Fructooligosaccharides: A Comprehensive Review on Their Microbial Source, Functional Benefits, Production Technology, and Market Prospects

Abstract

Fructooligosaccharides (FOSs) are carbohydrates of high nutritional value with various prebiotic properties. Optimizing their production process is of significant interest for expanding commercial-scale production. This review discusses the properties and potential applications of FOSs, addressing production challenges and providing an economic market analysis. Bibliometric analysis of data concerning the functional properties, production, purification, and applications of FOSs revealed an over 87% increase in the number of worldwide publications from 2012 to 2022, rising from 88 to 165. Furthermore, contributions from ninety-three countries were identified up to 2024, with Brazil ranking first, with 326 publications. Furthermore, Aureobasidium sp. and Aspergillus sp. have shown the best results for FOS production, with reported conversion in the order of 0.66 g FOS/g sucrose. Nevertheless, the formation of by-products or co-products requiring separation from the medium remains a challenge. Activated carbon, cation exchange resins, and zeolites are highlighted as key adsorbents, with the adsorption process achieving FOS purity exceeding 90%. Furthermore, membrane technology is identified as a more efficient and promising separation method. Addressing these limitations will facilitate the further expansion of the growing global FOS market, promoting a sustainable approach and their integration with biorefineries, which can enable the development of a wider range of value-added products.

1. Introduction

Recently, the growing consumer demand for safe and healthy food options has driven efforts to develop functional foods, including prebiotics [1]. In this regard, non-digestible oligosaccharides, such as fructooligosaccharides (FOSs), are considered a key ingredient for the development of such functional foods, since short-chain FOSs (polymerization degree ranging from 3 to 5) have been accepted and generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA) in 2000 [2].

FOSs are naturally occurring oligomers of fructose units linked by β-2,1-glycosidic bonds with a glucose unit attached to the terminal fructose unit of the fructan chain [3]. In the last decade, these compounds have attracted interest because of their potential health benefits. Classified as prebiotics, FOS promotes the growth and activity of beneficial gut microbiota, which can improve digestive health and overall well-being [4]. When consumed in appropriate amounts, FOSs offer numerous health benefits as follows: they are non-carcinogenic sweeteners with a low-calorie content (0.4 to 0.6 times less sweet than sucrose), making them a potential sucrose alternative; they can improve the absorption of calcium and magnesium in the gastrointestinal tract; they can lower cholesterol and triglyceride levels; they may help the body resist infections by reducing proinflammatory cytokines; they can activate the immune system; and they promote the synthesis of B-complex vitamins [3,4]. Due to the type of glycosidic bonds present, these compounds are not digested by human salivary or intestinal enzymes. Instead, they are metabolized by anaerobic bacteria in the intestine. This characteristic makes them suitable for consumption by individuals with diabetes [5].

The FOS market has grown significantly in recent years, especially after a study by Witkowski et al. reported that xylitol, another sweetener considered a key replacement for sucrose, may be associated with cardiovascular diseases, such as heart attack and stroke [6]. The world FOS market was worth USD 2.59 billion in 2022, with an expected compound annual growth rate (CAGR) of 8.8%, expected to reach USD 5.09 billion by 2030 [7].

FOSs have been extracted from various food sources, including onions and garlic, using methods like hot water extraction and ultrasonication [5]. However, most vegetables contain small amounts of FOSs, making industrial extraction difficult. Therefore, industrial FOS production is primarily achieved through the chemical or enzymatic hydrolysis of sucrose or inulin. Using biocatalysts is a promising approach because chemical processes often involve toxic chemicals, lack specificity, and typically require a high energy input [8]. The biotechnological production of FOSs is of significant interest. This can be achieved by using microbial enzymes FTases or microbial cells as whole-cell biocatalysts. Recent research has explored the usage of immobilized enzymes and cells to improve process efficiency and simplify purification [9]. This area is still under development, particularly considering the current global environmental concerns.

Although studies on FOS production, purification methods, or microorganisms used are available in the literature, these studies do not integrate this information, presenting it in a segmented manner across different works. In addition, results based on bibliometric analysis or a critical comparison of the technical–economic viability of FOS production by different methods are scarce. In this context, this review examines the properties and potential applications of FOS in the food and pharmaceutical industries. It also discusses production challenges, focusing on process technologies. Finally, it analyzes the economic and environmental aspects of FOS, providing a comprehensive overview of this compound as a functional food ingredient.

2. Functional Properties of Fructooligosaccharides

Fructooligosaccharides (FOSs) are carbohydrates with significant nutritional value and prebiotic properties, making them useful functional additives in food supplements, promoting human health [10,11]. Among their many applications, FOSs can be included in foods for people with diabetes and in dental products. They are considered non-cariogenic and may help reduce the risk of obesity because they are not fully metabolized by the body and have a low caloric value [12,13]. Furthermore, these sugars are a valuable option for replacing sweeteners in the food industry due to their favorable cost–benefit ratio, without negatively affecting the taste and consumer acceptance of the food [12,13].

FOSs exhibit diverse prebiotic and nutritional properties depending on their chemical composition, structure, and degree of polymerization, which are influenced by their production source [14]. Generally, FOSs promote the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus, due to their resistance to digestion by human enzymes [11,15,16]. The intestinal fermentation of FOSs yields several health benefits, including the modulation of the gut microbiota and intestinal transit, activation of lymphocytes and phagocytes with potential antitumor and antibacterial effects, and enhanced nutrient absorption. Further reported benefits include increased resistance to pathogenic microorganisms, the modulation of cell proliferation and differentiation, improved calcium absorption, antioxidant activity, and increased resistance to gastric acidity [12,16,17].

