2. Prebiotics
2.1. Traditional Prebiotics
Traditional prebiotics comprise carbohydrates that are predominantly resistant to digestion by mammalian enzymes
[18][39]. It was originally thought that these prebiotics were completely resistant to mammalian enzymes and reached the distal GIT intact; however, recent studies suggest there may be a degree of degradation of certain traditional prebiotics by brush border enzymes in the small intestine
[18][19][39,40]. Nonetheless, traditional prebiotics (beta-glucans, non-digestible oligosaccharides, inulin, pectin, and resistant starch) are particularly sensitive to degradation by bacteria in GIT, where they undergo fermentation, leading to the production of host-health-promoting by-products or metabolites
[20][41]. Through the fermentation of the prebiotic, beneficial bacteria obtain energy, which promotes their survival. Through the use of this mechanism, prebiotics selectively influence the composition of the GIT microbiota
[12][24]. These bacteria are beneficial to the host as, via the fermentation of the prebiotic substrate, they can produce health-promoting compounds including SCFA, such as acetate, propionate and butyrate, as well as organic acids such as lactate, succinate and pyruvate. These compounds exert multiple beneficial effects on the host energy metabolism
[21][22][23][24][42,43,44,45]. Although there are many health benefits associated with prebiotic supplementation, the satiety effect of prebiotic fibers must also be taken into consideration when choosing an appropriate inclusion rate, as high inclusion rates may result in a reduction in feed intake and subsequent performance
[25][26][46,47]. Each traditional prebiotic group exhibits distinct physical and chemical structural characteristics. In addition, there can be physical and chemical structural variations between two similar prebiotics due to differences in the source, extraction protocol and/or production procedure. Structural and chemical properties are crucial in relation to their bioactivity and effect on the GIT microbiota
[27][28][29][30,48,49]. It is worth mentioning that some of these bioactives have additional properties, such as antioxidant and anti-inflammatory properties
[30][31][32][33][34][50,51,52,53,54].
2.1.1. Beta Glucans (β-Glucans)
Beta-glucans are naturally occurring polysaccharides of D-glucose monomers linked through β-glycosidic bonds. β-glucans are cell wall components of yeast, algae, bacteria, mushrooms, and cereals such as barley and oats
[27][35][30,55]. β-glucans display a wide range of health-promoting properties, such as anti-inflammatory, antioxidant and prebiotic properties
[27][30][30,50]. The sugar component of β-glucans is predominantly pure glucose, except for in the case of laminarin, which also contains trace amounts of mannose
[36][56]. The characteristics of the different β-glucans, such as purity, linkage type, degree of branching, structure, solubility, and molecular weight, significantly impact their bioactivities
[27][37][30,57]. With different forms of β-glucans present in various sources, it is important to isolate the potential benefits of each, rather than grouping β-glucans under a single classification with collective properties. For example, the bonds found in bacteria are predominantly β(1–3) linkages, cereal β-glucans are predominantly β(1–3) and β(1–4) linkages, while in yeast, laminarin and mushrooms, the β-glucans bonds are β(1–3), with β(1–6) branches. Although yeast and laminarin consist of the same type of linkages, the ratio of bonds and branches and the structure of the β-glucans differs
[38][58]. Yeast β-glucans can also be utilized as potential encapsulating agents that can protect another bioactive from digestion, thereby increasing its bioavailability within GIT
[39][40][59,60]. β-glucan supplementation can improve pig performance by enhancing gut microbial composition
[27][41][30,61], improving gut morphology and barrier function
[42][33], and also improving immune status
[30][50] in pigs.
2.1.2. Non-Digestible Oligosaccharides
Non-digestible oligosaccharides (NDO), or functional oligosaccharides, make up a large proportion of the bioactives currently classed as prebiotics. The NDO are a group of oligosaccharides, typically 2–20 monomers in length, with β-links present among the units of monosaccharides. The NDO are distinguished by their monosaccharide composition, chain length, degree of branching, and purity. The NDO can be extracted directly from natural sources or produced via polysaccharide hydrolysis or enzyme processing
[43][78]. For example, xylo-oligosaccharide (XOS) and fructo-oligosaccharide (FOS) are obtained through the enzymatic degradation of xylan and inulin, respectively
[44][45][79,80]. Non-digestible oligosaccharides have both indirectly and directly beneficial effects on the host’s health. They indirectly benefit the host’s health by acting as a substrate for beneficial bacteria such as
Bifidobacteria and
Lactobacilli, thereby promoting their growth and enhancing the health benefits associated with these bacteria
[46][81].
