS ± SD P value SEM
Manganese
Intake of Mn, mg d−1 74.33c ± 0.317 70.50d ± 0.286 78.87b ± 0.160 81.05a ± 0.681  < 0.001 1.06
Faecal excretion of Mn, mg d−1 16.56 ± 0.848 16.67 ± 0.388 15.32 ± 0.813 16.89 ± 1.56 0.164 0.273
Urinary excretion of Mn, mg d−1 0.885a ± 0.028 0.771b ± 0.014 0.681d ± 0.008 0.772b ± 0.023  < 0.001 0.019
Absorption of Mn, mg d−1 57.77b ± 0.849 53.84c ± 0.380 63.56a ± 0.873 64.17a ± 1.81  < 0.001 1.13
Retention of Mn, mg d−1 56.88b ± 3.87 53.06b ± 2.39 62.88a ± 2.87 63.39a ± 1.81  < 0.001 1.14
ATTD of Mn, % 77.71b ± 1.13 76.35b ± 0.521 80.58a ± 1.04 79.15a ± 1.97 0.003 0.427
AID of Mn, % 73.59b ± 1.07 75.19ab ± 0.965 76.33a ± 2.24 77.78a ± 2.03 0.025 0.461
Retention:intake, % 76.52b ± 1.16 75.26b ± 0.538 79.71a ± 1.04 78.21a ± 1.97 0.002 0.521
Retention:absorption, % 98.47 ± 0.907 98.57 ± 0.804 98.93 ± 0.990 98.79 ± 0.048 0.123 4.05
Selenium
Intake of Se, mg d−1 0.351b ± 0.025 0.358b ± 0.002 0.396a ± 0.001 0.403a ± 0.034  < 0.001 0.006
Faecal excretion of Se, mg d−1 0.180a ± 0.019 0.178a ± 0.004 0.060c ± 0.016 0.083b ± 0.005  < 0.001 0.014
Urinary excretion of Se, mg d−1 0.238a ± 0.022 0.210a ± 0.008 0.218a ± 0.027 0.148b ± 0.024  < 0.001 0.010
Absorption of Se, mg d−1 0.172b ± 0.014 0.179b ± 0.003 0.336a ± 0.007 0.319a ± 0.006  < 0.001 0.020
Retention of Se, mg d−1 − 0.067d ± 0.014 − 0.031c ± 0.008 0.118b ± 0.027 0.171a ± 0.028  < 0.001 0.026
ATTD of Se, % 48.71c ± 3.29 50.08c ± 0.989 84.83a ± 1.56 79.32b ± 1.23  < 0.001 3.79
AID of Se, % 45.92c ± 2.10 47.90c ± 2.42 82.18a ± 1.60 77.42b ± 2.54  < 0.001 3.82
Retention:intake, % − 18.99c ± 3.95 − 8.54c ± 2.28 29.78b ± 6.71 42.53a ± 7.04  < 0.001 6.73
Retention:absorption, % − 39.01d ± 8.00 − 17.06c ± 4.63 35.12b ± 7.91 53.52a ± 8.02  < 0.001 9.84
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a–d Values within a row lacking without a common superscript letter are significantly different (P < 0.05).Data are least squares means for 6 replicate pens per diet.1 See Table 2.

Sodium, Potassium, and Magnesium balance

Sodium retention, as well as apparent total tract digestibility (ATTD), apparent ileal digestibility (AID), and retention as a percentage of intake (Retention: intake), were significantly higher in pigs from PC, FR, and BS groups compared to the NC group. The highest sodium excretion through feces and urine occurred in the NC group. Most potassium and magnesium balance indicators were significantly improved (P < 0.001) in the FR and BS groups compared to both control groups (PC and NC), while the lowest values for these minerals were consistently recorded in the NC group (Table 1).

