Fructooligosaccharides (FOSs): A Condensed Overview

Abstract

FOSs are short-chain fructose-based oligosaccharides with notable functional and health benefits. Naturally present in various fruits and vegetables, FOSs are primarily produced enzymatically or microbially from sucrose or long-chain fructans, namely, inulin. Enzymes such as fructosyltransferase, β-fructofuranosidase, and endoinulinase are typically involved in its production. The chemical structure of FOSs consists of an assembly of fructose residues combined with a glucose unit. The increasing consumer demand for healthy foods has driven the widespread use of FOSs in the functional food industry. Thus, FOSs have been incorporated into dairy products, beverages, snacks, and pet foods. Beyond food and feed applications, FOSs serve as a low-calorie sweetener for and are used in dietary supplements and pharmaceuticals. As a prebiotic, they enhance gut health by promoting the growth of beneficial bacteria, aid digestion, improve mineral absorption, and help regulate cholesterol and triglyceride levels. Generally recognized as safe (GRAS) and approved by global regulatory agencies, FOSs are a valuable ingredient for both food and health applications. This review provides an updated perspective on the natural sources and occurrence of FOSs, their structures, and physicochemical and physiological features, with some focus on and a critical assessment of their potential health benefits. Moreover, FOS production methods are concisely addressed, and forthcoming developments involving FOSs are suggested.

1. Introduction

Fructooligosaccharides (FOSs) can be described as short-chain carbohydrates built on a sucrose starting unit to which fructosyl residues are added [1,2,3]. For disambiguation [1,2,4], and according to IUB-IUPAC nomenclature [5], only oligomers with up to 10 monosaccharide units are considered as FOSs [1,2,4,6]. As a relevant member of the oligosaccharide family and generally regarded as safe (GRAS) by the Food and Drug Administration (FDA) [7,8], FOSs have gathered significant attention due to their functional properties and wide range of applications [9,10,11], such as in animal feed, dietary supplements, food and beverages, and the pharmaceutical industry [12,13,14]. As prebiotics with a low caloric value, FOSs are highly desirable in the food, pharmaceutical, and nutraceutical industries [15,16,17]. Moreover, FOSs are widely found in nature, namely, in fruits, vegetables, and cereals [4,17,18].

The growing interest in FOS production has been fueled by their acknowledged beneficial health effects that, besides the modulation of the human gut microbiota, also include the activation of the immune system and resistance to infections, improved mineral absorption, antioxidant features, and a lowering effect on serum lipid and cholesterol concentration [17,19,20,21]. This trend also aligns with the growing public awareness of the impact of diet on public health and the concomitant demand for natural products, functional foods, and low-caloric sweeteners [4,22,23,24]. As an outcome of all this and according to Grand View Research, the global market for FOSs was valued at EUR 2.5 × 10⁹ in 2022 and is expected to grow at a compound annual growth rate of 8.8% from 2023 to 2030 [12]. All things considered, it is understandable why the global market for FOSs is expanding, presenting challenges and opportunities for both academia and industries [4,12,25]. Some illustrative examples of FOS applications are summarized in Figure 1.

Besides their natural sources, FOSs can be produced by several methods, namely, the enzymatic hydrolysis of inulin and related high-molecular-weight fructans [32], enzymatic synthesis [33], microbial fermentation [25], chemical hydrolysis [34], and chemical synthesis [35], although, the latter is typically overlooked, since it is a complex, multi-step process that requires complex protection/deprotection steps due to the chemical susceptibility of substrates and products and often involves toxic chemicals that are incompatible with food standards [25,35,36,37,38]. Chemical hydrolysis is also hardly an option for FOS production, since careful control is required to prevent the excessive formation of fructose, and by-products, such as hydroxymethylfurfural, which is toxic for humans [39], are prone to be formed [34,40]. Enzymatic and microbial fermentation methods each have specific advantages and limitations, influencing yields, cost-efficiency, and environmental sustainability [20,21,25,33,41], as discussed later in this work.

This review aims to provide an overview of the key features of FOSs, contributes to the discussion of a consensual definition of FOSs, assesses FOS health benefits, and provides an updated perspective on FOS sources and production strategies, with a focus on the underlying principles and methodologies. Finally, this review intends to identify gaps in current knowledge and suggests prospective developments in the sustainable production of FOSs.

2. Structure, Properties, and Health Benefits

2.1. Chemical Structure

Some ambiguity can be found in the literature with respect to the definitions of the chemical structure and composition of FOSs. Hence, some authors state that FOSs can bear oligomers of both linear and branched structures [42]. These oligomers may consist solely of fructosyl (F) units or a combination of fructosyl units with a single terminal glycosyl (G) unit [2]. Other authors define an FOS as a combination of fructosyl units with a single terminal glycosyl unit arranged in a linear manner [40,43,44]. According to the latter definition, FOSs designate oligomers consisting of a small number of fructosyl units linked together by β-(2→1)-glycosidic bonds, with a terminal d-glycosyl residue linked to the fructosyl residue through a α-(1→2) glycosidic bond, also termed inulin–FOSs, since inulin shares the same structure, albeit with a degree of polymerization up to 60 [22,40,45,46,47,48]. Nobre and co-workers associate FOSs to fructans synthesized by fructosyltransferases [20]. Flores-Maltos and co-workers suggested that FOSs are mainly composed of chains of fructosyl units terminated by a glycosyl residue, which are all linked through β-(2→1)-glycosidic bonds [30]. Hill and co-workers assume FOSs are oligosaccharides composed of 2 to 10 fructosyl residues linked by either β-(2→1)- or β-(2→6)-glycosidic bonds, with or without a terminal d-glycosyl residue [49,50]. This enables the inclusion of other fructose-based oligosaccharides under the generalist FOS designation, besides inulin–FOSs, namely, those with fructosyl units are linked through β-(2→6) glycosidic bonds, termed levan–FOSs, which are also terminated by a d-glycosyl residue; those depicting either a β-(2→1) skeleton and β-(2→6) branches or β-(2→6) skeleton and β-(2→1) branches, termed graminans, which are mixed branched fructans; and those presenting an internal glycosyl residue contained between two fructosyl residues, termed neo-FOS (or neoseries). In this case, fructosyl-based chain elongation can involve mostly either β-(2→1) and β-(2→6) bonds, in which case FOSs are termed inulin neoseries or levan neoseries, respectively. A particular type of neo fructans is agavin, which is characterized by a high degree of β-(2→1) and/or β-(2→6) branching; oligofructans, composed solely of fructosyl units (F-series); and a fructan recently isolated from Radix Codonopsis (Dangshen), depicting a novel β-(2→3) linkage and an unusually high molecular weight (1.70 × 10³ kDa) [20,22,30,41,49,50,51,52,53,54,55]. The structures of the most common types of fructans are depicted in Figure 2. Overall, it can be suggested that FOS designation is primarily associated with inulin–FOS, since this is the prevalent type found both in nature and produced at a commercial scale [20,25,41,56].

