1. Introduction
Dogs, like cats, belong to the order of carnivores, so their main source of nourishment is made up of animal tissues. However, comparative studies of the nutritional needs, anatomical characteristics, and metabolic adaptations of these two species show how differently they have evolved from each other.
During evolutionary development, cats have always been obligated carnivores, but dogs, instead, are defined as opportunistic carnivores, developing eating habits more similar to those of omnivores. This is because dogs have lived in greater contact with man, gradually adapting to his type of diet. Most canine microbiome studies [
1,
2] looked to extruded kibble, representing up to 95% of the dry dog food market [
3]. Traditionally, the extrusion process requires a high content of carbohydrates (at least 30% starch in % dry matter—DM) in the form of vegetable ingredients. Recently, new alternative industrial processes allow the inclusion in the pet food market of reduced carbohydrate and increased protein-content kibble (no more than 15% starch in % DM and at least 25% protein in % DM) [
3].
It is well known that high-protein vs. high-carbohydrate diets generate different gut microbiome profiles. The maintenance of a healthy microbiome is affected by many other environmental factors; however, the changes they cause have less impact when compared to the alterations found in diseased animals. GI dysfunctions are mainly associated with gut dysbiosis. Acute and chronic diarrhea, obesity, metabolic diseases, cancer, and neurological dysfunctions have been associated with gut microbiome modifications [
3]. Similar to studies on healthy dogs, those on dogs with GI diseases also reported different taxa abundance percentages [
4]; however, most taxa consistently vary within the same disease phenotype.
Manipulations of the microbiome are often part of the treatment; several strategies have been proposed to improve and maintain the equilibrium of the intestinal microbiota, including probiotic supplementations. Currently, probiotics are also used to prevent or minimize symptoms due to the use of anti-inflammatory treatment [
5], acting on the frequency of diarrhea, the composition of the fecal microbiota, and/or markers of GI inflammation. Sometimes, the principal disadvantage of a probiotic supplementation is their low persistence in the canine gastrointestinal tract after finishing the probiotic supplementation [
6,
7]. A further approach is the use of synbiotics, in which the presence of prebiotic ingredients should support probiotic growth, increasing its proliferation within the GI tract and offering its own advantages [
8,
9]. However, the use of a combination of postbiotic and prebiotic supplementations in the canine diet has not yet been adequately explored.
The term postbiotic is derived from ‘biotic’, defined as ‘relating to or resulting from living organisms’, and ‘post’, a prefix meaning ‘after’; together, these terms suggest ‘after life’, that is, non-living organisms; postbiotic is a ‘preparation of inanimate microorganisms and/or their components that confer a health benefit on the host’ [
10]. Although the effects of postbiotics on the microbiota might be temporary, they can have direct and indirect antimicrobial activities. With regards to the direct antimicrobial activity, in vitro studies recognize compounds, such as bacteriocins, that have an antimicrobial action; on the other end, the indirect antimicrobial activity is associated with the transport of quorum-sensing (favoring the microorganisms communication) and quorum-quenching (capable of interrupting communication) molecules. Moreover, other studies highlighted the importance of other molecules like lactic acid that can be consumed by some members of the intestinal microbiota, resulting in SCFAs and butyrate production, which have beneficial functions [
11].
Postbiotics also perform local and systemic immunomodulatory functions by activating an immune response to fight infections and reduce the infection degree in the acute phase; some of the immunomodulatory microbial metabolites are branched-chain fatty acids and SCFAs. In addition, they improve the epithelial barrier functions; from Bifidobacterium spp. is derived substances promoting barrier function, reducing inflammation with mechanisms under study.
SCFAs found in postbiotics also have the potential to modify the function of the epithelial barrier, helping to keep it intact; they serve to increase transepithelial resistance and to stimulate the formation of the tight junction in some of the intestinal epithelial cells. Succinate, a bacterial intermediate in carbohydrate fermentation as well as a substrate for intestinal gluconeogenesis, appears to act on glycemic control, improving systemic metabolism.
Other research has also shown how postbiotics can modulate cognitive function and behavior using different neuroactive compounds, including neurotransmitters, such as serotonin, dopamine, acetylcholine, GABA, and various compounds that can bind to receptors expressed in the brain, such as serotonin. While the study of pre- and probiotics began several years ago, postbiotics have only recently entered the heart of scientific research; currently there are few works, especially in vivo studies, that have examined the effect of postbiotics and their possible combinations on the composition of the intestinal microbiota, representing another therapeutic strategy for modulating the gut microbiota of healthy and/or diseased animals.
