Maple Syrup and Bacillus velezensis based Supplement: in Vivo Study of the Impact on Growth Performance, Microbiota Composition, and Metabolic Activity in Weaned Piglets

Abstract

Post-weaning piglets often face digestive challenges and growth setbacks due to gut microbiota imbalances and increased susceptibility to infections and diarrhea. Various alternatives to antibiotic growth promoters have been proposed, including synbiotic supplements. Maple syrup is a source of prebiotic compounds, while Bacillus velezensis FZB42 is a nonpathogenic microorganism with probiotic potential. Therefore, this study investigated the effects of a fermented ingredient combining maple syrup and B. velezensis as a potential synbiotic. The focus was on growth performance, occurrence of diarrhea, short-chain fatty acid production, and intestinal microbiota composition in weaned piglets. A two-week experiment in a randomized complete block design was conducted with the following treatments: a negative control diet (NC); a positive control diet containing chlortetracycline hydrochloride (PC); a synbiotic supplement containing maple syrup and B. velezensis (SYN); and a freeze-dried maple syrup supplement (FMS). Compared to the NC group, SYN supplementation increased final body weight, average daily gain, and decreased the feed conversion ratio (p < 0.05). The results showed that dietary supplementation with SYN supplement increased butyric acid concentrations in the ileum compared to NC (p < 0.014) and increased acetic (p < 0.001) and butyric acids (p < 0.044) in the colon compared to PC treatment. Additionally, both maple syrup-based supplements favorably modulated the relative abundance of microbial taxa, increasing Oscillisibacter and reducing Campylobacter, among others. Our findings indicate that dietary supplementation with a synbiotic composed of B. velezensis FZB42 and maple syrup improved the growth performance of weanling piglets, increased acetic and butyric acid content in the colon and butyric acid in the ileum, and favorably modulated the intestinal microbiota.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00284-025-04514-5.

Introduction

The weaning phase represents a critical period in the developmental life cycle of pigs, often associated with challenges such as diarrhea, reduced growth rates, intestinal metabolic disorders, infections, and in some cases, early stage mortality [1, 2]. To mitigate these challenges, antibiotics were frequently administered to promote growth by reducing microbial load and inflammation, indirectly supporting immune function, and preventing postweaning illnesses in piglets, particularly diarrhea [3, 4]. Nevertheless, substantial concerns have arisen regarding the emergence of antibiotic resistance in both intestinal pathogenic and commensal bacteria, as well as the accumulation of drug residues in manure due to excessive antibiotic use [5, 6]. As a result, these repercussions extend beyond the individual animal receiving antibiotics, affecting broader human and animal populations [7]. In response, numerous countries worldwide have restricted the use of certain antibiotics in farm animals, prompting researchers to explore alternative strategies to enhance growth performance and immune function without adverse biological impacts. Current research investigates various potential alternatives in livestock management, including probiotics [8], prebiotics, synbiotics, acidifiers [9], and plant extracts [10]. Among these, probiotics have shown significant potential in improving nutrient digestion and absorption, strengthening immunity, balancing intestinal microbiota, and maintaining the integrity of the intestinal mucosal barrier [11]. A synbiotic is defined as ‘a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host’ [12].

Beyond sucrose, maple products are composed of diverse compounds, including a variety of sugars and nitrogenous organic matter, primarily ureides such as allantoic acid and allantoin [13]. Although these molecules have not yet been extensively studied for their probiotic or prebiotic properties, they can serve as the sole nitrogen source for certain microorganisms and may exert a selective influence [13]. Furthermore, maple syrup contains oligosaccharides such as neokestose, which may enhance its prebiotic potential. Due to their unique structural properties, these saccharides are resistant to mammalian enzymatic digestion, allowing them to serve as selective substrates for fermentation by gut bacteria [14]. Additional notable compounds in maple products include at least two prebiotic polysaccharides, specifically arabinogalactan and inulin [15, 16]. Arabinogalactans are widely used to promote colonic microbiota growth and their physiological benefits in humans have been recognized for decades [17]. They are now acknowledged as components of functional foods with health-promoting properties including immunity improvements [18, 19]. Moreover, maple products contain organic acids, vitamins, mineral salts, flavonoids, phytohormones, and phenolic compounds, all of which have the potential to beneficially modulate colonic microbiota and promote animal growth [20–22]. Collectively, these compounds highlight considerable potential for valorizing downgraded maple syrups as growth promoters in livestock, such as pigs. Additionally, in vitro assessments have shown that the digestibility of downgraded maple syrups is comparable to that of standard maple syrup, underscoring their potential for use as ingredients in nutritional supplements [23].