The number of fructosyl units that are reported in the literature for FOSs can be up to 60 [57], which corresponds to inulin, although a more common upper limit is 12 [2,25,58]. Yet according to IUBMB-IUPAC rules, the dividing line between oligosaccharides and polysaccharides is set at 10; hence, the degree of polymerization of FOSs should be between 3 and 9 [2,5,22].

2.2. Properties

As a result of their chemical structure, FOSs present appealing physicochemical, physiological, and sensory properties (

Table 1. Significant properties of FOSs and a brief perspective of their impact on food formulations and related goods.

Property	Comments	Reference
Physicochemical	Gelling/binding features: These features relate to compounds that promote cohesion/cross-linking of liquids/small particles, leading to the formation of a solid-like structure. Although not major gelling agents, FOSs can interact with strong gelling agents, e.g., alginate, gelatin, starch, and methylcellulose, contributing to gel formation, and/or act as a binding agent, impacting structure/shape and texture.	[60,61,62]
	Rheology: The incorporation of FOSs can impact the flow and deformation of materials under applied forces. Thus, due to its higher molecular weight, FOSs exhibit greater viscosity than sucrose, contributing to textural modifications of food products, such as bread and fruit juices.	[4,28,63,64]
	Stability: FOSs are stable under various temperatures and pH values, namely, within the normal pH range for foods (4.0 to 7.0). This feature enables FOSs to retain their probiotic features, enhance their shelf-life, and withstand acidic environments for targeted drug delivery.	[4,65,66,67]
	Solubility: FOSs are water soluble, around 80% at room temperature, which facilitates their incorporation in food products.	[4,30,68,69]
	Water binding: FOSs help to retain moisture, which can contribute to a softer texture of food products or other materials.	[63,70,71]
Physiological	Non-cariogenic: This relates to substances that do not cause tooth decay (cavities). FOSs are not metabolized in the mouth by bacteria such as Streptococcus mutans, one of the primary microorganisms accountable for tooth decay. Such bacteria metabolize sugars from food, yielding acids that erode the enamel on teeth, leading to decay over time. Replacing sugars by FOSs thus prevents the production of cariogenic compounds, resulting in healthier foods.	[4,72]
	Non-digestibility: the β-(2→1) osidic links of FOSs endure hydrolysis by human salivary and pancreatic enzymes of the host (specific for α-glycosidic bonds), hence reaching the colon undisturbed.	[7,49,73,74,75,76,77,78]
	Prebiotic effect: FOSs have been shown to modulate the gut microbiota, fostering the growth of beneficial microbial species, e.g., Bifidobacterium spp., Lactobacillus spp., and Faecalibacterium prausnitzii.	[29,33,43,49,75,79,80,81,82,83,84,85,86,87]
	Nutritional: This is related to the caloric value of foods, or the amount of energy produced when food is metabolized. The caloric value of FOSs, 1.0 to 1.7 kcal/g (4.2 to 7.1 kJ/g), compares favorably with that of dietary carbohydrates such as sucrose, glucose, and fructose, which have a caloric value of 3.9 kcal/g (16.3 kJ/g). Consuming such high caloric compounds without burning off excess calories ultimately results in fat accumulation. The incorporation of FOSs in food products contributes to a low caloric diet, hence to fat reduction.	[4,16,28,88,89,90]
Sensory	Organoleptic: This is related to sensory-apprehended food properties. FOSs are mild sweeteners, with a sweetness intensity of 30 to 60% that of sucrose, and FOSs are used to enhance the taste and sweetness of foods and other products, while reducing the amount of sugar.	[4,17,28,63,91,92,93]

The stability of FOSs is highly influenced by their environmental conditions; thus, for a given specific case, studies on their stability based on processing, preservation, and storage methods are advised [94,95].

), namely, ones related to their use in food and feed, where the dosage of FOSs in food products can be as high as 50% (w/w) [4,59].

2.3. Health Benefits

Health benefits associated with FOS consumption, typically about 0.8 g/day [4], have been vastly assessed [20,96,97,98,99,100]. The prebiotic role of these compounds is clearly the most prominent [15,20,29,57,81,84,85,86,98], as it is well aligned with the current definition of prebiotics as “a substrate that is selectively utilized by host microorganisms conferring a health benefit” [101]. FOSs are typically acknowledged to proceed to the colon undisturbed by the action of salivary and pancreatic enzymes [7,76], although in vitro studies have instead suggested that FOSs remain mostly unhydrolyzed by those enzymes [84,102]. FOSs are considered to resist the action of gastric acid [49,74], whereas it has instead been suggested that a small amount of the FOS is hydrolyzed by the stomach acid to generate fructose and glucose, which are adsorbed by the body [7]. Nobre and co-workers also reported minimal degradation of FOSs (<1.5% loss) after exposure to simulated harsh conditions of the gastrointestinal tract [103]. In a recent in vivo study, van Trijp and co-workers reported minimal degradation of a mixture of FOSs and galactooligosaccharides (GOSs) by the distal ileum bacteria. The authors ascribed this outcome to the low residence time in the small intestine that minimizes the microbial exposure to FOSs and GOSs [104]. Accordingly, Roupar and co-workers reported no growth of such bacteria in the ileum during the first 12 h of fermentation [105]. In any case, safe for a negligeable part (around 10%, based on studies with human volunteers), the ingested FOSs reach the colon [7,102,106], where they are fermented by the microbial gut community to most notably produce short chain fatty acids; SCFAs (acetate, propionate, and butyrate); and, to a minor extent, branched chain fatty acids, BCFAs (isobutyrate and isovalerate) and organic acids (lactate, pyruvate, and succinate) [7,29,102,107,108,109]. This fermentation results in changes in the microbial gut composition, which has, moreover, been shown to vary in an age-dependent manner and in their metabolic activities [105,110,111]. Typically, the genera Bifidobacterium and Lactobacillus were identified as those whose growth most benefits from available FOSs, since they are the most well adapted to FOS metabolization [76,79]. Nonetheless, FOSs can also be metabolized by pathogens present in the gut, such as strains of Bacteroides, Campylobacter, Clostridia, Enterobacter, Escherichia coli, Fusobacteria, Salmonella, and Shigella [20,29,107,110]. Still, beneficial microorganisms outperform pathogens in FOS consumption, leading to the competitive exclusion of the latter [79,107,110]. Moreover, produced SCFAs acidify the gut, thereby creating an unfavorable environment to pathogenic bacteria [20,79,112], albeit also hindering the growth of some beneficial microorganisms, e.g., Phascolarctobacterium, which impact other metabolic pathways, e.g., glucose [76,113]. Besides directly stimulating the growth of Bifidobacterium spp. and Lactobacillus spp., FOSs also foster the proliferation of SCFA-producing taxa, e.g., Faecalibacterium prausnitzii, one of the most plentiful butyrate-producing bacteria in the human intestinal tract whose relative abundance can be used as a biomarker of intestinal health in adults [80,114,115].