In vivo studies in mice have evaluated the nutritional properties of FOSs extracted from burdock, focusing on their effects on hypercholesterolemia and vascular inflammation [18]. These studies demonstrated reductions in total cholesterol, low-density lipoprotein cholesterol (LDL), and oxidized LDL, along with vascular protection and regulation of aortic pathways. Yuan et al. also investigated burdock-related FOSs in diabetic mice, examining antidiabetic effects and α-glucosidase inhibition [19]. Their findings indicated reduced blood glucose levels, improved glucose tolerance, and attenuated insulin spikes because of α-glucosidase inhibition, in addition to positive effects on cholesterol levels. These results suggest that burdock-derived FOSs may have potential applications in the prevention of cardiovascular diseases, hypercholesterolemia, and type 2 diabetes.

Ojwach et al. reported that FOSs synthesized by a partially purified fructosyltransferase from an indigenous coprophilic strain of Aspergillus niger XOBP48 exhibited antioxidant potential [20]. Their study employed assays such as DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (ferric iron reducing power), and radical scavenging assays. The results demonstrated the ability of the FOS to reduce and eliminate free radicals, indicating its antioxidant properties. Bandyopadhyay et al. evaluated the prebiotic properties of FOSs extracted from the tuber Dioscorea alata L., using chemical analyses, including high-performance thin layer chromatography (HPTLC), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS), in addition to in vivo and in vitro tests in mice [21]. The results revealed their prebiotic and antioxidant activities. The in vivo tests also showed a significant reduction in cholesterol levels and an enhanced immunological increase in mice, suggesting that this FOS possesses prebiotic and hypocholesterolemic effects. Hajar-Azhari et al. enzymatically synthesized FOS from sugar cane syrup and analyzed its prebiotic properties regarding the modulation of the human intestinal microbiota and production of short-chain fatty acids [22]. Their results revealed the increased growth of Bifidobacterium, stable growth of Bacteroides/Prevotella, and decreased growth of Clostridium histolyticum during 24 h of fermentation. The production of short-chain fatty acids, notably acetate, propionate, and butyrate, was also increased, demonstrating potential nutritional benefits.

Furthermore, FOS may enhance the growth of probiotics, creating a symbiotic mixture with potentially greater efficacy in improving gastrointestinal health. This combination has also demonstrated an angiotensin-converting enzyme inhibitory activity, which plays a role in blood pressure regulation [23,24]. In vitro studies by Dong et al. showed that combining FOS with Lactiplantibacillus plantarum reduced the pathogenic potential of Listeria monocytogenes, the causative agent of listeriosis, by inhibiting its growth [25]. Their study compared L. monocytogenes concentrations in the following four groups: FOS treatment, L. plantarum treatment, an untreated control, and a group receiving both L. plantarum and FOS. The combined treatment group exhibited reduced L. monocytogenes growth during storage at 10 °C and 25 °C, temperatures relevant to potential food contamination. Choi et al. reported that combining FOSs and probiotics may have preventative effects against neurodegenerative diseases when incorporated into foods like fermented soy milk [26]. Using SH-SY5Y cells, they found that L. plantarum 200655 and FOSs contributed to reducing lactate dehydrogenase release, thus promoting cell integrity and preventing oxidative stress-induced cell death.

In addition, Toporovski et al. investigated the effects of combining FOS with polydextrose on childhood constipation. Children received daily doses of the mixture as a food supplement for 45 days, during which fecal samples were analyzed [27]. The study found a significant reduction in constipation symptoms, suggesting that this combination may be a viable treatment option, with good acceptance among both parents and children. De Figueiredo, Ranke, and De Oliveira-Neto examined the influence of FOS and xylooligosaccharides on digestive enzyme hydrolysis, considering these prebiotics as potential nutrients for Bifidobacterium, Lactobacillus, and Salmonella typhimurium [16]. Their in vitro analyses demonstrated the resistance of these prebiotics to gastrointestinal enzymes and gastric acidity. Furthermore, the results indicated that FOS and xylooligosaccharides promoted the growth of beneficial bacteria, while not supporting the growth of the pathogenic bacterium Salmonella typhimurium, potentially inhibiting its proliferation.

Finally, the industrial and social interest in FOSs has increased due to studies reporting the potential of prebiotics in preventing and treating various intestinal health-related diseases. For instance, Lee et al. investigated the use of prebiotics for preventing and treating atopic dermatitis, a condition often associated with intestinal dysbiosis. Their findings suggest that prebiotic supplementation provides nutrients that promote the growth of beneficial bacteria, potentially mitigating this condition [28]. Similarly, Choi et al. and Wu et al. explored the benefits of combined prebiotics and probiotics supplementation in preventing and delaying cardiovascular diseases. They proposed that these supplements exert their effects by modulating the intestinal microbiota, thereby influencing cholesterol levels and reducing inflammatory responses [26,29].

3. Bibliometric Analysis of FOS

Interest in FOSs has grown substantially in recent decades. A bibliometric analysis was performed using VOSviewer^® (1.6.19) and Microsoft Excel^® (365), retrieving 2318 articles from the Scopus database. These articles covered a broad spectrum of topics related to FOSs, including their functional properties, production, purification, and application. The Scopus database was queried using the following criteria: TS = (“fructooligosaccharides”), time span = “from 1980 to 2024”, language = “English”, document type = “article, review or early access”. Here, TS represents the topic, meaning the search of the specified terms within the title, abstract, and keywords. The data extracted from Scopus was processed in Excel^® to categorize the documents, generating the data presented in Figure 1. This same dataset was then imported into the VOSviewer^® to construct the bibliometric network visualized in Figure 2.

Studies on FOSs began appearing in the 1980s. From 1983 to 2024, publication output on FOSs increased substantially (Figure 1A), rising from 88 articles in 2012 to 165 in 2022, an increase exceeding 87%. Early work, such as that by Hidaka et al., addressed the industrial production of FOS and its applications in humans and animals [30]. Among these applications, the prebiotic properties of FOS are prominent. Subsequent research has focused on FOS production and purification, with a continued increase in publications to the present day.