Nitrogen, Phosphorus, and Calcium balance

The majority of nitrogen balance indicators were significantly higher (P < 0.001) in the FR group than in the NC group. Both ATTD and AID values for nitrogen were significantly elevated in PC, FR, and BS groups compared to NC. Phosphorus and calcium showed significantly higher absorption, retention, ATTD, AID, Retention: intake, and Retention: absorption in the FR and BS groups relative to PC and NC groups. The NC group displayed the lowest balance values for phosphorus and calcium across all parameters (Table 2).

Copper, Zinc, and Iron balance

Copper absorption, retention, ATTD, AID, and Retention: intake were significantly increased in pigs fed FR and BS diets compared to both PC and NC groups. Conversely, pigs in the NC group had the lowest copper balance values, with the highest copper excretion. Zinc absorption and retention were significantly lowest in the NC group, whereas pigs from FR and BS groups demonstrated the highest ATTD, AID, Retention: absorption, and Retention: intake values for zinc. Urinary zinc excretion was highest in the PC group. Iron balance indicators were significantly improved in the FR and BS groups compared to both control groups (P < 0.001) (Table 3).

Manganese and Selenium balance

Manganese absorption, retention, ATTD, AID, and Retention: intake were significantly higher in pigs from FR and BS groups compared to PC and NC groups (P < 0.001). The highest urinary manganese excretion occurred in the PC group. Selenium retention, along with Retention: intake and Retention: absorption ratios, were significantly highest in the BS group, while selenium absorption was notably higher in both FR and BS groups compared to PC and NC groups. The highest ATTD and AID values for selenium were observed in the FR group (Table 4).

Mineral levels in blood plasma

Blood plasma from pigs in FR and BS groups showed significantly increased levels of phosphorus, copper, zinc, and iron compared to the NC group (P < 0.001). Plasma levels of phosphorus, calcium, and zinc in PC pigs were similar to those in other groups, while copper and iron were notably higher compared to NC pigs. No statistically significant differences were observed between groups for plasma sodium, potassium, magnesium, manganese, or selenium levels (P > 0.05) (Table 5).

Table 5.

Content of mineral elements in piglets’ blood plasma.

Item1 PC ± SD NC ± SD FR ± SD BS ± SD P value SEM
Sodium; mmol l−1 135.7 ± 8.76 135.1 ± 5.45 135.0 ± 4.14 130.7 ± 8.22 0.586 1.38
Potassium; mmol l−1 3.59 ± 0.761 3.90 ± 1.18 3.44 ± 0.233 3.52 ± 0.330 0.711 0.144
Magnesium; mmol l−1 1.24 ± 0.134 1.13 ± 0.233 1.08 ± 0.072 1.36 ± 0.431 0.277 0.054
Phosphorus; mmol l−1 2.91ab ± 0.424 2.07b ± 0.517 3.31a ± 0.595 3.59a ± 0.549  < 0.001 0.156
Calcium; mmol l−1 2.84ab ± 0.375 2.56b ± 0.253 2.96ab ± 0.136 3.11a ± 0.333 0.025 0.069
Copper; µmol l−1 19.92a ± 1.10 15.90b ± 1.56 18.71a ± 0.919 19.42a ± 0.818  < 0.001 0.389
Zinc; µmol l−1 12.02ab ± 0.716 10.81b ± 1.16 12.54a ± 0.688 13.78a ± 0.717  < 0.001 0.274
Ferrum; µmol l−1 11.93a ± 1.53 9.40b ± 0.866 13.05a ± 1.54 13.21a ± 0.683  < 0.001 0.39
Manganese; µmol l−1 0.025 ± 0.006 0.025 ± 0.011 0.022 ± 0.006 0.021 ± 0.005 0.700 0.001
Selenium; µmol l−1 0.059 ± 0.016 0.053 ± 0.013 0.059 ± 0.012 0.072 ± 0.008 0.087 0.003
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a–b Values within a row lacking without a common superscript letter are significantly different (P < 0.05).Data are least squares means for 6 replicate pens per diet.1 See Table 2.