Besides FOS composition, the effectiveness of FOS fermentation has been tentatively suggested to be strain-dependent rather than species- or genera-dependent [105]. Thus, Nobre and co-workers observed that the growth of the Lactobacillus strains depended on the FOS type (produced by Aspergillus ibericus; Aspergillus pullulans; and a commercial FOS preparation, Raftilose^® P95), whereas the growth pattern of tested Bifidobacterium strains was undisturbed by the substrate source [103]. On the other hand, Roupar and co-workers reported that the FOSs produced by A. ibericus stimulated Bifidobacterium growth (known as the bifidogenic effect). This behavior was ascribed to the presence of Bifidobacterium strains, which were different from those used in the work by Nobre and co-workers [105]. Conversely, neokestose has been shown to stimulate the growth of Bifidobacterium and Lactobacillus strains while inhibiting Bacteroides and Clostridium strains that are potentially harmful [116].

Most bacteria can use FOSs as carbon sources, but only a few can use long-chain fructans, e.g., inulin [105,107,117,118,119]. In particular, most Bifidobacterium spp. Strains ferment FOSs more effectively than highly polymerized inulin, favoring bifidobacterial populations [80,105,109,120]. Conversely, Roupar and co-workers suggest that overall, Lactobacillus shows no significant differences between FOSs and inulin [105], which could be related to the synthesis of extracellular fructofuranosidases by some Lactobacilli (e.g., Lactobacillus casei and L. paracasei), enabling them to hydrolyze inulin and other long-chain fructans into FOSs, which are then transported intracellularly for utilization [79,107].

Aiming to synthesize and critically evaluate available evidence on the impact of FOS supplementation in microbial gut composition, Dou and co-workers performed a systematic review and meta-analysis including eight studies performed between 1995 and 2022. FOS supplementation increased the number of Bifidobacterium spp. colonies compared to control groups, and doses of 7.5 to 15 g per day over more than 4 weeks resulted in more evident effects with good tolerability [29]. Moreover, while FOSs also promoted Lactobacillus spp. growth compared to a control group, the results were statistically less consistent [29]. These overall findings also align with the systematic review by Le Bourgot and co-workers, who considered 252 studies performed between 1997 and 2020 on the impact of FOS supplementation for infants and young children. The authors concluded that the number of Bifidobacterium spp. colonies increased as compared to control groups after 1 month of FOS supplementation at a dose of 4 or 5 g/L [121]. Also, while assessing the FOS supplementation given to adults aged 25 to 70 years, Mahalak and co-workers established that irrespective of the age group, FOSs fostered Bifidobacterium spp. growth and increased SCFA levels as compared to control groups, although the growth of Bifidobacterium spp. was most notable in the older adult group. The genus Bacteroides, Collinsella, Megamonas, and Ruminococcus showed minor (non-statistically significant) growth increases, with differentiated patterns depending on the age groups. On the other hand, the genus Bilophila, Odoribacter, and Oscillospira decreased after supplementation with FOSs but again with differentiated patterns depending on the age groups [110]. In addition to variables like FOS composition and microbial strains, discrepancies may arise between in vivo and in vitro studies on the prebiotic role of FOSs, since the latter cannot fully capture the complex gut microbiota interactions [107,110]. Moreover, a multi-omics analysis of the impact of FOS supplementation on the human gut microbiome confirmed common effects, such as the bifidogenic effect and a positive impact on the immune system, e.g., increased immunoglobulin A secretion. However, Kato and co-workers also reported wide inter-individual variability in both the gut microbiome and its response to FOSs. While acknowledging the small (eleven) number of individuals involved in their study, the authors suggest that FOS supplementation should be evaluated on an individual basis, since its health benefits are not alike to every individual [122].

Related to their prebiotic role and besides their impact on microbial gut composition, FOSs offer several health advantages (

Table 2. Selected health advantages of FOSs as related to their prebiotic role. In each case, a brief overview of the action mechanism and illustrative outcomes are provided.

Health Advantage	Comments
Mineral absorption	The intake of FOSs is intended to improve mineral (e.g., Ca, Zn, Mg, and Zn) absorption, thus contributing to bone health and reducing the risk of fractures and osteoporosis [96,124,125]. The mechanisms underlying the role of FOSs include the following: enhanced solubility and absorption, given the lower pH in the colon due to SCFAs [77,126]. SCFAs can stimulate the intestinal epithelium and increase its absorptive capacity [127,128] and contribute to the increased expression of calbindin-D9k, a calcium binding protein [126,129]. However, a recent review found conflicting clinical study results, as not all works suggest that FOS intake significantly increases calcium absorption [79].
Intestinal diseases	FOS supplementation has been found to alleviate the pathological immune response and prevent the impairment of the intestinal barrier in dextran sulfate sodium-induced acute colitis mice. This suggests that FOSs can help manage symptoms of inflammatory bowel disease by modulating gut microbiota and reducing inflammation [130]. FOSs can modulate inflammatory, oxidative, and immune activity in the gut, leading to a systemic response that improves overall health. Studies have shown that FOS supplementation can increase the number of Bifidobacterium spp. colonies, stimulate IgA secretion, and decrease proinflammatory cytokines [131]. SCFAs produced by the fermentation of FOSs play a critical role in regulating intestinal inflammation. These SCFAs, particularly butyrate, have immunomodulatory effects that can be used as a therapeutic approach in managing inflammatory bowel disease [132]. More specific details on the role of SCFAs in managing intestinal diseases can be obtained elsewhere [133].
Constipation	In elderly continuous ambulatory peritoneal dialysis (CAPD) patients, a 20 g/day FOS supplementation for 30 days significantly increased bowel frequency, softened stools, and accelerated the colonic transit time. FOSs were well tolerated, with only mild side effects like bloating and flatulence. Unlike traditional laxatives, FOSs can be easily integrated into one’s diet. Although further studies were suggested, given the small sample dimension, FOSs appeared to present a promising, well-tolerated alternative for managing constipation in CAPD patients [134].
Cancer	FOSs have been shown to induce a decrease in or even suppress colon tumor in animal models, which was tentatively ascribed to the stimulation of gut-associated lymphoid tissue due to the modulation of the colon microbiome [135]. A recent meta-analysis, which ultimately considered 17 case-control and 6 cross-sectional studies, established that the fecal concentrations of SCFAs, namely, acetic, propionic, and butyric acids, correlated inversely with both the risk and incidence of colorectal cancer [136]. Detailed insights into the positive impact of SCFAs in colorectal cancer can be found elsewhere [137].
Diabetes	FOSs are safe for individuals with diabetes given their scarce digestibility [79,125]. A meta-analysis by Costa and co-workers in 2012 suggested that the consumption of FOSs has a beneficial influence on glucose metabolism. Controversies related to this are associated with unsatisfactory methodologies/a small number of individuals enrolled in studies [138]. This outcome was somehow corroborated by a meta-analysis using animal models that evidenced the beneficial contribution of FOSs in the reduction in circulating postprandial glucose and insulin concentrations [139]. Garcia and co-workers reported the positive impact of controlled FOS administration in the composition of gut microbiota of type 2 diabetes patients, namely, the increased growth of Bifidobacterium spp. and Lactobacillus spp. [140]. Still, in a more recent review, Iatcu and co-workers again highlighted inconsistent findings—FOSs either had no effect or a positive effect on glucose metabolism and insulin levels—and a lack of the reproducibility of positive results in animal models and in vitro studies and in vivo studies in humans [141]. Besides individual variability, discrepancies may be related to different dosages, durations of administration, and combinations with other prebiotics.
Neurodegenerative conditions	Studies in animal models showed that FOS supplementation ameliorated cognitive deficits and pathological changes caused by Alzheimer’s disease by regulating the gut microbiota–GLP-1/GLP-1R pathway [142].
Liver diseases	Combined with galactooligosaccharides (GOSs), FOS supplementation proved effective in the treatment of individuals with steatotic liver disease associated with metabolic dysfunction and associated complications. Thus, insulin resistance, hyperglycemia, triglyceridemia, cholesterolemia, and IL-1β serum levels were reduced. Additionally, FOSs and GOSs modulated the lipogenic (SREBP-1c, ACC, and FAS) and lipolytic (ATGL) signaling pathways, reduced inflammatory markers, and enhanced the number of acetate-producing bacteria. Overall, the authors estimate that FOSs and GOSs mitigated this health condition by reducing the hepatic lipogenic pathways and the intestinal permeability through the gut microbiome–brain axis [143].
Anxiety and depression	Using animal models, FOSs and GOSs were shown to reverse symptoms of anxiety and depression. This improvement resulted from an increase in acetate-producing bacteria and intestinal permeability, which lowered chronic peripheral and central inflammation. Moreover, FOSs and GOSs fostered a decrease in proinflammatory cytokines [144].
Obesity	FOSs have been associated with the treatment and prevention of obesity [77,79,80]. This effect has been partly associated with their role in promoting satiety, thereby reducing meals and energy intake [145]; fostering weight loss and adjusting glucose regulation in overweight adults [146]; and suppressing hunger, albeit not significantly altering energy intake [147]. Despite some contradictory results, as Hess and co-workers reported a limited impact of FOS supplementation on acute satiety and energy intake [148], a systematic review by Ramos and co-workers suggests that FOSs influence satiety and lipid metabolism, namely through SCFAs produced during fermentation [149]. Inconsistencies may stem from individual variability, methodological details, dosages, and FOS compositions. Additionally, a recent review suggested that SCFAs exert epigenetic effects, potentially reversing metabolic and immune dysfunction caused by metabolic endotoxemia, thereby disrupting the cycle of obesity and inflammation [150].