In the present study, the authors evaluated whether two specific commercial dietary supplements (a combination of a postbiotic and prebiotics and a probiotic product) recommended for counteracting intestinal dysbiosis in dogs affected the quantity, composition, and metabolic activities (production of SCFAs) of canine gut microbiota after 6 h and 24 h of supplementation, compared to time zero.
The investigation was performed using an in vitro fermentation model simulating the dog intestinal tract. The in vitro model, also utilized in previous studies [
12,
13], offer several advantages, including dynamic sampling over time and high reproducibility, without the ethical issues that can arise in clinical human contexts.
2. Materials and Methods
2.1. Commercial Products Tested
Two commercial dietary supplements dedicated to counteracting intestinal dysbiosis in dogs were tested. Microbiotal cane
® (NBF Lanes S.r.l., Milan, Italy) are tablets with a purplish color, are odorless, and contain tindalized
Limosilactobacillus reuteri NBF 1 (formerly
Lactobacillus reuteri) DSM 32203 [
12], fructo-oligosaccharides (FOS), chicory inulin, microencapsulated butyric acid, and polyphenols from blood orange. NBF 1
® (NBF Lanes S.r.l., Milan, Italy) is a white powder, is odorless, and consists of the live probiotic bacterial strain
L. reuteri NBF 1 (formerly
Lactobacillus reuteri) DSM 32203 [
14].
2.2. Batch Culture Fermentations
A pilot fermenter (Applikon Fermentation System, Applikon Biotechnology, Delft, The Netherlands), stirred and pH- and temperature-controlled, was used to perform the anaerobic batch culture. The whole procedure was previously described by Belà et al. [
12].
The inoculum was prepared using fecal samples obtained from 3 adult healthy dogs following the procedure reported by Belà et al. [
12]. Subjects were fed a standard diet free of components that could influence the analysis; they had not consumed any antibiotics for at least one month before the study, had no history of GI disease, and were not regular consumers of probiotic/prebiotic supplements. The fecal samples were collected by the owners of the dogs, who gave their consent to offer the already naturally deposited stools. Ethical review and approval were waived for studies in which protocol requirement for the administration of a nutraceutical is covered by Directive 2010/63/EU of the European Parliament and by the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Fecal slurry (1%) was the inoculum of each batch culture fermentation. The latter were singularly supplemented and named as follows: Microbiotal was added with 0.5% (5 g) of the Microbiotal cane® supplement, and NBF 1 had, as inoculum, 109 CFU/mL of the NBF 1® supplement. The supplement concentration simulated the real dose recommended for use by the manufacturer.
The day before fermentation, the product chosen to be fermented and the fecal inoculum (fecal sample previously prepared and stored at −80 °C) were transferred from −80 °C into the cold room, while on the same day of fermentation, the saline solution, hemin, vitamin K, bile acids [
12], and the product to be tested were added to the medium just before starting fermentation.
The pH was adjusted at 6.80 ± 0.2 before inoculating the medium. The system was permanently gasified with N2 fed at 15 mL/min. The anaerobiosis, the temperature of 39 ± 1 °C, and the stirring (at 50/55 rpm) were kept for a period of 24 h, simulating the conditions of the canine intestine. During the fermentation, samples’ aliquots of 10 mL were collected at three specific time points: T0 (the beginning of fermentation), T6 (after 6 h of fermentation), and T24 h (after 24 h of fermentation). This latter time point was included to better understand the long-term fermentation changes on the microbiota, considering the model over a realistic time. Samples were stored at −20 °C and higher until be analyzed for bacterial enumeration and quantification of short-chain fatty acids.
2.3. Selective Enumeration of Bacteria Groups, Bacterial Profiling of the Fluid Microbiota, and SCFAs Determination
At each time point, two fermented fluid aliquots were used for bacterial enumeration/profiling and for SCFA quantification, respectively.
Real-time PCR was performed for the enumeration of selected bacterial groups, as described by Belà et al. [
12], after extraction of bacterial DNA [
15] from one aliquot of fermented fluid. The bacterial groups of interest were
Bifidobacterium spp.,
Lactobacillus spp.,
Bacteroides–Prevotella–Porphyromonas spp.,
Staphylococcus spp.,
Clostridium coccoides–Eubacterium rectale group, and Enterobacteriaceae. SYBR Green real-time PCR amplifications were performed using an iCycler iQ real-time detection system (Stratagene, La Jolla, CA, USA) associated with the MXP Software (2.0 version). All PCR experiments were carried out in triplicate, with a reaction volume of 20.6 μL, using iCycler IQ 96-well optical grade PCR tubes (Stratagene) covered with an iCycler optical cap (Stratagene).