Discussion

Studies indicate that supplementing pig diets with probiotics administered in various forms (e.g., spores, fermented substrates) can increase the apparent digestibility of nutrients and the retention of mineral elements, which contributes to limiting their excretion into the environment14–16. In the present study, Bacillus subtilis 87Y, administered both as spores (BS) and as a matrix of fermented rapeseed meal (FRSMb, FR group), significantly improved the retention of sodium, potassium, phosphorus, calcium, copper, zinc, iron, manganese, selenium, and nitrogen. These results are consistent with earlier reports regarding the effects of various probiotic strains (including Bacillus spp., Bacillus amyloliquefaciens, Enterococcus faecium) and probiotic blends on the efficiency of nutrient utilization in pigs14,17.

In the present studies, it was found that the apparent digestibility of phosphorus increased by 9% points, reflecting the cumulative effect of increased phytase activity and changes in the physicochemical environment of the intestine. In the piglets’ diets in the FR and BS groups, phytase activity was about twice as high as in the NC group, accelerating the hydrolysis of phytic acid and increasing phosphorus release. This phenomenon agrees with the findings of Czech et al.18 and Mosenthin and Broz19, who demonstrated that intensification of phytate degradation significantly improves the bioavailability of this element. In the case of the fermented rapeseed meal matrix, greater phosphorus retention resulted not only from higher phytase supply but also from its stabilization within the FRSMb structure. During fermentation, a protein-polysaccharide network is formed, protecting enzymes against degradation in the stomach and allowing their gradual release in the intestine20. Additionally, lowered water activity (< 0.75) promotes the stabilization of microbial spores and enhances their survival, as confirmed by Hong et al.8. These mechanisms allowed the preservation of phytase activity up to the small intestine, which is the primary site of mineral absorption. A similar increase in phosphorus utilization has been described previously in diets containing fermented components15,21.

In animals receiving Bacillus spp. as free spores (BS), an effect comparable to FRSMb was observed, albeit resulting from a different mechanism. Bacillus spp spores, due to their high resistance to low pH, pass through the stomach intact and germinate only in the small intestine, where they begin to synthesize phytase and proteolytic enzymes22,23. This enables effective hydrolysis of phytates and increased bioavailability of mineral elements, with spore activation directly in the intestine providing an effect comparable to that of enzymes stabilized within the FRSMb matrix.

Calcium retention in the FR and BS groups increased by as much as 187% compared to NC, greatly exceeding the typical 25–35% range reported after high-dose phytase supplementation24,25. Phytates bind calcium in poorly soluble complexes; thus, increasing its availability requires intensive hydrolysis of these structures. However, phytase activity alone does not explain such a high rise in retention, suggesting that additional factors are involved. Fermentation metabolites present in FRSMb, including short-chain fatty acids (SCFAs), lower the intestinal content pH, increasing the solubility of released calcium and supporting its transition to a more absorbable form. Both lower pH and SCFAs may also stimulate calcium transport via TRPV6 channels19,26. Combined with phytase protection in the FRSMb matrix, these mechanisms explain the observed effect. In the case of Bacillus spp. 87Y spores, increased calcium retention may result from their ability to colonize the small intestine and gradually release enzymes from germinating bacteria, supporting phytate breakdown and increasing calcium availability27.

An increased magnesium absorption (from 0.20 to 0.46 g/piglet/day) indicates that Bacillus spp. 87Y, in both the FRSMb matrix and as spores, modulated electrolyte transport. SCFAs, especially acetate and lactate, lower luminal pH in the intestine, increasing paracellular magnesium ion flow. The increase in Na⁺ and K⁺ retention supported Mg2+ transport dependent on Na⁺/K⁺-ATPase activity26,28. Thus, the fermented matrix acted not only as a bacterial carrier but also as a factor modulating electrolyte balance. Moreover, better magnesium utilization limited its excretion into the environment, which is ecologically significant, as excess Mg in soil disrupts ionic relationships and may impact nitrous oxide emissions29,30. Importantly, improved electrolyte absorption observed after the administration of Bacillus spp. spores alone suggests that their action was not limited to indirect effects from the matrix. After germination in the intestine, the spores could synthesize enzymes degrading magnesium-chelating compounds and modulate the expression of ion transporters such as TRPM6 and TRPM7, as well as activate signaling pathways in the intestinal mucosa, increasing both paracellular flow and transcellular transport of Mg²⁺, Na⁺, and K⁺ ions. As a result, a significant improvement in electrolyte retention was observed, further limiting environmental losses.