Bacillus species have demonstrated beneficial effects in enhancing digestive enzyme activity, strengthening intestinal health, and boosting immune function, ultimately leading to improved growth performance in pigs [24, 25]. For instance, Bacillus. velezensis DSM 15544, previously known as B. subtilis C-3102 [26], has been used in piglets with positive outcomes [27, 28]. The primary mechanism underlying these benefits is the creation of an anaerobic environment in the intestines upon germination, which promotes the growth of indigenous microbial Lactobacillus species while inhibiting the proliferation of pathogenic bacteria [29]. The incorporation of fermented feed components has been shown to improve the performance of weaned and growing pigs by increasing enzyme activity, short-chain fatty acid levels, and starch digestibility [30]. Moreover, metabolites derived from fermented feed exhibit antimicrobial, antioxidant, and immunoregulatory properties [31].

In previous research, buddy syrup (also referred to as VR5 or CT5), a maple syrup classified as inferior due to its undesirable flavor, was characterized and used as the basis for developing a supplement incorporating B. velezensis FZB42, aimed at animal feed [23, 32]. Bacillus velezensis FZB42 is a well-characterized strain with a history of safe use as biofertilizer and biocontrol bacteria in agriculture [33]. Using a simulated gastrointestinal tract, the TIM-1 system, the study demonstrated the ability of B. velezensis FZB42 to survive passage through the swine gastrointestinal tract conditions and reach the colon, supporting its potential as a probiotic strain [32]. Moreover, the species B. velezensis was deemed unlikely to pose a significant risk concerning antibiotic resistance in the domains of food fermentation or human application [34].

This study evaluated the effect of orally administering downgraded maple syrup and a formulation derived from downgraded maple syrup and containing B. velezensis FZB42 on piglet growth performance. Additionally, short-chain fatty acids (SCFAs) concentration in the ileum and colon were measured, and microbial communities in these regions were characterized through metataxonomic methods.

Materials and Methods

Supplement Production

The maple syrup used, downgraded for its buddy off-flavor, was kindly provided by PPAQ (Producteurs et Productrices Acéricoles du Québec, Québec, Canada). The main sugars present were measured by high-performance liquid chromatography and reported previously for this lot; sucrose (43.46% w/w), glucose (17.69% w/w) and fructose (11.33% w/w) [32]. The whey permeate was donated by a local industry (Québec, Canada). It was obtained by drying deproteinated sweet whey and contains a maximum of 89% (w/w) lactose, 9% (w/w) ashes, and a minimum of 2% (w/w) proteins as declared by the manufacturer.

The strain Bacillus velezensis FZB42 was purchased in lyophilized form from DSMZ (German Collection of Microorganisms and Cell Cultures, DSM 23117) and reconstituted as per the manufacturer instruction. Stock cultures in glycerol (40%) were prepared and preserved long-term at – 80 ℃ and activated by inoculation (1% v/v inoculum size) in Luria–Bertani Broth (LBB) (Fischer Scientific, Montréal, Canada) under agitation at 37 ℃ for 24 h. Strain identification of the stock culture was confirmed by Sanger sequencing of the nearly full length 16S rDNA gene at the Centre de Recherche du CHU de Québec (CHUL) platform before use in fermentation.

The maple syrup-based product containing B. velezensis, intended as a synbiotic (SYN), was obtained as fully described in our previous article [32]. Briefly, fermentation was carried out in a 30 L bioreactor (Biogenie, Quebec, Canada) with a 20 L working volume composed of 3.87 g/L maple syrup, 15 g/L of yeast extract (Fischer Scientific, Montréal, Canada) and a volume of inoculum of 5% v/v. The bioreactor was equipped with accessories, connected to a computer equipped with the iFix 3.5, Intellution software (MA, USA) controlling of pH, temperature, air flow, agitation, and antifoam. The temperature was maintained at 37 ℃, the oxygen level was maintained at a 75% saturation, and the pH level was controlled to remain at 7.

The fermented medium was combined with whey permeate in a 1:1 ratio, followed by stirring for 15 min to ensure the complete dissolution of the protective agent. The resulting mixture was sprayed using a laboratory-scale spray dryer (Büchi B-290, Flawil, Switzerland) that featured a control panel, an electric heating element, a peristaltic pump, two fluid nozzles, a drying chamber, and a cyclone composed of thick clear glass. Air, heated through an electric heating element, was directed in parallel with the atomized liquid within the drying chamber at a rate of 38 m³/h. Subsequently, the dried formulation was placed in hermetically sealed plastic bags and stored at 4 ℃. The viability was confirmed post-spray drying; viable counts were 2 × 10⁸ CFU/g.

The freeze-dried downgraded maple syrup (FMS) was obtained using a RePP model FFD-42-WS (Virtis, Gardiner, New York, NY, USA), The FMS was also transferred to hermetic plastic bags and stored at 4 ℃. Supplements were added in the feed before the beginning of the in vivo trial using a feed mixer.