Ninety-three countries/regions contributed to FOS publications between 1983 and 2024. Figure 1D presents a heat map illustrating the geographical distribution of these publications. Brazil is the leading contributor with 326 publications. Within Brazil, the State University of Campinas (Unicamp) has produced 72 papers, covering both the nutritional aspects (e.g., Pastore, G. M.) and FOS production (e.g., Maugeri, F.). China ranks second with around 268 papers, followed by the United States with 243. These top 3 countries account for approximately 28% of all articles.

Among the most prolific authors (Figure 1B), Fahey, G. C., and Ohta, A. stand out with 27 and 25 published articles, respectively, primarily focused on human health and nutrition. Conversely, Plou, F.J., and Singh, R. S., with 25 and 20 publications, respectively, predominantly focus on biotechnology. Figure 2 presents a bibliometric network generated using VOSviewer^®, illustrating author collaborations in FOS research. In this network, each node represents an author, and connecting lines represent co-authorships. Node size corresponds to the author’s citation count, highlighting Gibson, G. R. and Roberfroid, M. B., who authored the most cited article on the subject [31]. This seminal work introduced the concept of prebiotics in 1995.

The identification of author collaboration networks form distinct clusters plays a crucial role in understanding research dynamics. Grouping authors in bibliometric networks helps identify key research groups and their areas of focus. By organizing dispersed articles into structured categories, it simplifies the analysis of the research landscape. Clusters also reveal collaboration patterns among authors and institutions, highlighting research networks and cooperative efforts. Additionally, they uncover emerging fields and interdisciplinary links, offering strategic insights for projects and funding decisions. The blue cluster (left) is primarily composed of Japanese researchers, including Ohta, A., and Hirayama, M., who focus on the functional properties of FOS and their health applications [32,33].

The high density of this network indicates a well-established research group. The red cluster (center-right) consists of Chinese researchers, such as Zhang, Y. and Liu, Z., whose work centers on the biotechnology and industrial production of FOSs [34,35]. Their extensive collaboration is reflected in a substantial publication output. The green cluster (right) also comprised Chinese researchers like Chen, W. and Zhang, C., who investigate FOSs in functional foods and prebiotics, demonstrating their strong inter-member interaction [36]. The purple cluster (center-upper) is formed by researchers from the USA, including Fahey, G.C., whose research focuses on nutritional studies and the gastrointestinal health benefits of FOSs [37,38]. The yellow cluster (top-center) includes Brazilian and Portuguese researchers, such as Teixeira, J.A., who studied the bioprocesses and biocatalysis in FOS production [39]. The brown cluster (bottom-left) is made up of Spanish researchers, such as Plou, F.J., whose work emphasizes the biocatalysis and enzymatic methods for FOS production [40,41]. Central nodes represent authors with fewer collaborations, often from diverse research areas. Overall, the network analysis highlights the significant research community focused on FOSs’ biotechnological production, properties, and applications.

4. Microorganisms Potentially Producing Enzymes with Fructosyltransferase Activity

The demand for FOSs has risen due to the increased awareness of their probiotic properties and their use as supplements in functional foods for both humans and animals. This heightened demand has driven interest in more efficient industrial production processes, reflected in research efforts focused on identifying new microorganisms, the optimization of processes, and particularly in exploring enzymes produced by fungi [4,42].

Fungi are recognized for their capacity to produce and secrete a wide range of enzymes, owing to their role as decomposers. This characteristic has led to their designation as “biofactories”, with applications in various fields, notably food production [43]. FOSs are among the products of significant current research interest, synthesized through the enzymatic transfructosylation of sucrose, yielding molecules with probiotic properties [44].

Microorganisms, including bacteria and fungi, have been studied for their potential to produce enzymes that synthesize FOSs. Fungi are particularly prominent due to their capacity for both intra- and extracellular enzyme production. Fungal fructosyltransferases (FTases) are evolutionarily related to plant β-fructofuranosidase [4,45]. While some fungi produce FTase and FFase (fructofuranosidase), certain strains, such as Aspergillus japonicus and Aspergillus terreus, possess FFase exhibiting FTase activity. Notably, the activity of these fungal enzymes is dependent on the sucrose concentration. At low sucrose concentrations, FFase activity is predominantly hydrolytic, whereas at high concentrations, transfructosylation prevails [46,47,48].

FTases are primarily produced by fungi, notably within the genera Aureobasidium, Aspergillus, and Penicillium [49,50,51] (

Table 1. FTase-producing microorganisms.

Microorganism	Carbon Source	Enzymatic Activity	Yield/Conversion	References
Aureobasidium melanogenum (mutant D28)	Sugarcane molasses	2100.0 ± 61 U mL−1	0.58 g FOS g−1 molasses	[55]
Aureobasidium melanogenum 33	Sucrose	577.7 U mL−1	0.66 g FOSg−1 sucrose	[56]
Aspergillus niger ATCC 20611 (FV1-11CCFV1-7)	Sucrose	413.0 U g−1		[57]
		410.0 U g−1
Aspergillus niger ATCC 20611	Sucrose	310.0 U g−1
Aspergillus tamarii NKRC 1229	Sucrose	21.58 U g−1 intra 14.45 U g−1 mycelial	55.0%	[58]
Aspergillus tamarii Kita UCP 1229	Sucrose	1629.03 U gds−1		[59]
Aspergillus thermomutatus	Sucrose	6.5 U mg−1 protein—extra 0.5 U mg−1 protein—intra	62.5%	[60]
Aspergillus oryzae IPT 301	Sucrose	2100.0 U g−1		[61]
Penicillium citreonigrum URM 4459	Sucrose	22.05 U mg−1 370.67 µmol min−1		[62]
Aspergillus tubingensis XG21	Sugarcane molasses	558.3 U g−1	56.9%	[63]
Aspergillus oryzae S719	Wheat bran	155.4 U mL−1	586.0 ± 4.7 FOS L−1	[64]
Aspergillus niger sp. XOBP48	Sucrose	1.219.17 U mg−1 protein		[65]
Aureobasidium pullulans FRR 5284	Sugarcane molasses	23.6 U mL−1 (intra and extra)		[66]
Chrysonilia sitophila	Soybean meal Quinoa meal	3.98 U mL−1 (extra) 27.91 U mL−1 (intra)		[67]
Gliocladium virens	Orange peel Quinoa meal	0.32 U mL−1 (extra) 2.06 U mL−1 (intra)		[67]
Aspergillus oryzae DIA-MF	Aguamiel	1.347 U mL−1	9.06 g L−1	[68]
Aspergillus niger NRRL3	Carrot juice Carrot bagasse	90.82 U mL−1 244 ± 5.00 U mgprot		[10]
Penicillium citrinum	Banana peel Cane molasses	6.9 ± 0.15 U mL−1 min−1 7.3 ± 0.29 U mL−1 min−1		[42]
Aspergillus oryzae DIA-MF Aspergillus flavus	Aguamiel	1.59 U mL−1 0.27 U mL−1		[69]
Aspergillus welwitschiae FAW1	Artichoke/wheat bran/peptone	6.3 U mL−1		[17]
Aspergillus tamarii URM4634	Soybean bran and sucrose	26.10 U mL−1		[70]
Aspergillus labruscus ITAL 28.255	Rye flour and sucrose	1.81 U mL−1 (extra) 1.76 U mL−1 (intra)	150 mg mL−1	[71]
Aureobasidium pullulans ipe-3 KY618121	Sucrose	269.6 U/(g/L biomass)	123.2 g L−1	[72]
Aspergillus tamarii URM4634	Sucrose	50.90 U mL−1		[73]
A. tamarii URM4634	Soy bran	66.93 U mL−1		[74]