The efficiency of PCR amplification was optimized with a primer concentration of 500 ng/μL (pmol/μL), which was proved to be optimal for the amplifications of target sequences. The reaction mixtures contained 9.8 μL of iTaqUniversalSYBR Green Supermix (Bio-Basic, Amherst, NY, USA), 9 μL of distilled sterile water, and 0.4 μL of each primer (forward and reverse, DSMZ). Subsequently, 1 μL of DNA (or water for negative control) was added to the reaction mixture.
For each bacterial strain, the total bacterial copy number per organism was determined with 16S rRNA gene-targeted primers.
Amplification was performed with the initial temperature at 95 °C for 2 min. UniversalSYBR probe activation, 35–40 cycles of denaturation at 95 °C for 15–30 s, annealing temperature kept at the optimal temperatures for 20–60 s, extension at 72 °C for 30 s, and an additional incubation step at the same annealing temperature and length were performed to collect fluorescent data [
12,
16].
In addition, from the extracted DNA, high-resolution genotyping of the fermentation fluid microbiota was performed to comprehensively characterize the microbial composition of the fluid during that time. The 16S metagenomic analysis was carried out using a 16S next-generation sequencing (NGS) approach, as described by Micioni Di Bonaventura and colleagues [
17].
A second aliquot of fermentation fluid was used for the extraction and quantification of SCFAs, following the analytical procedure reported by Scortichini et al. [
18].
2.4. Statistical Methodology
The experimental fermentations were conducted in duplicate, and the analysis was repeated in duplicate. The data were expressed as the mean value ± standard deviation. One-way ANOVA and Tukey’s multiple comparison test were used to compare different time points within the same fermentation experiment and the two kinds of fermentations.
For the 16S NGS data, differences in relative abundances of taxa between the two types of fermentations were evaluated using the four multiple comparison test (R version 4.2.1), with a significance level of p < 0.05.
4. Discussion
The microbial ecology of the canine large intestine still attracts great interest from the scientific community. One of the focusing topics is the effects of specific compounds or microorganisms, like probiotics and prebiotics, on the canine intestinal microbiota. This in vitro study evaluated the potentials of two commercial dietary supplements, Microbiotal cane® and NBF 1®, to affect the canine intestinal environment in terms of microbiota composition and the production of SCFAs.
The predominant phyla in the fermentation fluid consistent with previous reports in dogs included Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria [
19], and the results from NGS reported taxonomic profile variation trends within the two fermentations.
The results from enumeration by real-time PCR showed that the fermentation of both supplements can significantly increase the amount of Lactobacillus spp., Bifidobacterium spp., and Bacteroides–Prevotella–Porphyromonas spp., whereas the amount of the Cl. coccoides–Eu. rectale group remained quite stable during the fermentative process. Staphylococcus spp. displayed an increase at 24 h of the process only, whereas Enterobacteriaceae increased during the fermentation time.
In detail, Microbiotal cane
® promoted a more immediate increase in
Lactobacillus spp. after the first 6 h of fermentation, whereas NBF 1
® promoted the increase at the end of the process only. The two supplements supported an increase in the
Bifidobacterium spp. counts only after 24 h. The fact that both
Lactobacillus spp. and
Bifidobacterium spp. increased in quantity during the fermentations of the tested supplements reveals the positive activities of Microbiotal cane
® and NBF 1
® on the gut canine microbiota, due to their capacity to increase bacterial groups involved in exerting beneficial effects on gut health. Like most intestinal bacteria, bifidobacteria are saccharolytic; they obtain carbon and energy through the fermentation of host and dietary carbohydrates. Bifidobacteria catabolize a variety of mono- and oligosaccharides released by glycosyl hydrolases acting on nondigestible plant polysaccharides or host-derived glycoproteins and glycoconjugates [
20]. Microbiotal cane
® consists of fructo-oligosaccharides (FOS) and inulin that are mixtures of fructose moieties linked by glycosidic bonds with a terminal glucose unit [
21]. FOS and inulin transit through the stomach and small intestine, where they are neither absorbed nor degraded, and reach the colon, where they are fermented by resident bacterial groups and promote the proliferation of bifidobacteria. Thus, FOS and inulin are effective prebiotics, defined as substrates, that are selectively utilized by host microorganisms, conferring a health benefit [
22]. The fermentations of oligo- and polysaccharides in the colon are the result of intestinal microbial metabolic activity. During transit through the large bowel, unabsorbed carbohydrates, such as FOS and inulin, are hydrolyzed to their respective sugars; these sugars are fermented to SCFAs and biomass by the complex bacterial population. SCFAs represent the most important source of energy for colonocytes; they can stimulate the growth of colorectal mucosal cells, retard mucosal atrophy, and decrease the risk of malignant transformation in the colon. Butyrate has been shown to have anti-cancer effects both in in vitro cancer cell culture systems and in in vivo animal model experiments. The associated mechanisms involve many signaling pathways, as well as anti-inflammatory actions [
23]. It stimulates mature colonocytes and inhibits undifferentiated malignant and stem cells [
24].