Experimental Design, Animals, Facilities, and Feedings

This experiment was carried out in the animal housing facility at the INAF (Institut sur la nutrition et les aliments fonctionnels, Québec, QC, Canada). Seventy-two pigs (Duroc × [Landrace × Large White], comprising 53 males and 19 females, aged 28 ± 1 days) with an average body weight (BW) of 8.57 ± 1.63 kg were purchased from a commercial farm (Coop Seigneurie, St-Anselme, QC, Canada). Piglets were weighed and allocated to nursery pens with a slatted plastic floor (0.67 m²/pig) per group of three according to their weight, to form three complete blocks with 4 repetitions of 2 pens for a total of 24 pens (6 pens by treatment). A five-day acclimatization period was implemented for all subjects. Throughout this period, animals were provided with ad libitum access to a basal diet (Sollio Agriculture, St-Hyacinthe, QC, Canada; Table 1) following the nutrient requirements of swine [35]. Subsequent to the acclimatization period, the piglets received experimental diets for a duration of 14 days. Each pen was randomly assigned to one of the following dietary treatments: 1) the basal diet (Table 1, NC negative control, 2) the NC diet containing chlortetracycline hydrochloride at a concentration of 110 mg/kg of feed (PC positive control, 3) the NC diet supplemented with maple syrup-based synbiotic 10 g/kg, which corresponds to a final concentration of B. velezensis amounting to 2 × 10⁹ CFU/kg of feed (SYN); and 4) the NC diet enriched with freeze-dried downgraded maple syrup at 10 g/kg of feed (FMS). The piglets were weighed at the beginning and end of the trial. Feed intake was evaluated daily per pen. Throughout the experiment, a mechanical ventilation system was used, the photoperiod was set to 12 h light, while the temperature was set at 21 ℃. Each pen was equipped with a stainless-steel feeder and a bowl drinker that allowed ad libitum access to feed and water during the trial period. Furthermore, animals originated from a herd that tested negative for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). During the course of the experiment, one pig assigned to the NC treatment died accidentally, and one pig assigned to FMS was found dead on the last day of a trial.

Table 1.

Composition and nutrient levels of the basal diet (air-dry basis)

Ingredient	Content (%)	Nutrient Level	Content1
Soft Wheat	10.00	Digestible energy, MJ/kg	16.86
Calcium Carbonate	1.06	Crude protein, %	20.27
Sodium Chloride (Salt)	0.50	Crude fat, %	7.16
Trace Elements & Vitamins Supplement	3.27	Calcium, %	0.89
DL-Methionine	0.20	Phosphorus, %	0.65
Vegetable Oil	5.00	Lysine, %	1.46
L-Threonine	0.18	Methionine, %	0.50
Corn	36.13	Cystein, %	0.32
Soybean Meal (Crude protein > 48%)	15.00	Threonin, %	0.96
Monocalcium Phosphate	1.00	Tryptophan, %	0.25
Hamlet Soy Protien (HP300)	14.88	Isoleucine, %	0.93
Whey Permeate	12.00	Valine, %	1.00
L-Tryptophan	0.03	Leucine, %	1.56
L-Valine	0.05	Phenylalanine, %	0.93
Lysine Sulfate (Lysine > 55%)	0.70	Tyrosine, %	0.73
Total	100.00

¹Calculated from [35] values

Sampling and Measurements

Individual weighing of the piglets was performed at the beginning and end of the experiment to measure body weight (BW) and calculate the average daily gain (ADG). Daily monitoring of feed consumption was performed within each pen, allowing the calculation of both the average daily feed intake (ADFI) and the feed-to-gain ratio (FCR). The diarrhea rate (%) was calculated as follows: total number of diarrhea piglets/(total number of piglets × test days) × 100. At the end of the experimental period (day 14), the piglets were first sedated by i.m. injections of glycopyrrolate (0.01 mg/kg BW) (Sandoz Canada Inc., Quebec, Canada) and Stresnil (4 mg/kg BW) (Janssen Animal Health, Merial Canada Inc., Quebec, Canada) and then euthanized by i.v. injection of sodium pentobarbital (106 mg/kg BW) (Bimeda MTC Animal Health Inc., Cambridge, Ontario, Canada). The entire small intestine was removed. The segment extending up to 50 cm cranially from the caecum was considered ileum [36].

Colonic digesta (at rectum level) and ileal digesta samples for bacterial DNA analysis were taken in PERFORMAbiome GUT tubes (DNA Genotek, Inc. Ottawa, ON, Canada) according to the manufacturer’s instructions. Colonic and ileal digesta samples were also taken for SCFA analysis in sterile tubes. All samples were collected and kept frozen at − 80 ℃until use.

Short Chain Fatty Acids Analysis

The colonic samples were treated as described by Laforge, Vincent [37]. Ileal digesta samples were subjected to a similar treatment with slight modifications: 1 mL of water was added per 500 mg of sample, followed by centrifugation at 18000 × g for 10 min at 4 ℃. Subsequently, SCFAs were quantified after liquid–liquid extraction using gas chromatography coupled to a flame ionization detector, adhering to the procedure delineated in Roussel, Chabaud [38].

DNA Extraction and Analysis

Total genomic microbial DNA was extracted from 250 mg of sample using the QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA quality and concentration were assessed using a NanoDrop 1000 spectrophotometer, purity was evaluated by determining the absorbance ratio at 260 and 280 nm (A260/A280) (Thermo Scientific, Wilmington, DE, USA).