). Among filamentous fungi, Aspergillus oryzae IPT-301 has been highlighted for its high transfructosylation activity [52]. Regarding FOS production, Aspergillus remains the leading genus, followed by Aureobasidium. Within Aspergillus, strain A. niger ATCC20611 is recognized for its high enzymatic productivity, while strain AS0023 is notable for industrial-scale FTase production for FOS synthesis [53,54]. It is important to note that enzymatic activity varies considerably across strains and is influenced by the carbon source in the cultivation medium. Some fungi produce two types of FTases, one of which exhibits particularly high transfructosylation activity [3].

Studies on fructosyltransferase production have traditionally focused on optimizing their physical and biochemical parameters. However, these modifications have inherent limitations [75]. Currently, with the increase in knowledge in molecular biology/genetic engineering, it has proven to be a strategy for the search for the microorganism’s transformation to increase enzyme production and consequently FOS production, not only in fungal species transformation but also in the genes used for expression in species such as bacteria and yeast [3].

The Aspergillus niger ATCC20611 strain was subjected to protoplast transformation, generating the FopA (A178P) variant. This variant demonstrated constitutive expression for thermostable β-fructofuranosidase, along with a 58% increase in β-fructofuranosidase activity, leading to a reduction in FOS synthesis time [57,76]. Similarly, Ademakinwa, Ayinla, and Agboola [77] obtained a group of chemically mutagenized low-enzyme-producing wild-type A. pullulans NAC8 strains. The subsequent optimization of the process parameters resulted in a 6-fold increase in extracellular and a 2-fold increase in intracellular fructosyltransferase activity compared to the wild-type strain.

Cuervo Fernandez et al. screened 17 filamentous fungal strains for their ability to produce FTase for FOS production [52]. Based on mycelial and extracellular enzymatic activities, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611, and strain IPT-615 were identified as the most promising. These strains exhibited high total fructosyltransferase activity, exceeding 12,500 units L⁻¹, with a favorable ratio of fructosyltransferase to hydrolytic activity [52].

The intracellular nature of enzymes complicates the development and scale-up of many processes. Conversely, enhancing enzyme secretion or expression in naturally secreting organisms simplifies this process. Coetzee et al. overexpressed two codon-optimized variants of the β-fructofuranosidase (fopA) gene from Aspergillus fjiensis under the control of strong promoters (alcohol oxidase (AOX1) or glyceraldehyde-3-phosphate dehydrogenase (GAP) in Pichia pastoris [78]. When the variants were cultured in shake flasks under GAP promoter control, the volumetric enzymatic activities of 11.7 U mL⁻¹ and 12.7 U mL⁻¹ were obtained, while the AOX1 promoter yielded 95.8 U mL⁻¹ and 98.6 U mL⁻¹, respectively, showing no significant difference between the codon-optimized variants. However, bioreactor cultivation under AOX1 promoter control resulted in significantly higher enzymatic activities as follows: 13,702 U mL⁻¹ (ATUM) and 2718 U mL⁻¹ (GeneArt^®), compared to 6057 U mL⁻¹ (ATUM) and 1790 U mL⁻¹ (GeneArt^®) under GAP promoter control. These results demonstrate that optimizing extracellular enzymatic activity through this strategy directly facilitates FOS production by simplifying downstream processing.

A study with an Aureobasidium melanogenum strain demonstrated that disrupting the CREA gene and encoding a glucose repressor led to a substantial increase in β-fructofuranosidase (FFAse) production. The engineered strain achieved an enzymatic activity of 2100 U mL⁻¹, significantly higher than the 600 U mL⁻¹ observed in the parental strain. More specifically, the activity was 2100 U mL⁻¹ in the disrupted strain and 600 U mL⁻¹ in the parental strain. Using whole cells of the modified strain, the bioconversion of 350 g L⁻¹ molasses yielded 0.58 g FOS per gram of molasses sugar within 4 h [55]

Guilarte et al. reported the successful mutagenesis of Aspergillus oryzae IPT-301 using UV irradiation to enhance β-fructofuranosidase and fructosyltransferase activities. Mutants were selected based on their resistance to stress conditions, including high temperatures and SDS concentrations [79]. Seven mutants exhibited a 1.5- to 1.8-fold increase in mycelial enzymatic activity compared to the parental strain, with IPT-747 showing the highest extracellular enzyme activity, 1.5 times greater than the original strain. The study highlights the potential of UV mutagenesis for improving FOS production and enzyme yields for industrial applications.