2.1.3. Inulin
Inulin is a naturally occurring non-digestible carbohydrate that belongs to the class of dietary fibers known as fructans
[47][101]. Inulin is a polymer that contains both oligosaccharides and polysaccharides. It is a type of fructan mixture that can be found in a wide variety of plants. However, in its industrial use, it is most commonly extracted from chicory roots
[48][102]. Inulin is generally a linear chain comprising one terminal glucose molecule and a chain of fructose units linked by β(2–1) bonds
[47][101]. Inulin’s fructan composition and the number of monomer units, referred to as the degree of polymerization, varies depending on the source
[49][50][103,104]. The degree of polymerization of inulin can range from approximately 2 to 60
[51][105]. The FOS is obtained via the enzymatic hydrolysis of inulin, reducing the degree of polymerization
[45][52][80,106]. The degree of polymerization has a direct influence on the physical properties of the compound. The higher the degree of polymerization of inulin is, the greater its gel-like behavior will be, with longer chains having lower solubility.
When included in sow diets, inulin increases litter performance and improves the antioxidant status of the sow
[53][109]. Inulin has been utilized in weaned and grower pig diets to varying degrees of success
[54][55][56][57][110,111,112,113]. At an inclusion rate of 4%, inulin increases
Lactobacilli and
Bifidobacteria, and reduces the presence of harmful
Clostridium spp. and members of
Enterobacteriaceae in the intestinal microbiota of grower pigs
[55][56][111,112]. However, at an inclusion rate of 3%, inulin does not alter the number of
Lactobacilli,
Bifidobacteria,
Enterococci,
Enterobacteria or bacteria of the
Clostridium Coccoides/
Eubacterium rectale-group in the duodenum, jejunum or caecum
[57][113]. The use of short-chain inulin, long-chain and a 50:50 mixture of both all exerted similar effects on the GIT microbiota of pigs in the post-weaning/grower phase, increasing the total number of
Lactobacilli and
Bifidobacteria, particularly in the mucosa-associated microbiota
[56][112].
2.1.4. Resistant Starch
Resistant starch is a non-digestible carbohydrate defined as the fraction of starch that resists digestion in the stomach and small intestine and acts as a substrate for bacterial fermentation
[58][117]. There are four types of resistant starch: resistant starch type 1 (RS1) which is found in grains and cereals; RS2, which is found in starch foods, such as banana and potato; RS3, which are retrograded starches that occur when cooking and cooling starchy foods; and RS4, which are man-made chemical resistant starches
[59][118]. A meta-analysis including results from 24 published studies involving RS2 concluded that there is a negative relationship between the RS2 inclusion rate and pH in the large intestine and that increasing RS2 levels promotes fecal
Lactobacilli and
Bifidobacteria in pigs
[60][119]. The optimal inclusion rate to achieve these results is suggested to be 10–15%
[60][119]. However, this meta-analysis included studies of pigs covering a broad range of start weights (4.6–105 kg). Resistant starch may be particularly effective in the post-weaning phase, inclusion rates of 0.5–14% raw potato starch improves post-weaning fecal scores
[61][62][63][120,121,122]. The microbiota was not analyzed in the study utilizing an inclusion rate of 0.5%
[62][121]; however, an inclusion rate of 5% increases the presence of
Clostridia in feces
[61][120], and rates of 7 and 14% increase
Lactobacilli and
Bacteroides prevalence in the colon
[63][122].
2.1.5. Pectin
Pectin is a plant cell wall polysaccharide that can be utilized by bacteria in the GIT, but which is indigestible to mammalian digestive enzymes. It is present in the cell wall of fruits, vegetables, and legumes
[64][127]. Pectin is a large component of the dietary fiber fraction of feedstuffs such as beet pulp, citrus pulp, and soybean hulls. Citrus and apples are common sources of pectin for use in pig diets
[65][66][67][68][69][128,129,130,131,132]. The molecular structure of pectin varies depending on its source. The three major pectin structures are homo-polygalacturonate, rhamnogalacturonan I (RGI) and rhamnogalacturonan II
[70][133]. The degree of methyl esterification, the composition of neutral sugars, the degree of branching, and the presence of amide groups all influence the effects of pectin on the microbiota
[71][134]. The cumulative production of the total SCFA and propionate is largest in fermentations of pectin with high methoxyl
[71][134]. The influence of the wide-ranging structural variations present in pectin are reflected in its effects in vivo in terms of variability of findings.