The microbial community was analyzed by Illumina next-generation 16S rRNA gene amplicon sequencing as in N’guyen et al. [39]. Briefly, taxonomic analyzes were assessed by sequencing the 16S bacterial rRNA gene in the V3-V4 region using the amplification primers 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′- GACTACHVGGGTATCTAATCC-3′) [40] adapted to incorporate Illumina Nextera transposon-based adapters (Illumina, United States) and a sample barcode sequence that allows multiplexed sequencing. High-throughput sequencing was performed at the CHU de Québec-Université Laval (Québec, QC, Canada) on a MiSeq platform using 2 × 300 bp paired-end sequencing (Illumina, United States).

Microbiota Bioinformatics Analysis

Sequencing results were analyzed using the DADA2 library [41] to identify amplicon sequence variants (ASV) with the R programming language using the following parameters: trimLeft = c(25, 21), truncLen = c(275, 275). Taxonomic identification of ASV was performed using BLAST against the 16S RefSeq Nucleotide sequence records (33175[BioProject] OR 33317[BioProject], downloaded on November 8, 2024) with Geneious Prime 2025.0.2 software (geneious.com). The following BLAST parameters were applied: custom BLAST, Max E-value: 1e-50, and the top 5 results were retrieved. The lowest taxonomic rank of the best result was used. In case of ambiguity, the higher taxonomic rank was assigned. The species rank was only assigned in cases of nonambiguity and 100% pairwise identity. The results were manually reviewed to ensure accuracy of taxonomic assignment.

Statistical Analysis

The impact of feed treatment on growth parameters, incidence of diarrhea, and SCFA amounts was evaluated using a mixed model wherein blocs and pens were included as random factors. Post hoc multiple comparisons were conducted using Tukey’s HSD test. A p-value < 0.05 was deemed to indicate a statistically significant difference. The statistical analyses were executed using Minitab (eBase Solutions Inc., Vaughan, ON).

The impact on the composition of the intestinal microbiota was conducted using the Web-based tool MicrobiomeAnalyst [42]. The input data consisted of tab-delimited plain text files, including ASV tables containing read counts of each ASV across samples, a metadata file describing sample group information, and a custom taxonomy table with taxonomic rankings. The data was filtered to eliminate low quality features, with criteria applied for low-count filtering (minimum count 4, prevalence in samples 10%) and no low-variance filtering. Data normalization was carried out using total sum scaling. Rarefaction curves were used to assess sequencing coverage. Alpha diversity, including Chao1 and Shannon indices, was calculated based on unfiltered data. The differences in alpha diversity between the treatment groups were evaluated in JMP Pro 18 (SAS Institute Inc., Cary, NY) using a mixed model that included pen and replicate groups as random factors, with a significance threshold of p < 0.05. Beta-diversity was computed in the MicrobiomeAnalyst interface by performing a principal coordinate analysis (PCoA) using the Bray–Curtis distance metric on ASV. PERDISP and PERMANOVA were performed to test for differences between treatment groups. To assess differences in relative abundances of the bacterial phylum, class, and genus between groups, pairwise comparison tests were performed with the multifactor analysis option, using covariate adjustment for blocks of replicates and pens and a negative binomial model. Statistical significance was further adjusted for all comparisons using the false discovery rate method in JMP Pro 18.

Results

Growth Performance

The administration of supplements to weaned piglets resulted significant differences in growth performance (Table 2). A clear trend was observed in the performance of the treatments, ranking in descending order as follows: PC, SYN, FMS, and NC. Compared to the NC treatment, supplementation with the maple-based B. velezensis synbiotic led to a significant increase in body weight (19.11 vs. 17.16 kg), ADG (0.644 vs. 0.521 kg/d), and FCR (1.912 vs. 2.271), without affecting ADFI (Table 2). However, these outcomes did not significantly differ from those of the PC group, although they were slightly lower on average. The results for FMS supplementation were intermediate between the NC and SYN treatments, yet no significant differences were detected. Furthermore, no significant differences in diarrhea rates were observed across all groups (PC 2.78% ± 1.79; NC 5.56% ± 4.68; SYN 5.16% ± 3.50; FMS 5.95 ± 4.45).

Table 2.

Effects of treatments on growth performance in piglets

Parameters	Treatments (Mean ± SEM)				SEM	ANOVA p-value
	NC	FMS	SYN	PC
Initial body weight, kg	10.05 ± 1.14	10.10 ± 1.14	10.71 ± 1.14	10.67 ± 1.14	1.141	0.342
Final body weight, kg	17.16 ± 1.55b	17.75 ± 1.55ab	19.11 ± 1.54a	19.25 ± 1.55a	1.552	0.017
Average Daily Gain, kg	521 ± 49c	548 ± 51bc	599 ± 49ab	644 ± 49a	0.494	0.007
Average daily feed intake, kg	1 104 ± 31	1 089 ± 31	1 116 ± 31	1 092 ± 31	0.319	0.502
Feed/gain ratio	2.271 ± 0.165a	2.107 ± 0.173ab	1.912 ± 0.165bc	1.703 ± 0.165c	0.165	0.012