). More detailed information can be found elsewhere [17,79,123].

3. Natural Occurrence and Production Methods

3.1. Natural Occurrence

The major natural sources of fructans are plants, as summarized in

Table 3. A brief overview of the main sources of fructans and corresponding titers [16,30,40,77,96,97,151,152,153,154].

Plant	Titer (Average % in w/w)
Chicory roots	22.9
Candy leaf (Stevia rebaudiana)	15.0
Jerusalem artichoke tubers	13.6
Yacon	13.2
Garlic	5.0
Onions	4.3
Asparagus	2.5
Bananas	2.5
Wheat	2.4
Tomatoes	1.8
Barley	0.2

. In spite of such diversity, the preferred commercial source of FOSs are chicory roots, with candy leaf, Jerusalem artichokes, and yacon as the most promising alternatives [16,30,52,97]. Fructans can also be found in some fungi, archaea, and bacteria, e.g., Bacillus, Brenneria, Haloarcula, Lactobacillus, Leuconostoc, Pseudomonas, Streptococcus, and Zymomonas. However, these cases involve a degree of polymerization up to 1 × 10⁵ and molar weights from 10³ g/mol to 10⁸ g/mol [16,52]; hence, they are out of the scope of this work.

The type of fructan and the degree of polymerization vary significantly depending on the plant [155], the storage organ [77,154,156], or the age of the plants [157], as exemplified in

Table 4. Fructan types and some examples of their natural sources and their degree of polymerization. Data highlight the diversity of the degree of polymerization depending on factors such as the specificity of the source or the growth stage/age of the source. Further details/complementary information can be found elsewhere [16,25,52,159].

Fructan Type	Source and Degree of Polymerization (DP)
Inulin	Candy leaf, Stevia rubidian (Bert.) Bertoni (root): $\overline{DP}$ 1 = 28 [156]
	Candy leaf, Stevia rebaudiana (Bert.) Bertoni (stem, two extracts): $\overline{DP}$ = 12 (inulin-rich extract); DP < 6 (FOS-rich extract), $\overline{DP}$ = 4.5 [154]
	Chicory, Cichorium intybus L.: $\overline{DP}$ = 28.67 [160]
	Dahlia, Dahlia decorative: 19 < $\overline{DP}$ < 23 [161]
	Jerusalem artichoke tubers, Helianthus tuberosus L.: $\overline{DP}$ ≈ 10 (0 days of flowering); $\overline{DP}$ ≈ 18 and DPmax 2 = 19 (50 days after flowering); $\overline{DP}$ ≈ 8 (80 days after flowering) [158]
	Yacon, Smallanthus sonchifolius: 3 < DP < 7 [162]; 2 < DP < 10 [151]
Levan	Paenibacillus sp.: $\overline{DP}$ = 18 [163] 3
	Transformed sugar beet, Beta vulgaris L: PpFT1 transformants, DP > 40; PpFT2 transformants 3 < DP < 40; transformants were obtained with timothy (Phleum pratense) 6-SFT genes [164]
	Gomphrena marginata: DP ≈ 40 [165]
Graminans	Wheat grains, Triticum aestivum L. var. Homeros: DP < 5 [166]
	Polygonatum cyrtonema Hua: 5 < DP < 10 [167]
Neo-inulin	Asparagus (Asparagus officinalis L.) [168], red onion (Allium cepa var. viviparum (Metz) Mansf.) [169]: DP = 3 (neokestose)
Neo-levan	Tupistra chinensis Baker rhizome; DP = 20 [170]
	Oat {Avena spp.): DP = 4 (6G,6-kestotetraose) [171]
	Lolium perenne: DP = 8 [172]
Agavin	Agave durangensis: DP > 10 [173]
	Agave spp.: DP ≤ 9 (two- to four-year-old plants), DP ≤ 70 (10- to 12-year-old plants); A. salmiana spp. crassipina: DP ≤ 50; Agave tequilana variety cenizo: DP ≤ 70 [157]
Unnamed, novel α-D-fructofuranosyl-(2→3)-β-d-fructofura- nosyl linkage	Dangshen, Codonopsis pilosula (roots, Radix Codonopsis): DP ≈ 9.4 × 103 (estimated)

¹ $\overline{DP}$: Average degree of polymerization; ² DP_max: maximum degree of polymerization; ³ a rare example of a microbially produced fructan with a DP close to that typical of fructans with plant origins.