The ultraviolet mutagenesis of Aspergillus oryzae ZT65, selected for fructosyltransferase overexpression and osmotic resistance, yielded the mutant A. oryzae S719. Using the extracellular crude enzyme from this mutant with 900 g L⁻¹ sucrose, FOS production reached 586.0 ± 4.7 g L⁻¹ (65% yield) in a 50 L bioreactor, achieving a productivity of 29.3 g L⁻¹ h⁻¹ over 20 h. This productivity doubled that obtained with 500 g L⁻¹ sucrose [72].

Mao et al. evaluated FOS synthesis using a novel fructosyltransferase (FT-A) from Aspergillus niger TCCC41686, where its cDNA was expressed in Pichia pastoris [80]. This FT-A demonstrated potential for industrial FOS production, achieving a yield of 67.05 ± 3.12% within 70 min, maintaining over 60% enzymatic activity between 50 and 80 min. Similarly, Yang et al. performed heterologous expression using A. niger YZ59 (CICIMF0901), obtaining approximately 57% yield (343.3 g L⁻¹) during 2 h [81].

Yeasts have been widely used in recent years due to their ease of manipulation and fermentation. Saccharomyces cerevisiae is a particularly strong candidate for gene expression due to its well-established genetic background, ease of handling, and fully sequenced genome [82].

Amorim et al. investigated expression in Saccharomyces cerevisiae, transforming the yeast to express the mutant FTase inv gene (L196) under the constitutive GPD promoter [83]. Galactose addition, induced gene expression, and enzyme production were verified via protein gel. A native 22-amino acid signal sequence facilitated enzyme secretion. The constitutive expression of the mutant FTase inv gene (L196) under the GPD promoter simplified the process and reduced the costs associated with induction, enhancing its suitability for industrial applications.

5. Production of Fructooligosaccharides

FOSs can be produced through the enzymatic transfructosylation of sucrose, a reaction that follows a ping-pong bi-bi mechanism. Initially, sucrose binds to the enzyme, releasing glucose and leaving fructose bound as an intermediate. Subsequently, a second sucrose molecule or an existing FOS molecule accepts the fructose moiety, yielding 1-kestose (GF₂), 1-nystose (GF₃), or 1F-β-fructofuranosylnystose (GF₄) [84,85].

During fermentation, the secreted enzymes can catalyze the transfructosylation of sucrose in the culture medium, producing FOSs. Nevertheless, this approach has been infrequently reported in the last five years. A more common method involves reacting sucrose with fructosyltransferase extracted from fermented broth or biomass under optimal pH, temperature, and concentration conditions. The use of immobilized enzymes is an effective and economical approach for large-scale FOS production [86].

5.1. Production of Fructooligosaccharides via Fermentation

FOS can be obtained by the following two main methods: submerged fermentation (SmF) and solid-state fermentation (SSF). Regardless of the chosen method, precise control over the culture medium’s composition and operational conditions (e.g., pH, temperature, agitation, aeration) is crucial for maximizing FOS production. The following sections discuss the recent advances in SmF and SSF for FOS production, focusing on the influence of key parameters on FOS productivity, yield, and concentration.

5.1.1. Submerged Fermentation

Liquid culture media must provide the nutrients necessary for microbial growth and metabolism. Synthetic fermentation media are chemically defined nutrient mixtures designed to support microbial fermentation processes with precision and consistency. These media are formulated with specific carbon sources (e.g., glucose or sucrose), nitrogen sources (e.g., ammonium salts or nitrates), essential vitamins, trace minerals, and other growth factors to meet the exact nutritional needs of the microorganisms. This precise formulation allows for reproducible results and enhanced metabolic control. Synthetic media also reduce contamination risks, simplify downstream purification, and enable the detailed monitoring of nutrient utilization and metabolic activities [87].

Sucrose is frequently used as a carbon source for FOS synthesis in shake flasks and bioreactors [60,88,89]. At low concentrations, sucrose primarily supports cell growth by providing energy. However, excessively high concentrations can lead to enzymatic inhibition [90,91]. This inhibition can be attributed to the formation of hydrogen bonds between sucrose molecules, which hinder their interaction with the enzyme’s active site. Higher sucrose concentrations also increase the likelihood of non-productive binding to enzyme subsites. When sucrose binds to these non-productive subsites, it prevents other sucrose molecules from accessing the productive subsites [51,92].

KO et al. conducted a fermentation to produce FOS in shake flasks and a bioreactor using sucrose concentrations of 100, 200, 300, and 400 g L⁻¹ [93]. The highest FOS productivities achieved in shake flasks and the bioreactor were 3.69 g L⁻¹ h⁻¹ (at 300 g L⁻¹ of sucrose) and 3.91 g L⁻¹ h⁻¹ (at 400 g L⁻¹ of sucrose), respectively. Cell growth inhibition was observed at 400 g L⁻¹ of sucrose in shake flasks.

Nitrogen is essential for the synthesis of proteins and other cellular components. Common organic and inorganic sources include yeast extract, peptone, urea, sodium nitrate (NaNO₃), and ammonium salts. While several studies combined yeast extract and NaNO₃ to produce high amounts of fungal FTase [49,94,95], Nobre’s group showed that the addition of yeast extract into the culture medium did not influence FOS production for Aureo-basidium pullulans CCY 27-1-94 [96], Aspergillus ibericus MUM 03.49 [89], and Penicillium citreonigrum URM 4459 [97] in shake flasks. In these studies, only NaNO₃ acted as a nitrogen source.

Potassium dihydrogen phosphate (KH₂PO₄) acts as both a phosphorous source and a pH buffer in culture media. During fermentation, the production of organic acids and ion release can cause pH fluctuations [87,98]. These pH changes can alter amino acid charges, affecting protein structure and potentially denaturing enzymes [99,100]. KH₂PO₄ concentrations are typically maintained below 5 g L⁻¹ to avoid inhibiting FTase activity [44,74]. Nevertheless, studies using Response Surface Methodology (RSM) by Nobre et al. demonstrated that for A. pullulans CCY 27-1-94, varying KH₂PO₄ concentration from 4.0 to 8.0 g L⁻¹ had no statistically significant effect on FOS concentration, yield, or productivity [96].