Means with different superscripts in the same row differ significantly (Tukey’s HSD test p < 0.05). SEM standard error of the mean

SCFAs Analysis

Within the ileum digesta, the concentrations of isobutyrate, valerate, isovalerate, and hexanoate were predominantly below the detectable or quantifiable thresholds (Supplementary Data S1). No significant differences were observed between dietary treatments for acetate (p = 0.246) or propionate (p = 0.491). However, a significant difference was identified for butyrate (p = 0.017, Fig. 1), with the concentrations in the SYN group being higher compared to those in the NC group (p = 0.014). Regarding the colonic content of SCFAs (Fig. 2), acetate concentrations varied among groups (p = 0.003). SYN treatment significantly increased acetate concentration compared to the PC group (p = 0.001). For butyrate, a significant difference was observed between the SYN and PC treatments (p = 0.044). No significant differences were found for hexanoate (p = 0.357), isobutyrate (p = 0.745), isovalerate (p = 0.810), or valerate (p = 0.177). Propionate showed only a marginal difference (p = 0.063).

Fig. 1.

Effect of maple syrup and a maple syrup-based synbiotic on ileal short-chain fatty acids in weaned piglets. Violin plots show distributions and horizontal bars indicate the mean. NC Negative control group, PC Positive control group, SYN synbiotic treatment group, FMS Freeze-dried maple syrup treatment group. *p < 0.05

Fig. 2.

Effect of maple syrup and maple syrup-based B. velezensis synbiotic on piglet colon short-chain fatty acids. NC Negative control group, PC Positive control group, SYN synbiotic treatment group, FMS Freeze-dried maple syrup treatment group. Violin plot shows the data distribution and horizontal bars indicate the mean of each group. *p < 0.05, ***p < 0.001

Microbial Composition of the Ileal and Colon Digesta

For the ileal samples, the summation of the ASV table yielded a total of 3,783,601 reads, with a mean of 54,051 reads per sample, ranging from 36,180 to 88,820. After the final data filtering step, 113 ASVs remained (Supplementary Data S2). In contrast, for the colonic samples, the ASV table summation accounted for a total of 1,558,121 reads, with an average of 22,258 reads per sample, ranging from 12,865 to 30,465. The total number of features remaining post-filtering was 472 (Supplementary Data S2). Rarefaction curves indicate adequate sequencing coverage for all samples (Supplementary Fig. S1).

The microbiota composition differed between the ileum and colon, with the ileum harboring fewer genera (Fig. 3) than the colon (Fig. 4). The dominant genera also varied considerably between the two sites. In the ileal digesta, regardless of treatment conditions, Terrisporobacter was the most prevalent genus, followed by Streptococcus, Lactobacillus, and Clostridium. In contrast, in the colonic compartment, Prevotella was the most prevalent genus, followed by Limosilactobacillus, Segatella, and Roseburia.

Fig. 3.

Ileal microbiota composition in weaned piglets. NC Negative Control group, PC Positive Control group, SYN Synbiotic Treatment group, FMS Freeze-Dried Maple Syrup Treatment group

Fig. 4.

Microbial relative abundance in the colon of weaned piglets. NC denotes the negative control group, PC represents the positive control group, SYN signifies the synbiotic treatment group, and FMS indicates the freeze-dried maple syrup treatment group. Genus < 10000 total reads threshold are grouped as ‘Others’

Assessment of microbiota alpha diversity across treatments using the Chao1 and Shannon indices revealed no significant treatment effects on community diversity in either the ileum or the colon (Fig. S2). Beta-diversity variation was consistent across treatment groups, indicating no significant differences in dispersion (PERMDISP: ileum p = 0.219 and colon p = 0.221) at either sampling site. However, marginal differences were observed in beta diversity in the ileal microbiota (PERMANOVA, r = 0.069 and p = 0.054) (Fig. S3a), while significant differences were detected in the colonic microbiota (PERMANOVA, r = 0.055 and p = 0.013; (Fig. S3b). Pairwise PERMANOVA analyses revealed significant differences in beta diversity between the PC and the NC groups (FDR adjusted p = 0.046), as well as between the PC group and both SYN and FMS treatments (FDR adjusted p = 0.018 and 0.006, respectively).

A differential analysis of taxonomic groups was conducted at the bacterial genus, class, and phylum levels (Supplementary data S3). At the genus level, several taxa exhibited significant differences between treatments (Fig. 5). Specifically, compared to the NC group, the SYN and FMS treatments modulated five and three genera in the ileum, and 31 and 35 genera in the colon microbiota, respectively. The most pronounced differences were observed in the ileum microbiota for Potamosiphon, Romboutsia, and Actinobacillus, which were less abundant in the SYN group than in the NC group. Romboutsia was also significantly less abundant in the FMS group compared to the NC group. In the colon, the most notable effects of the SYN treatment compared to the NC group included an increase in the abundance of Paraprevotella and a decrease in Galbibacter and Succinivibrio.