. It must be pointed out that the degree of polymerization and polydispersity also depends on the harvesting time [157,158]. Moreover, fructans with a lower degree of polymerization were observed in the sprouting and flowering phases, whereas fructans with a higher degree of polymerization phases were observed in the dormancy phase in several plants [59]. Finally, obtaining and consequently identifying extracts depends as well on the technique used, as highlighted by Lopes and co-workers [156].

Fructan synthesis from sucrose in plants involves the action of various fructosyltransferases. The process starts with sucrose: sucrose 1′-β-d-fructosyltransferase, 1-SST (EC 2.4.1.99) transferring a fructosyl residue from a sucrose molecule to the C-1 of fructosyl in another sucrose molecule, forming 1-kestose. (2→1)-β-d-fructan:(2→1)-β-d-fructan 1-β-d-fructosyltransferase, 1-FFT (EC 2.4.1.100) then transfers one fructosyl residue from a fructan to 1-kestose, yielding 1-nystose. This process is repeated, resulting in chain elongation and the formation of inulin-type FOSs with an increasingly higher degree of polymerization. Conversely, the synthesis of levan-type FOSs begins with sucrose: [6)-β-d-fructofuranosyl-(2→1]n α-d-glucopyranoside 6-β-d-fructosyltransferase, 6-SFT or levansucrase (EC 2.4.1.10) transferring a fructosyl residue from a sucrose molecule to the C-6 of fructosyl in another sucrose molecule, forming 6-kestose. The repetitive transfer of fructosyl residues to 6-kestose by 6-SFT leads to chain elongation and a higher degree of polymerization. Neo-FOS synthesis begins with 1F-oligo[β-d-fructofuranosyl-(2→1)-]sucrose 6G-β-d-fructosyltransferase, 6G-FFT (EC 2.4.1.243) transferring a fructosyl residue from 1-kestose to the C-6 glucosyl residue of sucrose to yield neokestose. The latter can be elongated through the transfer of fructosyl residues by either 1-FFT or 6-SFT inulin neoseries or levan neoseries of fructans, respectively. The synthesis of graminans begins with 6-SFT transferring a fructosyl residue from sucrose to the C-6 of 1-kestose to produce bifurcose. Chain elongation and branching proceeds through the action of 6-SFT and 1-FFT [51,52,174]. As for agavins, biosynthesis begins with fructosyl residues which are transferred to neokestose by 1-FFT, 6-SST, and 6-SFT, resulting in chain elongation and branching [51,175].

The content and degree of polymerization of fructans in plants can also be impacted by the action of fructan exohydrolases (FEHs), since, unlike microorganisms, plants lack endohydrolases [176,177]. Fructan breakdown occurs during harvesting, storage, and sprouting [178,179] as a response to stress and bacterial infections [177,180,181,182,183]. The most common FEHs are β-(2→1)-d-fructan fructohydrolase, 1-FEH (EC 3.2.1.153), (2→6)-β-d-fructan fructohydrolase, 6-FEH or exolevanase (EC 3.2.1.154), and β-d-fructan fructohydrolase, 6&1-FEH or exoinulinase (EC 3.2.1.80), which hydrolyze fructan molecules at the terminal non-reducing (2→1)- and (2→6)- and both (2→1)- and (2→6)-linked β-d-fructofuranose residue, respectively, with the release of fructose [155,176,180,184,185,186]. 6&1-FEHs have also been noted to present a strong affinity for small graminans [155]. More recently, Ueno and co-workers isolated a novel fructan exohydrolase, 6G & 1-FEH, that preferentially hydrolyzes β-(2,6) linkages between fructose and glucose in neokestose [187]. The seasonal mobilization of fructan reserves, with impacts on the degree of polymerization, is well-acknowledged and has been associated with corresponding differentiated levels of FEH activity [18,183,188,189]. While studying fructan accumulation in wheat, Meguro-Maoka and Yoshida established that the expression of FEH genes was related to the regulation of water-soluble carbohydrate accumulation from autumn to early winter and fructan consumption when covered by snow [190]. Such findings were further corroborated by Krivorotova and Sereikaite, who correlated seasonal changes in fructan exohydrolase activity in three Jerusalem artichoke cultivars, with fluctuations in the total fructan and FOS content. Accordingly, the authors suggested harvesting Jerusalem artichoke tubers in autumn before enzyme activity peaks, if a high degree of polymerization fructans are envisaged or in late autumn/over winter, if short-chain FOSs, which are preferably fermented by beneficial gut microbiota, are desired [154,191].

Alongside native plants, transgenic techniques have been implemented towards the tailor-made production of FOSs, which may involve either the modification/improvement of the metabolic pathway of the host culture or the introduction of the fructan synthetic pathway. The latter has been often carried out in common crops consumed by humans as food or animals as feeds, as detailed elsewhere [52,185].

3.2. Production Methods

3.2.1. Extraction from Natural Sources

Fructans can be extracted from natural sources using water or aqueous alcohol, such as methanol or ethanol [2,68]. When stored in organs, such as bulbs or roots, FOSs are easily extracted and processed into purified products, since there are no contaminants [77]. Hot or boiling water extraction is the most used method for fructan recovery from plants, after which the raw-extracted inulin can be concentrated, refined (by e.g., flocculation), decolorized by ion exchange, and spray-dried [52,192,193]. Yet such a conventional approach is prone to cause oligosaccharide degradation, given the required high temperature and long extraction time [52,192]. To overcome these drawbacks, auxiliary/alternative extraction methods have been evaluated, such as microwaves, pulsed electric fields, ultrasounds, and ultrafiltration [17,52]. In a recent work, Garcia-Villalba and co-workers compared hot water, ultrasound, microwave, and simultaneous ultrasound–microwave for the extraction of fructans from agave flour. The authors concluded that the simultaneous ultrasound–microwave extraction method provided the best compromise considering outputs such as time duration, energy consumption, eco-friendly solvents, and yields [173]. When addressing the extraction of fructans from Eremurus spectabilis root powder, Pourfarzad and co-workers also observed that ultrasound extraction outperformed hot-water extraction [194]. On the other hand, when comparing the yield after extraction from burdock using water at either room temperature or at 100 °C under reflux and ultrasound-assisted extraction, de Marins and co-workers reported the highest yield for hot-water extraction [195]. Still, they also observed that the extraction method influenced the degree of polymerization of the recovered fructans. Thus, the lowest degree of polymerization (DP) was observed for ultrasound-assisted extraction, followed by room-temperature and hot-water extraction. Nonetheless, Zeaiter and co-workers were able to extract inulin-type fructans with a high degree of polymerization from artichoke wastes by ultrasonication [196]. Ruiz-Aceituno and co-workers reported that pressurized liquid extraction outperformed microwave-assisted extraction when recovering fructans from artichoke external bracts when both techniques were performed at 60 to 75 °C [197]. Rivera and co-workers reported that similar extraction yields were obtained when pulsed electric field-assisted extraction was compared with hot-water extraction, although in the former method, an inulin-type fructan with a higher degree of polymerization was obtained [198]. Sánchez-Madrigal and co-workers opted for enzyme-assisted extraction to recover fructans from a wild sotol plant. Using a commercially prepared enzyme, Pectinex Ultra SP-L, the authors obtained a product rich in fructans with a degree of polymerization of 8 to 10 [199]. Demirci and co-authors combined microwave-assisted extraction with ultrafiltration to improve the extraction yield and purity of inulin recovered from artichoke powder [200]. Finally, Shalini and co-workers combined enzyme-assisted extraction, using a commercially prepared hemicellulose with a rapid solvent (70% methanol) extractor system at room temperature to recover FOSs from garlic [201]. This strategy increased the extraction yield by 32%, as compared to conventional hot-water extraction. Moreover, subsequent purification of the extract with activated charcoal resulted in a final product that was 94% pure in 1-kestose. These examples illustrate the feasibility of using methods other than conventional hot-water extraction for the recovery of fructans. Overall, these methods allow for FOSs yields of up to 30%, suggesting that further improvements are needed [17]. Moreover, despite being available in plenty of plants, the FOS titer is often relatively low, except for chicory and agave (e.g., around 5 to 10% FOS and 15 to 20% inulin in chicory roots [16]), which, combined with their season-limited nature, makes their large-scale production more amenable by enzymatic and microbial methods, either by hydrolysis or by synthesis [16,20,40,41,51,202].