Human in vivo trials have established that the addition of FOS or inulin to the diet leads to an increase in bifidobacteria [
25], and several studies have described in vitro fermentation of FOS by human fecal cultures [
26].
A different situation concerns
Bacteroides–Prevotella–Porphyromonas spp., which increased during the fermentation of Microbiotal cane
®, especially at the end of the process, whereas it remained quite stable at the end of NBF 1
®’s fermentation, with respect to the starting point, even if an initial decrease was detected after 6 h. Among dominant beneficial bacteria, there are several species of
Bacteroides that metabolize polysaccharides and oligosaccharides, providing nutrition and vitamins to the host and other intestinal microbial residents. The specific molecular interactions responsible for the beneficial and detrimental effects of
Bacteroides species in humans [
27] are not yet clear. It is already known that some pathogenic bacteria belong to this group, but current studies highlight that
Bacteroides–Prevotella–Porphyromonas spp. also releases some beneficial metabolites, like SCFAs. Therefore, the fermentation of Microbiotal cane
® causing an increase in this bacterial group cannot be considered a negative result.
The NGS data confirmed, in part, the modulation trend of the bacterial groups detected by real-time PCR. In addition, the presence of Fusobacteriaceae was highlighted with a notable existence of the
Fusobacterium genus at 24 h in both fermentations. It is noteworthy that this bacterial group has a positive aspect, being prevalent in the guts of healthy dogs who typically spend a lot of time outside [
28].
Both supplements exerted a stabilizing effect on the Cl. coccoides–Eu. rectale group, to which numerous harmful and pathogenic species belong.
The Enterobacteriaceae family includes genera and species of prevalent fecal origin (
Escherichia,
Salmonella, and
Shigella), environmental localization (
Budvicia,
Buttiauxella,
Citrobacter,
Enterobacter,
Erwinia,
Klebsiella,
Providencia,
Serratia, etc.), and mixed localization (
Citrobacter freundii,
Enterobacter aerogenes, and
E. cloacae;
Klebsiella ozenae,
K. oxytoca, and
K. pneumoniae; and
Proteus spp.). Most of these bacterial species, which naturally inhabit human and animal intestines [
29], have been detected in the two fermentation fluids by the NGS analysis.
The fermentation of the two supplements also promoted a non-significant increase in the Staphylococcus spp. after 6 h, becoming significant at the end of the process. Staphylococci are bacteria that commonly live on the skin, throat, and intestines of people and animals without causing problems; however, in particular conditions, they can penetrate the human/animal body and develop infections that are sometimes mild, other times serious. It is important to remember that the intestinal transit in dogs is much faster than in humans, and the intestinal fermentation usually resolves in the first 6–12 h. In the present study, what happens after 24 h of fermentation was also observed, but in dogs, such a long fermentation process never occurs. Consequently, the fact that staphylococci increased above all at the end of the 24 h of fermentation and not in the first 6 h can be considered a positive result.
The acetic, propionic, and butyric acids were the only ones to increase after 24 h of fermentations and contributed to lowering the intestinal pH, reducing the growth of pathogenic species and influencing the intestinal microbiota composition [
30]. Acetic acid derives from the metabolisms of
Bifidobacterium spp. and
Lactobacillus spp. [
31]; the significant increase in this carboxylic acid at 24 h can be related to the increases in lactobacilli and bifidobacteria at that point. Among the metabolites released by the lactobacilli, butyric acid was also produced in a modest amount, especially at the end of the fermentation with Microbiotal cane
®. On the other hand, butyric acid constitutes a major energy source for colonocytes, and it is partially metabolized by hepatocytes for gluconeogenesis with propionic acid [
32]. The production of propionic acid was detected at a low level only after 24 h of fermentation for both supplements.