The reduction in zinc and copper excretion, by > 60% and approximately 40%, respectively, following Bacillus spp. 87Y use (as spores and in the FRSMb matrix) highlights a marked improvement in their intestinal utilization. Improved retention of these trace elements results from the simultaneous activation of several interacting mechanisms. A key role is played by the hydrolysis of phytates, releasing Zn²⁺ and Cu²⁺ ions from poorly soluble complexes18,31. Surfactin, a biosurfactant synthesized by Bacillus spp., is also important, as it increases cell membrane permeability and can intensify divalent ion absorption32. Another mechanism is related to the presence of levan (~ 4% DM), a prebiotic polysaccharide which stimulates the growth of lactic acid bacteria producing SCFAs capable of forming soluble metal complexes33. Levan also enhances adhesion of bacteria, including Lactobacillus, to the intestinal epithelium, strengthening barrier integrity34. This combination of probiotic, prebiotic, and enzymatic action makes FRSMb a functional synbiotic35,36. These findings align with the observations of Domżał-Kędzia et al.37 and Konkol et al.38, indicating a significant role for fermentation metabolites in increasing trace element bioavailability. Administration of Bacillus spp. spores alone (BS) also improved Zn and Cu retention through rapid germination, local enzyme and signaling molecule production, and modulation of intestinal transporters39.

A similar mechanism was observed in the case of iron, its retention in the BS and FR groups was three times higher than in NC, indicating a highly effective improvement of its bioavailability. Chelating properties of Bacillus spp. secondary metabolites, including surfactin, stabilize Fe2+ ions in more bioavailable forms40. Additionally, exopolysaccharides and SCFAs modulate the intestinal redox environment, promoting Fe³⁺ reduction to Fe²⁺ and increasing its absorption41. These effects support the activity of antioxidant enzymes such as GPx, which may enhance immune status42.

Higher plasma concentrations of phosphorus, calcium, and the trace elements copper, zinc, and iron in piglets after Bacillus spp. 87Y (FR and BS) supplementation confirm the effectiveness of their release and absorption, and improved bioavailability. Physiologically, these values, within the reference range43, may support enzymatic functions, metabolic processes, and antioxidant mechanisms. The beneficial impact of probiotics on macroelement bioavailability and their plasma concentration has been previously described44, though some studies indicate variable effects depending on strain, dosage, and environmental factors.

The exceptionally favorable effect of Bacillus spp. 87Y, as both spores and FRSMb matrix, is also significant from an environmental perspective. Nitrogen and phosphorus emissions per body weight gain dropped to values congruent with Farm-to-Fork strategy targets, aiming to reduce mineral losses by 20% by 203045. The reductions achieved here exceeded typical values noted after high-dose phytase use46. For every 1000 piglets, this limits N and P losses over 42 days by 26 kg and 3.6 kg, respectively, reducing the area needed for slurry spreading. Nitrogen excretion was reduced as a result of both phytase activity and protease production, which break down feed proteins into more absorbable forms, thus improving dietary nitrogen utilization47. The proposed nutritional solution aligns with the EU “Farm to Fork” strategy goals of reducing nutrient losses by 20% by 203048. Another important environmental aspect is the potential partial substitution of soybean meal with fermented rapeseed meal, reducing reliance on imported protein and lowering the carbon footprint of production49.

Despite the demonstrated benefits, this study covered only a single growth phase of piglets. Further research should include different production stages and direct measurements of greenhouse gas emissions (methane, nitrous oxide, ammonia) to reliably assess the environmental impact of Bacillus spp. 87Y, both as spores and within the fermented FRSMb matrix.