Microorganisms require specific metal ions for optimal growth and metabolic activity. These ions are crucial for various cellular processes, including osmoregulation, pH homeostasis, enzyme activation, protein synthesis, DNA replication, transcription, respiration-based energy generation, and stress responses [101,102,103]. While the impact of these ions on FOS production has not been extensively investigated recently, salts such as MgSO₄.7H₂O, KCl, MnCl₂, and FeSO₄.7H₂O are consistently included in synthetic culture media at low concentrations [20,50,89,97].

Although synthetic media offer certain advantages, they often fail to replicate the intricate nature of complex media, frequently resulting in lower FTase and FOS production [13,104,105]. A comparative analysis of FOS production in aguamiel and a modified Czapek-Dox medium showed that the FOS yield in aguamiel reached 20.30 g L⁻¹ within 24 h of fermentation, doubling the yield obtained in the synthetic medium. Khatun et al. developed a synthetic medium designed to closely mimic the chemical composition of sugar cane molasses, as previously determined [66]. Although they did not directly measure FOS production, their findings revealed higher intra- and extracellular transfructosylating activities in the agricultural medium, reaching 68.7 U mL⁻¹ and 54.9 U mL⁻¹, respectively.

Regarding operational conditions, the authors in [96] used an experimental design to maximize FOS production by A. ibericus. Varying temperature (25–35 °C) and pH (5.5–6.5) showed positive effects for both factors. The model predicted a maximum FOS yield of 0.56 at an optimal temperature of 37 °C and pH of 6.2. The same research group conducted a similar study with P. citreonigrum, investigating pH (4–6), temperature (25–35 °C), and fermentation time (36–60 h). Statistical analysis revealed pH had no significant effect, while fermentation time had a positive effect, and temperature had a negative effect. Elevated temperatures can denature enzymes by disrupting their three-dimensional structure. A subsequent experimental design, focusing on the significant factors, indicated optimal values of 28 °C and 61 h for temperature and fermentation time, respectively.

Prolonged fermentation can lead to the consumption of FOS as a carbon source. Muñiz-Márquez et al. observed a rapid decrease in FOS concentration after 48 h, coinciding with sucrose depletion [106]. After 72 h, FOS was totally depleted. These findings are consistent with Nobre et al., who reported a 61% reduction in FOS concentration between 24 and 60 h [88].

Controlling fermentation time is also necessary for managing FOS mixture composition. Initially, FTase generates 1-kestose, resulting in a higher concentration compared to other FOS molecules. However, GF₂ becomes more susceptible to transfructosylation, leading to its conversion to GF₃ or hydrolysis to sucrose [88,93,97]. While FOS mixtures rich in 1-kestose are more soluble and slightly sweeter, making them suitable for food applications, those with higher DP molecules (GF₃ and GF₄) provide more sustained prebiotic benefits due to their slower fermentation by gut bacteria. Nobre et al. showed that over a 24 h period (38–62 h), FOS composition shifted from 66% GF₂, 33% GF₃, and 1% GF₄ to 51% GF2, 46% GF₃, and 1% GF₄ [97]. This trend is consistent with other studies by this research group [88,97,107].

Combined aeration and agitation provide sufficient oxygen for aerobic microbial growth, reproduction, and metabolite secretion. Aeration supplies oxygen into the fermentation broth, while agitation enhances oxygen transfer by reducing bubble size, thereby increasing the gas–liquid interfacial area [13,108,109].

In shake flasks, agitation speeds of 150–200 rpm are typically used to achieve high FOS production [46,110,111]. Nevertheless, bioreactors require higher agitation speeds (150–900 rpm) and aeration rates (0.75–1.0 vvm) to overcome increased broth viscosity and mass transfer limitations at larger scales and higher biomass concentrations [89,93,96,97,112].

Maiorano et al. fermented A. oryzae IPT-301 in an 8 L bioreactor to investigate the effects of agitation speed (400 and 800 rpm) and aeration rate (0.5, 0.75, and 1.0 vvm) on biomass, extra- and intracellular At, and FOS production [61]. Their results showed that increasing the agitation speed boosted biomass production by up to 70%. Moreover, a higher agitation speed induced a morphological shift from mycelial to pellet form. This morphological change may explain the higher extracellular At levels observed at 800 rpm across all aeration rates, consistent with previous reports [112,113,114]. Aeration rates also influenced transfructosylating activity, with 0.75 vvm yielding the highest activity. Higher aeration rates reduced transfructosylating activity by approximately 35%, potentially due to oxygen inhibition. Consequently, the combination of 800 rpm agitation and 0.75 vvm aeration resulted in the highest FTase activities, corresponding to the highest FOS concentrations.

5.1.2. Solid-State Fermentation

SSF involves cultivating microorganisms on solid supports, which can be either inert or insoluble substrates that may serve as a source of energy and nutrients for microbial growth [115]. The absence or near absence of free water mimics the natural habitat where fungi are better adapted to grow, since their hyphal growth facilitates solid matrix colonization, and their good osmotic tolerance supports cellular integrity [10,116]. Aspergillus species and agricultural waste residues are the most employed for FOS or enzyme production [10,59,117,118]. Using a mixture of polyurethane foam (inert support) and the sap of Agave salmiana as solid substrate and A. oryzae BM-DIA, Michel et al. [69] produced 0.5 g L⁻¹ FOSs, comprising mainly of 1-kestose. Guerra et al. [10] used carrot bagasse as solid support for A. niger NRRL3 growth and enzyme production. They observed a transfructosylation activity of 10 U mL⁻¹ after 4 days of fermentation at 30 °C. Further substrate supplementation with nitrogen and metal traces increased the transfructosylation activity for 90 U mL⁻¹. Similarly, nitrogen supplementation in a solid substrate containing a mixture of artichoke and wheat bran promoted a 9-fold increase in the transfructosylation activity of Aspergillus welwitschiae [17].