Fig. 5.

Comparative analysis of genus abundance across treatments in ileum and colon microbiota. NC Negative control group, PC Positive control group, SYN Synbiotic treatment group, FMS Maple syrup treatment group, Log2FC Log2 fold change of normalized read counts. Only statistically significant differences are reported (FDR adjusted p < 0.05)

Within the colon microbiota, the genera Butyricicoccus, Campylobacter, Coprococcus, Holdemanella, Oscillibacter, and Ruminococcus exhibited consistent and statistically significant differences in abundance between the SYN and FMS groups relative to both NC and PC groups. The abundance of Campylobacter was lower in the SYN and FMS groups compared to the PC and NC groups, with significantly greater reduction in the SYN group than in the FMS group. This effect was also evident at the class (Epsilonproteobacteria) and phylum (Campylobacterota) levels (Fig. S4). Similarly, Coprococcus and Holdemanella were less abundant in the SYN and FMS groups than in the NC and PC groups, though their abundance was higher in the SYN group compared to the FMS group. In contrast, Butyricicoccus, Oscillibacter, and Ruminococcus were more prevalent in the microbiota of piglets receiving maple syrup and synbiotics. Furthermore, Butyricicoccus was more abundant in the FMS group, while Oscillibacter and Ruminococcus exhibited similar levels in both the SYN and FMS groups.

The results suggest that dietary supplementation with a synbiotic formulation composed of B. velezensis and maple syrup can enhance the growth performance of weaned piglets, increase the acetic and butyric acid levels, and favorably modulate the gut microbiota.

Discussion

The prevalence of diarrhea in piglets, akin to many other animals, is attributed to alterations in the composition of intestinal microbiota caused by pathogen invasion, environmental stressors, and the use of broad-spectrum antibiotics [43]. Consequently, diarrhea represents a complex challenge and a major contributor to economic losses within the pig farming industry [44]. Weaning, a primary stressor in the piglet’ life, can lead reduced growth performance, immune dysfunction, disruptions in the intestinal microbiota, and long-term effects on the structural and physiological integrity of the intestinal mucosa [45, 46]. Historically, antibiotics have played a critical role during this period. However, concerns over antimicrobial resistance in pathogens, along with the emergence and advancement of alternatives such as probiotics, have gradually reduced reliance on antibiotics [47]. Previous research has demonstrated the beneficial effects of probiotics containing various Bacillus species in improving growth performance and controlling diarrhea in weaning piglets [48, 49]. The present study investigated the impact of a synbiotic formulation based on maple syrup and the potential probiotic B. velezensis FZB42, on post-weaning piglets. The findings revealed that the synbiotic intervention significantly increased final body weight, ADG and FCR, compared to the NC group during the study period. Since these results were not significantly different from those observed with antibiotic treatment, the synbiotic formulation emerges as a promising alternative. These findings align with previous research involving Bacillus subtilis C-3102 [28, 50, 51] and Bacillus licheniformis [49, 52–54]. In the absence of a significant effect of the FMS treatment on growth parameters compared to the NC group, it appears that the inclusion of B. velezensis is essential for enhancing the digestibility, availability, or assimilation of feed nutrients.

The study demonstrated that the synbiotic formula significantly increased the butyric acid concentrations in the ileum compared to the NC group, and elevated both acetic and butyric acid levels in the colon relative to the antibiotic treatment. SCFAs play an active role in nutrient metabolism and immune function, while also regulating the composition of the intestinal microbiota [55]. They are produced through the fermentation of oligo or polysaccharides by anaerobic bacteria in the large intestine [46]. SCFAs serve not only as the principal source of energy for colonocyte proliferation but also play key role in modulating the host’s immune and inflammatory responses while influencing the microbial community composition [55]. Furthermore, SCFAs contribute to the establishment of an acidic environment in the intestine, thereby inhibiting the proliferation of certain pathogenic bacteria [56]. SCFAs, generated from the fermentation of indigestible carbohydrates, have been shown to positively influence the growth of newborn mice [57]. Among them, butyrate is particularly known for promoting cellular growth by supplying energy to colonic epithelial cells and modulating immune responses [58]. The administration of probiotics has been shown to increase SCFA concentrations, which can benefit the intestinal environment in piglets [59]. Probiotics enhances SCFA production, particularly acetic acid, which has the potential to mitigate post-weaning diarrhea in piglets [59]. Similarly, the direct supplementation of B. subtilis and B. licheniformis has been reported to increase acetic and butyric acid concentrations in weaned piglets [52, 58, 60, 61]. Our study supports these findings, confirming the ability of B. velezensis FZB42 to enhance SCFA concentrations. Since Faecalibacterium and Butyricicoccus have been identified as key butyrate-producing genera in pigs [62], and their levels were elevated in the SYN group compared to the PC group, it is plausible that the synbiotic effect on digesta butyrate composition is mediated through these microorganisms.