Summary

Supplementation of piglets with Bacillus subtilis 87Y, administered as free spores or in a matrix of fermented rapeseed meal, significantly improves macro- and trace element retention and reduces their environmental excretion. The form of administration determines the mode of action: free spores enhance mineral bioavailability through intestinal germination, local enzyme and metabolite production, and beneficial effects on the microbiota, while the fermented matrix provides a synergistic effect by enzyme stabilization and prebiotic action. The results clearly confirm the validity of both forms of Bacillus subtilis 87Y as effective strategies to improve mineral utilization in swine. To fully assess their environmental potential, further studies covering subsequent production phases and real measurements of greenhouse gas emissions are necessary.

Methods

Ethical approval

We confirm that all animal procedures were carried out in accordance with relevant institutional, national, and international guidelines and regulations concerning the care and use of animals for scientific purposes. All methods were reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org).All animal procedures were approved by the Local Ethics Committee on Animal Experimentation at the University of Life Sciences in Lublin, Poland (approval no. 50/2018, dated April 1, 2018).

Preparation of fermented rapeseed meal (FRSMb)

Fermented rapeseed meal (FRSM) was produced using Bacillus subtilis strain 87Y. Rapeseed meal was sterilized to eliminate indigenous microorganisms. Bacillus subtilis 87Y was cultured overnight in lysogeny broth (LB) at 37 °C to an optical density (OD600) of approximately 0.1. The bacterial suspension was mixed evenly into rapeseed meal, achieving a final moisture content of 50%. Solid-state fermentation was conducted at 37°C for 24 hours with periodic aeration to maintain aerobic conditions. Post-fermentation, FRSMb was dried, ground, and stored appropriately until usage.

Bacillus subtilis 87Y

The Bacillus subtilis 87Y strain was isolated from the earthworm Eisenia fetida50. The culture was prepared as described51. The FRSM was prepared in modular (6 modules) solid state fermentation bioreactor with maximum 5 m3 total working volume.

Animals and experimental design

The experiment involved 384 weaned pigs (Yorkshire × Danish Landrace), 28 days of age, equally divided by sex among four dietary groups. The positive control group (PC) received a standard diet with all feed additives recommended for the specific growth phase, whereas the negative control group (NC) was fed the same basal diet without any additives, including enzymes, acidifiers, ZnO, probiotics, prebiotics, or antioxidants. The FR group was given the NC diet supplemented with 8% fermented rapeseed meal (FRSMb) containing Bacillus subtilis 87Y, replacing a portion of the extracted soybean meal. The BS group received the NC diet with the addition of 0.1% Bacillus subtilis 87Y as free spores.

To facilitate the transition to post-weaning nutrition, experimental diets were introduced prior to weaning to promote early adaptation to new feed components. At the start of the experiment, initial body weights (mean ± SD) were as follows: PC = 9.70 ± 0.92 kg; NC = 9.56 ± 0.80 kg; FR = 9.62 ± 0.94 kg; BS = 9.66 ± 1.07 kg. Animals were individually identified, weighed, and assigned to treatments to achieve homogenous groups, with each group comprising six replicate pens of 16 pigs (eight barrows and eight gilts) per pen. The study continued until pigs reached approximately 30 kg of body weight.

The experimental period lasted 50 days and covered the interval from weaning to the end of the nursery phase. The following were evaluated: production parameters, digestibility of mineral components, retention of macro- and microelements, blood biochemical parameters, and excretion levels of biogenic elements in feces.

Experimental diets

The nutritional composition of the diets was formulated according to NRC13 standards, except for zinc in the NC, FR, and BS groups, where its sole source was the base feed ingredients.