In SSF, the proper adjustment of the moisture level is required during substrate preparation to avoid contamination and low diffusion of nutrients and oxygen [119]. The best level of moisture for the growing cells strongly depends on the type of substrate used for SSF. This occurs because, at the same moisture level, different amounts of water can be available to the metabolic functions of the microorganisms [120]. The calculation of the Water Absorption Index (WAI) and Critical Humidity Point (CHP) is a relevant method for assessing this information [121]. While the WAI indicates the amount of water absorbed by a support-substrate, the CHP provides the percentage of this absorbed water that is bound to the substrate and is unavailable for microbial metabolism. Therefore, substrates with a high WAI and low CHP are ideal for SSF, as they provide sufficient water for microbial growth without compromising substrate structure [120,121]. De La Rosa et al. [117] assessed the WAI and CHP in several agro-industrial wastes for FOS production. The highest WAI was exhibited for sugarcane bagasse (7.83 g gel g⁻¹ dry matter) and no statistical difference was observed for the CHP. As a result, SSF using sugarcane bagasse supplemented with aguamiel resulted in the highest FOS production of 7.64 g L⁻¹ after 12 h. For FOS production, moisture values typically range between 50% and 70% [69,116,118,122].

Achieving high FOS yields in SSF depends on the control and optimization of the operational parameters. In SSF, in addition to the parameters discussed for SmF, particle size is a critical factor. Controlling particle size is necessary to balance the surface area for microbial growth and the void space, facilitating oxygen and heat transfer [123].

De la Rosa et al. [117], using sugarcane bagasse and Aspergillus oryzae DIA-MF, maximized FOS production by optimizing particle size (un-sieved and 1 mm), moisture (50 and 70%), and spore concentration (2 × 10⁶ and 2 × 10⁷ spores g⁻¹). Statistical analysis showed that only the interactions between inoculum and particle size and between inoculum and moisture had a significant effect on FOS production. The highest FOS concentration (3.57 g L⁻¹) was obtained by treatment 7 (unspecified).

Batista et al. implemented an experimental design to investigate the effects of sucrose concentration (160–240 g L⁻¹), temperature (25–35 °C), spore concentration (10⁵–10⁷ spores mL⁻¹), moisture content (60–80%), and photoperiod (0–24 h) on FTase production by A. tamarii Kita UCP1279 [59]. The moisture content and photoperiod significantly affected FTase production, with higher levels favoring transfructosylation activity. Some fungal strains are sensitive to continuous light exposure. Simulating light and dark cycles can influence microbial growth and enzyme production by affecting exocytosis and potentially activating specific enzyme-encoding genes [69,118].

In contrast, Guerra et al. [10] reported that FFase production by A. niger NRRL3 was significantly influenced by inoculum size. The highest FFase production (120 U mL⁻¹) was observed with 1 × 10⁶ spores mL⁻¹. This was attributed to the intraspecific competition between fungal cells at high inoculum sizes and insufficient FFase production at lower inoculum sizes. Additionally, they evaluated the effect of incubation time on FFase production, which reached a peak of 117 U mL⁻¹ on day 4.

Despite these studies, research focusing on the optimization of parameters to maximize FOS production in SSF remains scarce. A deeper understanding of these parameters is essential for addressing SSF scale-up problems, such as gradients in temperature, pH, O₂, substrate availability, and moisture [124]. Additionally, a previous economic scale-up analysis demonstrated that SSF is the most cost-effective and environmentally friendly alternative compared to SmF using free or immobilized cells [119]. These advantages are likely due to the higher volumetric productivity, lower energy consumption, reduced contamination risks, and improved utilization of agro-industrial waste residues as a cost-effective feedstock of SSF [123].

5.2. Production of Fructooligosaccharides Using Whole Cells and Soluble FTase Extracts

After fermentation, FTase can be obtained from the fermented broth (soluble FTase) or the biomass (whole cells). To optimize FOS production, the enzyme’s operational (e.g., pH, temperature, and reaction time) and kinetic parameters are determined. Ojwach’s group outlined a detailed protocol for FOS production using this approach in three studies [20,66,125].

The following subsections detail how each operational parameter affects FOS production, categorized by enzyme source. First, it is necessary to clarify the terminology used in the literature to describe the enzyme.

FTase produced during fermentation can be intracellular (i-FTase), secreted into the culture medium (extracellular FTase or e-FTase), or bound to the microbial biomass (mycelial FTase or m-FTase).

Choukade and Kango introduced a methodology for isolating e-FTase, i-FTase, and m-FTase from A. tamarii NKRC 1229 [58]. They isolated e-FTase from the fermented broth using a precipitation technique. To obtain i-FTase and m-FTase, they initially ground mycelia under liquid nitrogen to lyse the cells. The resulting ground mycelia were resuspended in a buffer solution and centrifuged. The supernatant was collected as i-FTase, while the remaining biomass underwent further processing. This biomass was resuspended in the same buffer solution and sonicated to release m-FTase into the medium.

Nevertheless, other studies [126,127,128] have relied on biomass sonication to lyse cells and extract the enzyme, referring to the extract as i-FTase.

Furthermore, using biomass as a biocatalyst presents challenges. While it contains both i-FTase and m-FTase, the specific contribution of intracellular proteins to FOS production remains unclear. Consequently, the terms “whole cells”, “m-FTase,” and even “i-FTase” are often used interchangeably to describe this biocatalyst [3,58,89,129,130].

Therefore, due to this lack of consensus in the literature, we use the term “whole cells” when mycelia are used as a biocatalyst and “soluble FTase” when e-FTase, i-Ftase, or m-FTase are used as soluble enzymes.

5.2.1. Soluble FTase

Fungal FTases optimally produced FOS at 50–55 °C and pH 5.5–6.0 [80,99,129,131]. A notable exception is the FTase from A. oryzae DIA-MF, which produced 31.01 ± 3.42 g L⁻¹ FOS at 30 °C, a 6-fold increase compared to the yield observed at 50 °C [132].