The intestinal microbiota plays a crucial role in nutrient utilization, SCFA production, immune system enhancement, and resistance to harmful bacteria [63], and it is well established that both antibiotics and probiotics can be incorporated in animal feed to regulate the diversity and composition of gut microbiome [64]. Microbial community diversity is often used as a key metric to assess the stability and health of the gastrointestinal ecosystem [65]. In the colonic region, significant variations in microbiota beta diversity were observed between the PC group and the other dietary interventions. Broad-spectrum antibiotics are known to have a profound impact on gut microbiota composition. Specifically, chlortetracycline has been previously documented to alter the diversity of the piglet microbiota [66]. In this context, while the antibiotic-treated group exhibited a shift in microbiota compared to the NC group, the maple-based supplemented groups did not induce a comparable shift in beta diversity compared to the NC group. This suggests that the symbiotic formulation exerted a more targeted effect on the gut microbiota compared to the broad-spectrum antibiotic.

Previous studies have demonstrated that Bacillus species can significantly influence the diversity of intestinal microorganisms in piglets [67]. Additionally, Bacillus species contribute to enhancing and strengthening digestive and immune functions in piglets [68, 69]. Among the observed differences between the SYN and FMS groups, a notable shift was detected in the Terrisporobacter genus, a key member of the intestinal microbiota In the SYN group, Terrisporobacter increased in the colon, whereas it decreased in the FMS group compared to the NC group. This genus has been reported to include emerging anaerobic pathogens capable of acetogenesis through the degradation of various carbon sources, such as xylose and cellobiose while also inducing oxidative stress [70, 71]. However, the degradation of these carbon sources has been shown to improve feed digestibility, thereby promoting weight gain [72, 73]. Therefore, this difference, along with other microbiota modulations, may explain the differences, albeit non-significant, between the SYN and FMS treatments. Moreover, Bacillus species have been reported to play a critical role in helping piglets reestablish a healthy microbiota and regulate potential pathogens by disrupting the quorum sensing mechanisms of harmful microorganisms [74]. However, our results do not point in this direction as a specific mechanism of B. velezensis FZB42. So far, there is no evidence for activity against animal pathogens among compounds produced by B. velezensis FZB42, as testing was limited to other microorganisms, mostly phytopathogens [33]. Nevertheless, this perspective should be investigated to help document the strain’s probiotic potential.

The modulation effects on microbiota were sometimes similar for both maple-based treatments in comparison to the NC and PC groups. The use of maple syrup as a prebiotic source have enhanced beneficial effects by modulating the intestinal microbiota, improving the nutrient digestion and absorption, or strengthening the immune system of the animal [75]. Previous research on maple products has demonstrated a positive modulation of the gut microbiome in mice. The use of maple syrup as a substitute for sucrose altered the composition of the intestinal microbiota in mice subjected to a high-fat, high-sucrose diet [76]. Furthermore, a study involving a synbiotic containing maple sap, inulin, and two strains of probiotics was effective in mitigating antibiotic-induced disruptions of the mouse microbiota [77]. In a human clinical trial, replacing a small amount of refined sugar with maple syrup led to a significant reduction in major cardiometabolic risk factors, along with specific alterations in the gut microbiota [78].

Within the ileal microbiota, the abundance of Romboutsia was reduced in both SYN and FMS treatment groups compared to the NC group. Previous studies have suggested that Romboutsia species may be involved in the catabolism of glucose and fructooligosaccharides [79]. Notably, the abundance of Romboutsia varies between different pig breeds and has been associated with fat accumulation [80, 81]. Obese Jinhua pigs exhibit enhanced meat quality, which is attributed to increased intramuscular fat content compared to lean Landrace pigs. Fecal microbiota transplantation studies of these breeds in mice have shown an increase in Romboutsia abundance in mice receiving microbiota from Obese Jinhua pigs [81]. Consequently, the supplementation of pig feed with a maple syrup-based additive may contribute to modulating the gut microbiota in a way that could potentially improve meat quality.

In the colon, several key genera exhibited similar responses to both maple-based interventions. Notably, the abundance of the genus Campylobacter was reduced in both maple syrup-based supplementations compared to the NC and PC groups. Campylobacter is strongly associated with diarrhea in post-weaning piglets [82, 83]. However, the occurrence of diarrhea remained constant across treatments and was minimal throughout the experiment, likely due to the improved health status under the breeding conditions. Conducting the experiment in commercial environments or implementing a challenge test against Campylobacter would be valuable in confirming the effects observed in this study.