The composition of the diet for the control and experimental groups was as follows: wheat (PC = 543.6 g; NC = 548.9 g; FR = 521.9 g; BS = 546.9 g); barley (300 g); full-fat soybean (20 g); soybean meal (PC = 66.23 g; NC = 65.9 g; FR = 7.13 g; BS = 65.8 g); soybean oil (PC = 8.13 g; NC = 6.52 g; FR = 12.98 g; BS = 8.28 g); fish meal (20 g); lysine (PC, NC and BS ~ 7.8 g; FR = 8.74 g); threonine (PC, NC and BS ~ 3.2 g; FR = 3.55 g); methionine (~ 2.07 g); monocalcium phosphate (PC, NC and BS ~ 5.5 g; FR = 4.78 g); limestone (~ 8 g); sodium chloride (~ 4.6 g).

Each diet included 3 g/kg of a 0.25% vitamin-mineral premix, supplying per kg: vitamin A – 11,250 IU; vitamin D3–2,250 IU; vitamin E – 90 mg; DL-α-tocopherol – 81.81 mg; vitamin E biologically active – 33.52 IU; vitamin K – 2.25 mg; vitamin B1–2.25 mg; vitamin B2–5.64 mg; vitamin B6–3.39 mg; vitamin B12–0.027 mg; pantothenic acid – 12 mg; niacin – 22.5 mg; biotin – 0.113 mg; choline – 500 mg; folic acid – 0.90 mg; Fe – 112.5 mg; Cu – 150 mg; Mn – 78.75 mg; Se – 0.45 mg.

The PC group also received 3.5 g kg−1of feed additives containing (per kg): 0.160 g Zn as ZnO (78% purity); 3 g of a blend of propionic, orthophosphoric, lactic, and formic acids; 0.1 g of 500 FTU phytase (E. coli origin); 0.15 g protease; 0.050 g of endo-1,3(4)-β-glucanase and endo-1,4-β-xylanase; and 0.20 g Saccharomyces cerevisiae.

Phytase activity (FTU kg− 1) in feed mixtures: FRSMb = 115.2; PC = 1423; NC = 623; FR = 1395; BS = 1100. Phytic phosphorus ranged from 2.50 g kg− 1 in PC to approximately 3 g/kg in NC, FR, and BS. Lactic acid content was: FRSMb = 40.3 g kg− 1; PC and NC ~ 17.2 g kg− 1; FR = 19.5 g kg− 1; BS = 18.1 g kg− 1.

All diets contained similar crude protein (~ 16.2%), crude fat (PC/BS: ~3.2%; NC: 3.1%; FR: 3.8%), and crude fiber (PC/NC/BS: ~3.4%; FR: 3.8%). Table 6 presents the mineral content in the feed mixtures and FRSMb.

Table 6.

The mineral content in the feed mixtures and FRSMb.

Mineral elements FRSMb PC NC FR BS
Sodium (g) 2.27 2.13 2.13 2.13 2.13
Pottasium (g) 5.66 5.44 5.60 5.45 5.45
Magnesium (g) 1.34 1.23 1.59 1.24 1.24
Calcium (g) 6.99 6.50 6.16 6.16 6.16
Phosphorus (g) 13.01 6.06 5.32 5.47 5.32
Copper (mg) 0.006 4.41 4.38 4.76 4.46
Zinc (mg) 0.053 142.9 43 47 47
Ferrum (mg) 0.542 69 69.2 78.5 78.5
Manganesium (mg) – 24 24.1 26.1 26.1
Selenium (mg) – 0.348 0.351 0.305 0.306
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All animals received ad libitum access to feed and water throughout the study.

Sample collection

During the experiment, samples of the experimental feed and FRSM were collected twice from each group to determine nitrogen and mineral content (phosphorus, calcium, magnesium, zinc, copper, ferrum, manganum, selenium).

Apparent Total Tract Digestibility (ATTD) was determined using the conventional method. From day 66 to 75 of life, including a 5 days adaptation period and a 4-day collection period, six boars per group (one from each pen) were individually housed in metabolic cages to collect feces and urine. Feces were retained on a plastic mesh beneath each pen, and urine was collected into plastic containers. Each day, 10 mL of 10% sulfuric acid was added to bind ammonia. Feces and urine were collected and weighed daily.