3.2.2. Enzymatic Production by Hydrolysis

This approach involves the hydrolysis of plant fructans, such as inulin, with a high degree of polymerization (up to 60) [16,40,45]. This involves the use of β-fructofuranosidases, which cleave β-(2→1) fructofuranosidic bonds, and levanases, which cleave β-(2→6) fructofuranosidic bonds [16]. The former, more specifically endoinulinases, 1-β-D-fructan fructanohydrolase (EC 3.2.1.7), which catalyze the endohydrolysis of (2→1)-β-D-fructosidic linkages, are typically used in the production of FOSs from inulin. Thus, fructooligosaccharides with various lengths (degree of polymerization from 2 to 7 and $\overline{DP}=4$), e.g., inulobioses, inulotrioses, inulotetraoses, and inulopentaoses, besides glucopyranosyl-(fructofuransoyl)_n-fructose-type molecules, are released by random hydrolysis on the internal glycosidic bonds of the inulin chain [20,56,203,204]. Endoinulinases can be sourced from bacteria (e.g., Bacillus sp.), molds (e.g., Aspergillus sp.), and yeasts (e.g., Kluyveromyces sp.) [32,45]. The degree of polymerization is influenced by several reaction parameters, as summarized in Table 5. The enzyme source influences the hydrolysis outcome, affecting the type and amount of products formed [20,56,204]. Thus, as summarized by Martins and co-workers [40], the endo-inulinase from Xanthomonas oryzae No. 5 mainly produces FOSs with a degree of polymerization over 5, while the same enzyme from Pseudomonas sp. No. 65 primarily yields FOSs with a degree of polymerization ≤ 3. Thus, combining different endo-inulinases may allow for tailored FOS production based on specific needs.

The hydrolysis of inulin produces higher yields and greater FOS purity compared to synthesis, with an 81% yield for the former compared to 55% yield for latter. During FOS synthesis, high amounts of glucose are released, which inhibits enzyme activity. Moreover, synthesis leaves mono- and disaccharides in the final product. Hydrolysis requires very careful control of the reaction conditions and may also lead to the formation of several unwanted by-products. In both cases, removal of the by-products is required, thus increasing costs and complexity [20,40,41,205]. Still, the use of engineered endoinulinases has been shown to improve efficiency of FOS production, with yields of up to 90%, as summarized elsewhere [40]. Examples of commercially available FOSs that are obtained through inulin hydrolysis are Raftilose^® P95, which contains FOSs with a degree of polymerization between 2 and 8 and $\overline{DP}=4$ [206], and Fibrulose F97, whose FOS consists of 70% with a degree of polymerization between 2 and 10 while a maximum of 5% has a degree of polymerization exceeding 20 and $\overline{DP}=5.5$ [46,207]. Further examples and details can be found elsewhere [16,20,46].

Table 5. Some reaction conditions affecting the degree of polymerization (DP) of products from inulin hydrolysis [32,206,208,209].

Reaction Conditions	Comments
Enzyme concentration	Higher enzyme concentrations generally lead to a higher rate of hydrolysis, resulting in shorter FOS chains (lower DP).
Reaction time	Longer reaction times can lead to a further breakdown of the FOS, reducing $\overline{DP}$.
Temperature and pH	The temperature and pH of the reaction influence enzyme activity and stability. Optimal conditions can maximize enzyme efficiency and control the DP of FOSs.
Substrate concentration	Higher substrate concentrations may lead to a higher FOS yield but also to a broader distribution of DP due to varying enzyme–substrate interactions.

Table 5. Some reaction conditions affecting the degree of polymerization (DP) of products from inulin hydrolysis [32,206,208,209].

Reaction Conditions	Comments
Enzyme concentration	Higher enzyme concentrations generally lead to a higher rate of hydrolysis, resulting in shorter FOS chains (lower DP).
Reaction time	Longer reaction times can lead to a further breakdown of the FOS, reducing $\overline{DP}$.
Temperature and pH	The temperature and pH of the reaction influence enzyme activity and stability. Optimal conditions can maximize enzyme efficiency and control the DP of FOSs.
Substrate concentration	Higher substrate concentrations may lead to a higher FOS yield but also to a broader distribution of DP due to varying enzyme–substrate interactions.

Although endoinulinases are typically employed in inulin hydrolysis [14,205], Huazano-Garcia and López used an endoinulinase, either isolated or combined with an exoinulinase, to produce branched FOSs from agavin [210]. Endoinulinase degraded 31% of agavins in 48 h, producing ~20% branched FOSs, while the inulinase mixture hydrolyzed 33% in 90 min but yielded only 10% branched FOSs. This outcome aligns with the acknowledged decrease in prebiotic activity observed when endo- and exoinulinases are used together, since this results in the release of non-prebiotic sugars like glucose, fructose, and sucrose. The removal of these sugars requires costly separation operations, e.g., nanofiltration or ion exchange chromatography [14].