Among the various taxa affected, the relative abundance of Coprococcus was reduced in the SYN and FMS groups compared to NC and PC groups. This genus has been documented to have a negative association with body weight in pigs [84]. In the aforementioned study, Oscillospiraceae (synonym Ruminococcaceae) was identified as having a positive association with body mass index, aligning with the present findings, which indicate an increased relative abundance of Ruminococcus and Oscillisibacter in response to FMS and SYN. Furthermore, in post-weaned piglets, both Coprococcus and Ruminococcus were associated with increased expression of barrier proteins, while Oscillisibacter showed a positive correlation with the overexpression of enzymatic and barrier proteins [85]. Moreover, a favorable correlation was observed between Oscillisibacter and productivity metrics, such as the FCR. Similar results were noted for Butyricicoccus, which also increased in response to FMS and SYN. In fact, these two genera exhibited significant responsiveness to a diet enriched with fructooligosaccharides [85]. Additionally, in another study, Oscillisibacter was found to be significantly elevated in high-ADG pigs compared to low-ADG pigs [80]. Lastly, a species of Oscillisibacter, namely Oscillibacter valericigene, was identified as an integral component of the pig microbiota, playing essential roles in maintaining organ indices, intestinal epithelial barrier function, intestinal mucosal morphology, and host metabolism [86]. Together, both the existing literature and the results presented herein suggest that the piglet microbiota may be beneficially modulated by fructooligosaccharides present in maple syrup used. Abundance and types of fructooligosaccharides in maple syrup has not been extensively documented and could vary between regular and downgraded syrups. If the effects are specific to the type of maple syrup used, i.e., buddy syrup, supply may be of concern in the future owing to climate changes [87].

Limits of the Study

The work presented in this study demonstrated the effects of maple syrup supplementation in piglets, in lyophilized form and as a fermented supplement containing B. velesensis FZB42, a potential probiotic. However, a control group with only the bacteria without maple syrup was not included in the design. Therefore, the effects of the strain on its own cannot be confirmed. Moreover, the experimental design was limited in terms of replications and may have lacked the power to detect smaller effects, such as the modulation of the microbiota by the FMS treatment on weight gain, which were not significant in terms of productivity parameters. Furthermore, our study focused on the acute post-weaning phase, and the 14-day duration was chosen to capture early physiological and microbial responses and evaluated a single dosage, which may not provide a comprehensive understanding of the dosage or long-term effects. Finally, since different breeds exhibit distinct metabolic traits and microbiota composition [81, 84, 86], examining only one breed may not fully capture the variations that could influence the study outcomes. Further experiments are needed to address these limitations and confirm the potential of maple syrup and B. velesensis FZB42-based supplements on piglets, as well as to further document the probiotic properties and safety of the strain for animal use.

Conclusion

Collectively, the findings presented indicate that dietary supplementation with a maple syrup-based synbiotic containing B. velezensis FZB42 significantly improved outcomes for weaning piglets in terms of body weight, ADG, and FCR. Additionally, colonic acetic and butyric acid content was enhanced, accompanied by a targeted positive modulation of the intestinal microbiota. In some cases, supplementation with freeze-dried maple syrup alone was sufficient to induce measurable changes in beneficial gut microbiota members, demonstrating its functional properties and prebiotic potential. Notably, there was effective control of the pathogenic genus Campylobacter. These findings provide novel insights into the beneficial effects of maple syrup-based supplements and B. velezensis in post-weaning piglets and may represent a viable alternative to growth-promoting antibiotics.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ADFI

Average daily feed intake

ADG

Average daily gain

ASV

Amplicon sequence variant

BW

Body weight

DNA

Deoxyribonucleic acid

FCR

Feed conversion ratio

FDR

False discovery rate

FMS

Freeze-dried maple syrup

NC

Negative control

PC

Positive control

PCoA

Principal coordinate analysis

PCV2

Porcine circovirus type 2

PRRSV

Porcine reproductive and respiratory syndrome virus

SCFA

Short-chain fatty acids

SEM

Standard error of the mean.

SYN

Synbiotic

Author Contributions

Conceptualization, G.D., F.G. and I.F.; methodology, G.D., F.G., M.F. and I.F.; validation, G.D., L.L-V., M.F., F.G. and I.F.; formal analysis, G.D., F.G. and M.F.; investigation, G.D. and L.L-V.; resources, G.D., L.L-V., F.G., M.F. and I.F.; data curation, G.D., L.L-V. and M.F.; writing–original draft preparation, G.D.; writing–review and editing, G.D., L-L.V., M.F., F.G. and I.F.; visualization, G.D. and M.F.; supervision, M.F., F.G. and I.F.; project administration, F.G. and I.F.; funding acquisition, I.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Conseil de Recherches en Sciences Naturelles et en Génie du Canada (CRSNG), grant number RDCPJ-52377318, the Consortium de recherche et innovations en bioprocédés industriels au Québec (CRIBIQ), grant number 2017-032-C29, and by the Producteurs and Productrices Acericoles du Quebec.

Data Availability

The sequencing data used to support the findings of this study is available at NCBI bioproject PRJNA1258565.

Declarations

Competing Interests

The authors declare that they have no competing interests.

Ethical Approval

This in vivo trial was approved by our Institutional Animal Care Committee (#2021-756). The animals were cared for according to a recommended code of practice and procedures reviewed by the Institutional Animal Care Committee in accordance with the Canadian Council on Animal Care (CCAC) guidelines on the care and use of farm animals in research [88].

Consent for Publication

All authors have read and agreed to the published version of the manuscript.