2.2. Novel Prebiotics
2.2.1. Proteins, Hydrolysates, Peptides, and Amino Acids
The interaction between proteins and the GIT microbiota has been intensely investigated in recent years; although certain modes of action have been suggested, the exact mechanisms remain unclear. Generally, protein digestion and absorption occur in the small intestine, leaving small fractions of protein to transit into the large intestine. Hence, there is a scarcity of amino acids available to bacteria in the distal GIT and competition exists for residual peptides and amino acids among different bacterial groups. This scarcity limits the growth of bacteria and different strains can have specific amino acid requirements
[72][73][74][139,140,141]. Recently, the reduction in crude protein levels in the diet of pigs in the post-weaning period has been an area of major research focus
[75][142]. The objective is to reduce the quantity of undigested dietary protein and excess endogenous nitrogen that arrives in the large intestine and is fermented by potentially pathogenic nitrogen utilizing bacteria, thereby reducing their proliferation and the production of toxic metabolites
[76][77][143,144]. However, reducing dietary crude protein also reduces the amino acid availability for the beneficial GIT bacteria that utilize amino acids to proliferate and produce host-health-prompting metabolites
[78][145].
The extent of hydrolysis and absorption of ingested proteins and amino acids in the GIT prior to reaching the large intestine means that the supplementation of protein or amino acids for the purpose of promoting the growth of beneficial bacteria in the colon is far from optimal, as only small fractions of the supplemented protein or amino acid will be available in the large intestine. However, new techniques for shielding these peptides and amino acids from degradation and absorption have been developed. An example of this is the use of a prebiotic galacto-oligosaccharide (GOS), conjugated with a protein, lactoferrin hydrolysate, that has been pre-hydrolyzed by pepsin
[79][138]. In an aqueous solution, these combinations are suggested to form helical structures, with the GOS component acting as the outer layer with the protein components stored within
[80][147]. This particle structure is suggested to protect the protein from digestive enzymes in the stomach and small intestine, making it indigestible and unabsorbable. The particles are then subjected to digestion by bacteria in the large intestine as the outer layer undergoes fermentation, thereby releasing the inner protein component and making it available to the bacteria
[79][80][138,147].
Although the conjugation was suggested to be a key step in the success of the study in
[79][138], other studies have had positive results when casein hydrolysates are simply supplemented in combination with yeast β-glucan in both sows
[81][82][136,137] and weaned pigs
[40][82][60,137]. Interestingly, when supplemented alone, these bioactives have minimal effect, suggesting that a form of natural encapsulation occurs when supplemented together, allowing the yeast β-glucan to act as bioactive carrier for the casein hydrolysate
[40][60]. Maternal supplementation with the bioactive combination of the β-glucan and casein hydrolysate increases the abundance of the phylum
Firmicutes, including
Lactobacillus and
Christensella, in the sow feces, while increasing cecal and colonic abundance of
Lactobacillus and cecal abundance of
Christensella in the offspring at weaning time
[81][136]. Maternal β-glucan and casein hydrolysate supplementation also increases the abundance of
Lactobacillus, decreases the abundance of
Enterobacteriaceae and
Campylobacteraceae, and increases butyrate production in the offspring 10 days post-weaning
[82][137]. The casein hydrolysate used in these studies has an established anti-inflammatory effect
[83][148].
The amino acid composition of the casein hydrolysate may play a part in the beneficial effects seen with its supplementation. Casein hydrolysate contains a wide range of different amino acids; the profile varies depending on the degree of hydrolysis and enzymes used
[84][149]. For example, in
[85][150], glutamate and glutamic acid (21%), proline (10.2%), leucine (8.7%) and lysine (7.3%) contribute to 47.2% of the amino acid mass of the casein hydrolysate utilized. The role of amino acids in the diet stretches beyond their function as protein building blocks. They act as energy substrates and signaling molecules and can be metabolized into biologically active compounds, which can promote GIT health
[86][151].