Levanases can also be used to produce short-chain levan–FOSs by hydrolyzing levan [16,211,212]. This approach entails the use of exolevanases and endolevanases, (2→6) (2→6)-β-d-fructan fructanohydrolases (3.2.1.65), which promote the inner random hydrolysis of (2→6)-β-d-fructofuranosidic residues to yield levan–FOSs with degrees of polymerization between 2 and 10 [185,203]. Still, exolevanases are prone to reduce FOS yields, as they favor the accumulation of fructose, and endolevanases are hard to obtain; hence, this approach is currently limited. Still, if adequately sourced, endolevanases may produce levan–FOSs with a specific chain length [211,212,213,214]. Moreover, levan can also be hydrolyzed by levanbiose-producing levanase, (2→6)-β-d-fructofuranan 6-(β-d-fructosyl)-d-fructose-hydrolase, or- 6-levanbiohydrolase (EC 3.2.1.64), which promotes the release of disaccharide residues as levanbiose, 6-(-β-D-fructofuranosyl)-D-fructose, from the end of the chain, thus providing further alternatives to the production of levan–FOSs [203,213]. Levan can also be degraded by levan fructotransferase (EC 4.2.1.16) to yield di-D-fructose-2,6′:6,2′-dianhydride (DFA IV), an alternative sweetener composed of two fructose residues [213,215].

3.2.3. Enzymatic Production by Synthesis

The enzymatic synthesis of FOSs involves fructosyltransferase activity, typically exhibited by inulosucrase, sucrose:(2→1)-β-d-fructan 1- β-d-fructosyltransferase (EC 2.4.1.9), levansucrase, and by some invertases, β-d-fructofuranoside fructohydrolase (EC 3.2.1.26), when operating under conditions with a high-substrate concentration [16,216]. Enzymes exhibiting fructosyltransferase transfer the fructosyl unit from sucrose to various acceptors (sucrose or FOSs), producing fructans of different molecular weights based on enzyme specificity. This process generates FOSs such as 1-kestose, nystose, and fructofuranosyl nystose, namely when inulosucrases and invertases are involved [20,40,45,47]. Levansucrase mainly synthesizes FOSs with β(2→6) linkages, producing 6-kestose and related β(2→6)-linked FOSs. However, some levansucrases also produce 1-kestose and neokestose [217,218,219]. Thus, unlike in FOS productions by enzymatic hydrolysis, only glucopyranosyl-(fructofuransoyl)_n-fructose type molecules are obtained [20,40,45,47]. Invertases are easily available from bacillus, molds, and yeasts, e.g., Bacillus spp., Aspergillus niger, and Saccharomyces cerevisiae, respectively [14,20,25,41,45], and inulosucrases can be obtained from bacteria and molds such as Lactobacillus spp. and Aspergillus spp., respectively [52,204], and levansucrases can be obtained from several bacteria, e.g., Bacillus spp., Lactobacillus spp., and Pseudomonas spp. [52].

It is not surprising that invertases are able to synthesize FOSs, since plenty of hydrolytic and transfructosylating enzymes operate through a double displacement mechanism. Thus, firstly the anomeric carbon of the fructosyl moiety in the fructose donor, e.g., sucrose, is attacked by Asp-60, resulting in the formation of a covalent enzyme–fructose intermediate. Afterwards, the leaving group, e.g., glucose, is protonated by the residue Glu-292. In the second stage, the latter residue deprotonates the acceptor molecule, water for invertases or sucrose/fructan for fructosyltransferases, the type of hydroxyl group of the acceptor molecule influencing the glycosyl linkages of the FOS molecule [25]. Molecular docking studies involving hydrolytic and transfructosylating enzymes of fungal origin shared a similar protein–ligand interaction profile and noted that the total FOS production yield correlates with the combined affinity energies of kestose, nystose, and fructosyl–nystose, indicating that higher ligand affinity leads to increased FOS production. Moreover, findings provide key insights into the molecular basis of FOS synthesis and offer a basis for optimizing FOS production and target the production of a specific FOS [220]. Accordingly, molecular docking studies and molecular dynamics simulations were combined with (semi)rational designs to produce engineered transfructosylating enzymes that are able to specifically synthesize FOSs with a degree of polymerization up to 5 [221], or neo-kestose, 6,6-nystose and 1,6-nystose) [222].

The enzymatic synthesis of FOSs results in a product with a lower and narrower degree of polymerization (within 2 and 4 and $\overline{DP}=3.6$), hence clearly short-chain FOSs, compared to enzymatic hydrolysis [41], but again, the composition of the final product depends on the enzyme as well as on the operational conditions (Table 5) [16,25,52,216]. Moreover, the enzymatic synthesis of FOSs from sucrose ensures consistent quality, is unaffected by seasons or locations, and benefits from sucrose being a widely available, low-cost substrate [56].

Commercially available short-chain FOSs are produced by fructosyltransferases and include, e.g., Actilight^® 950S, which contains 1-kestose (37.1%); nystose (47.7%) and fructosyl-nystose (15.2%) [223]; and Meioligo^®, which contains 1-kestose (36%), nystose (51%), and fructosyl-nystose (9%) [115]. Further examples can be found elsewhere [14,20,30,204].

Additionally, efforts to further improve the efficiency of enzymatic FOS production are on-going, namely, at the process level, modelling kinetics and protein engineering, e.g., through the design of recombinant enzymes, abridging either hydrolysis [37,56,224,225] or synthesis [216,226,227,228,229,230]. Within this scope, a fructosyltransferase from A. niger expressed in Pichia pastoris displayed high substrate affinity, stability, and efficiency in FOS synthesis. Thus, an FOS yield of 67% was achieved, which bested other reported fructosyltransferases, highlighting its potential for industrial use. This behavior was partially attributed to amino acid variations outside conserved domains [231]. An engineered β-fructofuranosidase from A. fijiensis expressed in P. pastoris was compared to the native enzyme towards the production of FOSs from white and brown sugar, as well as from refinery and A-molasses (from sugar mill). Optimal temperature and enzyme dosages were identified to achieve an FOS composition similar to a commercial product. The recombinant enzyme displayed superior efficiency, as the reaction time was reduced by 28% and 25% for white and brown sugar, respectively. Refinery molasses did not yield the target FOS composition, unlike A-molasses, with the native enzyme achieving a 6% shorter reaction time when compared to the recombinant enzyme. The low pH of refinery molasses (4.5, compared to an optimum pH between 5.0 and 5.5 for the enzymes) and impurities present in the stream were associated with poor enzyme performance. The engineered enzyme displayed increased thermostability, which enhanced product productivity by reducing the reaction time by 19% at a pilot scale, using white sugar as the substrate. Despite the potential of the engineered enzyme for FOS production, the susceptibility to impurities were limiting, highlighting the need for further improvements, e.g., process optimization and/or further enzyme engineering [232]. A different approach was suggested by Wan and co-workers [233]. In this case, FOS synthesis was enhanced by the heterologous expression of glucose oxidase and peroxidase in A. niger. These enzzymes were detected in the mycelium due to their successful fusion with the native β-fructofuranosidase domain. The multi-enzyme mycelia enabled a direct sucrose conversion without further purification, as the by-product glucose was oxidized by glucose oxidase and peroxidase, which removed the potentially toxic hydrogen peroxide that was formed. The high FOS yield (71.0%) achieved with this whole-cell system suggests that this approach presents a cost-effective method for high-purity FOS synthesis. Process optimization towards improved FOS synthesis using a commercially produced fructosyltransferase was presented by Niu and co-workers [234]. The reaction was performed at high sucrose concentrations (800 g/L) and at a high temperature (65 °C), leading to a 155.9% increase in the transglycosylaton rate and a 113.5 increase in volumetric productivity when compared to commercial methods. The authors highlighted that working with such concentrated systems enhanced enzyme thermostability, which aligned with the mitigation of high-viscosity issues and substrate solubility in concentrated systems. Further examples and details can be found elsewhere [14,20,28,40,235]