Feces were dried at 105 °C for 3–5 h to determine the drying coefficient, then reweighed and homogenized. A 50 g sample from each daily fecal portion was combined to form one pooled sample per pig. Urine collections were pooled and 50 mL composite samples were prepared and stored at 4 °C until analysis.

On the final day of digestibility testing, blood samples were collected from the same pigs into heparinized tubes, centrifuged at 1400×g for 10 min at 4 °C to obtain plasma, and stored at -20 °C for mineral analysis.

To assess Apparent Ileal Digestibility (AID), diets were supplemented with 2 g kg− 1 SiO₂ as an indigestible marker. On day 77 of the study, six randomly selected boars from each experimental group (the same animals previously used in the ATTD trial) were slaughtered in a licensed commercial slaughterhouse in accordance with the provisions of Council Regulation (EC) No. 1099/2009 of 24 September 2009 on the protection of animals at the time of killing. The entire procedure was carried out by qualified personnel following applicable technological and veterinary standards to ensure the highest possible level of animal welfare and to minimize pre-slaughter stress.

Animals were transported under conditions designed to reduce stress and, after a resting period, were stunned using electrical stunning to induce immediate loss of consciousness. Exsanguination was then performed by severing the major blood vessels, in accordance with standard industrial slaughter procedures. All activities were conducted in compliance with current national and EU legislation regarding animal welfare and food safety. Approximately 100 mL of digesta from the last 100 cm of the distal ileum was collected, dried, and stored frozen for chemical analysis.

Chemical analysis

The feed and FRSMb were analyzed for nitrogen content using the Kjeldahl method52. The content of minerals in all biological matrices (FRSMb, feed, feces, urine, ileal digesta, and blood plasma) was determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, iCAP Series 6500 Duo, Thermo Scientific, USA). Prior to analysis, samples were subjected to microwave-assisted wet digestion using a Berghof Speedwave system (Eningen, Germany), equipped with temperature and pressure monitoring for each Teflon digestion vessel (type DAP100). Approximately 0.5 g of sample was digested with 6 mL of 65% HNO₃ and 1.5 mL of 30% H₂O₂.

All analyses were performed in duplicate.

Calculations

Apparent Total Tract Digestibility (ATTD) and Apparent Ileal Digestibility (AID) for nitrogen and minerals (phosphorus, calcium, magnesium, zinc, copper, ferrum, manganum, selenium) were calculated as follows:

ATTD (%) = [(daily intake - daily fecal excretion)/daily intake] × 100.

AID (%) = 100–100 × [(SiO₂ in diet × nutrient in ileal content)/(SiO₂ in ileal content × nutrient in diet)]

Nutrient retention (g/day for N, P, Ca, Mg; mg/day for Zn, Cu, Fe, Mn, Se) was calculated as:

Retention = Intake - (Fecal Excretion + Urinary Excretion).

Statistical analysis

Numerical data were statistically analyzed using the STATISTICA 13.3 PL software package (v.13.3; TIBCO Software Inc. 2017, Paolo Alto, CA, USA, http://statistica.io). Prior to analysis, data distribution normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was evaluated with Levene’s test. For variables meeting the assumptions of normality, a one-way analysis of variance (ANOVA) was performed. When significant differences were detected, Tukey’s HSD post-hoc test was applied to compare means between groups (NC, PC, FR, BS). For variables that did not meet the assumptions of parametric tests, the Kruskal-Wallis test with multiple comparisons using Dunn’s method was applied. Results are presented as mean ± standard deviation (SD). Differences were considered statistically significant at p ≤ 0.05 and highly significant at p ≤ 0.01.

Author contributions

A.C., B.N.D. , L.W. designed the study, conducted the experiment, and analyzed the data. A.C. , B.N.D. prepared the initial draft of the manuscript. M.L., L.W. , A.L. critically revised and edited the final version. A.C., L.W. , A.L. contributed to the interpretation of the results. M.L. , A.C. approved the final version of the manuscript for publication.

Funding

This research was funded by National Centre for Research and Development, Poland, grant number POIR.01.01.01-00-1106/20.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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