Tryptophan is an amino acid that has received increased attention over the past number of years due to the beneficial effects of the metabolites produced via the bacterial tryptophan metabolism in the GIT
[87][88][89][90][152,153,154,155]. Tryptophan metabolism by the GIT microbiota is a source of aryl hydrocarbon receptor (AhR) ligands, with AhR being recognized as having important roles in the regulation of intestinal homeostasis, as reviewed in
[91][156]. Microbiota-derived AhR ligands are typically indole derivatives, such as indole-3 ethanol (IE), indole-3 pyruvate
[92][157], indole-3 aldehyde (I3A) and tryptamine (TA)
[93][158]. These ligands can stimulate the AhR, leading to enhanced intestinal barrier function
[94][95][159,160] and reduced inflammation
[96][161].
The potential benefits of enhancing the abundance of tryptophan-metabolizing bacteria in the GIT microbiota is a promising strategy with which to stimulate the AhR and promote intestinal homeostasis. Increasing tryptophan content in weaned pig diets has been shown to improve average daily feed intake (ADFI) and average daily gain (ADG)
[87][152]. In the cecum and colon, tryptophan supplementation enhances alpha (α) diversity, increases
Prevotella, Roseburia, and
Succinivibrio genera, reduces
Clostridium sensu stricto and
Clostridium XI, increases indole-3-acetic acid and indole, and induces AhR activation
[87][152]. In the jejunum, tryptophan supplementation reduces the abundance of
Clostridium sensu stricto and
Streptococcus and increases the abundance of tryptophan metabolising
Lactobacillus and
Clostridium XI. This study also reported enhanced intestinal barrier function and the secretion of host defence peptides
[88][153].
2.2.2. Nucleotides
Nucleotides are organic molecules that serve as precursors of DNA and RNA. Nucleotides have been recently suggested as an “overlooked prebiotic” that could potentially play a role in shaping the composition of the microbiota
[97][37]. Interestingly, in vitro, nucleotides promote the growth and secretion of the biofilm of the probiotic
Lactobacillus casei, while also enabling the crude extract of
Lactobacillus casei to resist the biofilm formation of the pathogenic bacteria
Shigella [97][37]. In mice, nucleotide supplementation promotes microbial diversity, while nucleotide-free diets enriched pathogenic bacteria, such as
Helicobacter, and decreased beneficial bacteria, such as
Lactobacillus, in feces
[97][37]. In chickens, yeast nucleotides increase α diversity and the abundance of
lactobacillus in the ileal microbiota
[98][172]. Research investigating the effect of nucleotide supplementation on the pig’s microbiota is sparce. Nucleotides are present in the sow’s milk and may contribute to the establishment of the offspring’s microbiota
[99][100][173,174]. Oral supplementation of nucleotides to pigs pre-weaning does not affect α diversity, but increases the fecal abundance of
Campylobacteraceae and decreases
Streptococcaceae at weaning
[100][174]. However, the product utilized in this study, SwineMOD
® (Prosol, Madone, Italy), also contains yeast glucans which likely contribute to the effects on the microbiota
[100][174]. Maternal nucleotide supplementation is associated with positive effects on offspring GIT health parameters, such as inflammation, intestine morphology and diarrhea occurrence
[101][175]. However, the question of whether supplementing nucleotides in the maternal diet leads to alterations in the nucleotide composition of the milk and subsequently in the composition of the offspring’s microbiota remains to be answered.
Supplementing a pure nucleotide blend to 3-day-old weaned pigs results in dramatic changes in the colonic microbiota, reducing the
Firmicutes:
Bacteroidetes ratio and increasing the relative abundance of beneficial bacteria such as
Faecalibacterium,
Blautia and
Prevotella [102][176]. Furthermore, the pure nucleotide blend increases the level of the SCFA acetic acid, isobutyric acid, isovaleric acid and valeric acid in the colon
[102][176]. A nucleotide-rich yeast extract increases cecal
Lactobacillus and colonic
Clostridium cluster
IV, and decreases cecal
Enterobacteriaceae and colonic
Enterococcus spp. when supplemented to pigs for the initial two weeks post-weaning
[103][177].