3.2.4. FOS Production by Microbial Fermentation

Microbial production of FOSs typically involves the use of either prokaryotes (e.g., Bacillus spp., Erwinia spp., and Lactobacillus spp.) or eukaryotes (Aspergillus spp., Aureobasidium spp., and Penicillium spp.) with fructosyltransferase activity [20,25,204]. This approach makes it possible to bypass the enzyme recovery and purification stage and its associated costs and to produce FOSs in a single step [20,202]. On the other hand, the microbial production of FOSs is often associated with relatively low yields, e.g., 0.3 to 0.6 (w/w) [20], and low purity $\approx 50\%$ (w/w) [20], given the complexity of the fermentation media; the difficulty of maintaining optimal conditions for both microbial growth and enzyme activity, which require precise control of factors such as pH, temperature, and nutrient availability; and the dependence on pure sucrose [14,202,204]. To overcome these downsides, some alternatives have been implemented, namely, the use of whole-cell inactive microorganisms, thus ruling out the coordination of conditions for proper microbial growth and enzyme activity [20]; the design of recombinant strains to increase FOS yields from sucrose, achieving high production efficiency [233,236]; the use of low-cost substrates, e.g., aguamiel and molasses, as alternatives to pure sucrose [237]; the use of co-cultures to consume the small saccharides remaining in the fermentation medium, thus increasing FOS purity in the mixture [202]; or a combination of the former [238].

Submerged fermentation is the preferred approach to implement the microbial production of FOSs, although some promising results (e.g., FOS $\approx 90\%$ (w/w)) have been obtained using solid state fermentation [239]. Still, scaling-up solid-state fermentation is challenging, especially given the gradients in inoculum, moisture, oxygen, pH, and temperature moisture inside the reactor [20]. Nutraflora^® is a short chain FOS complex (degree of polymerization from 3 to 5 [240]) produced by fermentation [204].

Extensive details on the microbial production of FOSs can be found elsewhere [20,25,47,204]. A detailed quantitative assessment of the different approaches for FOS production regarding titers, productivity, yields, and purity can be found in a recent review, which suggest that the use of engineered enzymes is the most effective method, all things considered [20].

4. Conclusions

Since their emergence as a new product in circa 1980, the demand for FOSs has been steadily rising, and market predictions confirm this trend for the near future. This pattern is intertwined with growing evidence and consumer awareness on the impact of a balanced diet in health and wellbeing, especially with the impacts of the gut microbiome on the latter. Consumers have thus become more receptive to a holistic approach to wellbeing and follow a low carbohydrate, high-fiber, and proactive diet. FOSs fit into this pattern, given their physicochemical, physiological, and sensory properties that ease their incorporation in various formulations in food, feed, and medical areas, thereby fueling their prebiotic, low glycemic, and textural enhancement potential. The successful application of FOSs in these areas has also fostered research in their occurrence in natural sources, structure, and synthetic mechanisms. These efforts have provided further insights into types of FOSs that have already been identified, while prompting the quest for novel FOSs from natural sources, not only increasing product diversity but also requiring increasingly complex methodologies for proper isolation and characterization.

Overall, the incorporation of FOSs in diets has provided health benefits, yet some findings are occasionally inconclusive or even contradictory. Such outcomes can be associated with lack of standardization in the performed studies given the diversity of FOSs used, the frequency of administration, the dosages and duration of the studies, the nature of the target population, and the sample size. This could be overcome through the use of a consistent set of parameters, adequate statistical frameworks, and large-scale clinical trials. Data also highlight the individuality of the microbiome and how this can impact its modulation by FOSs and prompt individual and specific responses to diets. Properly addressing and incorporating such a vast array of information requires proper big data management. Moreover, further knowledge of the impact of FOSs on specific gut microbiota strains; structure–function relationships; the selective effects of FOSs in systemic health conditions, e.g., metabolic disorders, mental health (via the gut–brain axis), and immune modulation, is also required. The use of omics technologies, such as metagenomics and metabolomics, can contribute to filling these gaps. The knowledge gathered is expected to foster the development of targeted FOS products tailored to specific health needs, such as personalized nutrition, a rapidly emerging market.

The availability of FOSs from natural sources is rather limited to cope with market demands, due to several factors, among them, the poor titer, seasonality, and relatively poor yields of the extraction methods. Moreover, a uniform composition of the obtained product is barely obtained. Among the implemented production methods, enzymatic- and microbial-based methods stand out, all favoring the production of inulin FOSs. Enzymatic methods rely either on hydrolysis or synthesis. The former typically rely on endo-acting enzymes, namely endoinulinulases, to hydrolyze long-chain fructans and to yield FOSs with a relatively high degree of polymerization, e.g., above 5 (but always under 10). Careful control of the reaction is required to prevent excess hydrolysis, and the final product is prone to vast diversity. Enzymatic synthesis relies on enzymes with fructosyltransferase activity and usually relies on sucrose as the substrate and yields short-chain FOSs, e.g., degree of polymerization 3, which are more rapidly digested by beneficial microbial gut strains. Although the final product has a more consistent composition than those produced by enzymatic hydrolysis, yields can be relatively low, and excessively generated glucose is often inhibitory, and the cost of sucrose adds to the final product, a drawback that can be overcome using suitable agro-industrial by-products. Overall, enzymatic methods can be jeopardized by their enzyme cost, reduced stability, low activity, and sensitivity to inhibitors, e.g., high substrate/product concentrations. Some of these hurdles can be addressed by screening microbial strains for more effective enzymes and/or engineering microbial strains to overexpress the required genes, e.g., endoinulinase or fructosyltransferase, to improve their tolerance to a high degree of substrates/products, and to enhance stability. The microbial production of short-chain FOSs reduces costs, as enzyme recovery and purification are avoided, yet downstream processing is typically more complex when compared to enzymatic methods. Additionally, the optimal conditions for microbial growth may differ from that of FOS synthesis, although the latter can be mitigated if inactive microorganisms are used. Again, genetically engineered microorganisms with improved activities/stability are foreseen to overcome the highlighted limitations. Finally, the use of immobilized enzymes/whole cells can enable continuous production and favor constant product compositions, avoiding the variability of batch productions.

The future of FOS research and applications lies at the intersection of scientific innovation, sustainability, and consumer-driven demands for functional and natural products. By addressing current challenges and exploring novel avenues, FOSs can become a cornerstone in the development of health-promoting ingredients and sustainable production practices.