2.2.3. Polyphenols
Polyphenols are secondary metabolites in plants and are particularly abundant in fruits, vegetables, grains and teas
[104][179]. Polyphenols have established antioxidant and anti-inflammatory activities, as reviewed in
[105][180]. Besides that, polyphenols have antimicrobial activity and can modulate the GIT microbiota when included in the diet of pigs
[106][181]. As mentioned, polyphenols are deemed to have ‘prebiotic-like properties’ as they possess microbiota-modulating abilities when included in the diet. However, it is not clear if polyphenols are utilized directly by bacteria in the GIT, which is a requirement to be classed as a prebiotic, and so they are currently classed as “prebiotic-like.
Polyphenol supplementation has been associated with increases in the abundance of beneficial
Lactobacillus [106][107][181,185],
Bifidobacteria [107][185] and
Prevotella [106][181] and decreases in abundance of harmful
Streptococcus and
Clostridium [108][186]. Feeding polyphenol-rich plant products to weaned pigs reduces the abundance of harmful bacteria, including
Streptococcus and
Clostridium, without affecting the abundance of the beneficial bacteria,
Lactobacillus and
Bifidobacterium [108][186]. However, supplementation can also lead to an increase in the pH of the feces and a reduction in the concentration of SCFA
[108][186]. The decrease in SCFA noted may be a result of a decrease in
Bacteroidetes abundance, which is a primary contributor to SCFA and promote a balanced microbiota, as polyphenol supplementation can decrease
bacteroidetes in colonic digesta of weaned pigs
[106][181]. The reduction in SCFA concentration and increase in the pH of the feces noted in
[108][186] indicates a reduction in bacterial fermentation in the GIT. This is not a desirable effect as SCFA plays an essential role in the regulation of metabolism, the immune system, and cell proliferation in the GIT, while the increase in pH is not desirable as a lower pH in the intestine can help to limit the growth of pathogenic bacteria
[109][110][111][187,188,189]. A combination of functional amino acids (arginine, leucine, valine, isoleucine, cysteine) with a polyphenol-rich extract from grape seed skins reduces microbial diversity.
3. Stimbiotics
The concept of a stimbiotic was proposed in
[11][23], where the authors suggested that certain bioactives, that were classed as prebiotics, may not be exerting their beneficial effects in the mode of action expected under the definition of a prebiotic
[11][23]. Hence, they proposed a new class of bioactive which they termed “Stimbiotics”. When stimbiotics are included at low inclusion rates they promote increases in SCFA production disproportionally greater than if they were merely substrates for fermentation
[11][112][23,194]. It is suggested that stimbiotics are pump primers, where they signal to fiber fermenting bacteria to increase their activity and thereby promote an increase in fiber fermentation. An example of a stimbiotic is XOS, which consists of chains of xylose linked by β(1–4) bonds
[113][195]. The XOS is effective at modulating the GIT microbiota and improving performance when included in the diet of weaned pigs at inclusion rates as low as 0.02%
[114][31]. Inclusion rates for NDO prebiotics, such as FOS, GOS and mannan oligosaccharide (MOS), can vary but are generally much higher than this, in the region of 0.1–0.2%
[46][112][81,194]. Even at 10 or 20 times lower inclusion rates, stimbiotics can exert a greater effect on certain fiber fermentation parameters than certain prebiotics
[112][194]. The use of XOS at a 0.007% and 0.01% inclusion rate has minor effects on the GIT microbiota and performance but overall results from trials suggest a higher inclusion rate of 0.02% or 0.04% to be more effective
[114][115][116][117][31,196,197,198]. Stimbiotics are a relatively new concept and although a proportion of these bioactives have been studied as prebiotics, studies investigating their effect at the low inclusion levels associated with stimbiotic activity are limited, especially in the case of maternal supplementation where it is yet to be studied.
4. Conclusions
The importance of the GIT microbiota is becoming increasingly evident, particularly with the strict new restrictions on antibiotic and antimicrobial use. Therefore, modulating the GIT microbiota through dietary intervention is a crucial area of exploration that can enhance animal health by increasing the production of host-health-promoting metabolites and limiting the proliferation of pathogenic bacteria. The benefits of prebiotic use illuminates their status as an intriguing bioactive group that can potentially act as alternatives to antibiotic and antimicrobial use on pig farms, particularly in the post-weaning phase. The benefit of prebiotics is evident. However, given the broad range of traditional prebiotics, combined with the growing list of newly classed novel prebiotics, the most effective prebiotics at the different stages of development need to be clarified. Particular attention should be placed on direct comparative research into different prebiotics and inclusion rates at critical periods of development.
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