For industrial FOS production, a broad enzyme thermal stability profile is desirable. Higher temperatures can mitigate contamination risk, reduce viscosity, and enhance substrate solubility. Conversely, lower temperatures can decrease storage and transport costs [133].

A. oryzae S719 FTase retained about 89% of the activity after 2 h of incubation at 55 °C and about 50% after 12 h. At 25 °C, it maintained approximately 70% of its activity for 24 h [126]. FTase from A. oryzae IPT-301 retained up to 96% of its activity at temperatures below 35 °C for 1 h. However, a sharp activity decrease was observed at higher temperatures, with only 44.26% of the initial activity remaining at 65 °C [129].

A. niger strains have demonstrated superior thermostability. The TCCC41686 strain showed no loss of initial activity for 1 h of incubation at 40 and 50 °C and retained over 80% of its residual activity after 24 h at the same temperatures [80]. Similarly, the MH445969 strain retained nearly 90% of its residual activity between 40 and 60 °C after 6 h of incubation [20].

These A. niger strains also exhibited enhanced pH stability compared to A. oryzae strains. The FTase from A. niger TCC41686 retained more than 90% of its initial activity after 24 h of incubation across a pH range from 4.0 to 11.0 [80]. The enzyme from A. niger MH445969 was stable between pH 4.0 and 10.0 for 6 h, retaining approximately 98% of its residual activity [20]. In contrast, while A. oryzae S719 retained more than 90% of its initial activity at pH 4.0–11.0 for 2 h, A. oryzae IPT-301 showed a rapid decline in residual activity at pH values above or below 6.0 [49,129].

Metal ions interact with amine and carboxylic acid groups in proteins, leading to various effects, including enhanced stability, altered enzymatic activity, and the potential disruption of protein function [134,135].

Studies on fungal FTase have examined the effects of monovalent, divalent, and trivalent cations. Notably, Mg²⁺ and K⁺ at concentrations of 5 mM or 10 mM consistently enhanced catalytic activity, whereas heavy metal ions such as Hg²⁺, Ag⁺, and Pb²⁺ caused enzyme denaturation at all tested concentrations [20,80,131]. The effects of Cu²⁺ and Fe²⁺ varied, ranging from enzyme denaturation to increased activity [20,131].

The ion Ca²⁺ at 10 mM increased the relative activity of A. niger MH445969 FTase by 7.72% [20]. Ca²⁺ is also commonly included at 1 mM in sucrose solutions to enhance FOS production by bacterial FTases [128,136]. Recent mutational analyses of conserved calcium-binding residues in bacterial FTase have further demonstrated the role of Ca²⁺ in enzyme stability, activity, and product profile [69].

Mao et al. investigated the effect of varying A. niger TCCC41686 FTase concentrations on FOS production under optimized pH, temperature, and sucrose conditions [80]. Increasing the FTase concentration from 40 to 400 U increased FOS yield from 60% to 80%. The highest FTase concentration (400 U) also accelerated FOS production, achieving maximum yield within 30 min. Han et al. reported a maximum FOS yield of 64% after a 4 h reaction using 12 U of purified A. oryzae S719 FTase [131]. They noted that this represented the fastest reported FOS production time using non-recombinant FTase. Rapid product recovery is economically advantageous due to reduced energy and material costs.

Kinetic analysis provides crucial insights into enzyme–substrate interactions and the rates of enzyme-catalyzed reactions. The Michaelis–Menten model is frequently applied to kinetic studies of soluble FTases from fungi and bacteria. In this model, Km represents the substrate concentration at which the reaction velocity is half-maximal, while Vmax denotes the maximum reaction rate under substrate saturation. Reported Km values for FTases from various microorganisms include 442 ± 9 mM for A. niger TCCC41686 [80], 350 ± 130 mM for L. reuteri 121 [136], and 310 mM for A. oryzae S719 [131]. The enzymes from A. oryzae IPT-301 [129] and A. niger MH445969 [21] exhibited higher affinities for sucrose, with Km values of 146 mM and 79.51 mM, respectively. Among these microorganisms, A. niger TCCC41686 presented the highest Vmax of 6.55 g L⁻¹ min⁻¹ [81].

Catalytic efficiency, measured by the ratio of the turnover number (kcat) to Km, is another frequently reported kinetic parameter. It reflects the enzyme’s ability to efficiently convert a specific substrate, in this case, sucrose into FOS. A high catalytic efficiency indicates that the enzyme can effectively produce FOSs even at low sucrose concentrations.

The FTases from L. reuteri 121 and A. niger TCCC41686 demonstrated catalytic efficiencies of 2.2 mM⁻¹ s⁻¹ and 6.8 mM⁻¹ s⁻¹, respectively [80,126]. These values were higher than the 0.11 mM⁻¹ s⁻¹ reported for the FTase from A. oryzae S719 [131]. This disparity likely explains the need for higher sucrose concentrations (500 g L⁻¹) in FOS production using A. oryzae S719, as compared to L. reuteri 121 and A. niger TCCC41686 (~150 g L⁻¹).

5.2.2. Whole Cells

The whole-cell immobilization of enzymes offers advantages over using soluble enzymes. The cellular environment provides protection, enhancing enzyme stability and reducing operational costs by simplifying downstream processing and enabling cell recycling. For example, Zhang et al. used Y. lipolytica cells to produce 185 g L⁻¹ of FOS in a batch process. These whole cells were recovered by centrifugation and reused for nine consecutive batches with no significant decrease in FOS production [128]. Furthermore, after 60 days of storage at 4 °C, the cells retained their enzymatic activity, yielding 183.4 g L⁻¹ of FOS.

In contrast, Garcia et al. reported a 50% reduction in the transfructosylation activity of whole cells from A. oryzae IPT-301 after ten successive one-hour cycles [137].

Several studies have focused on optimizing operational parameters for FOS production using whole cells. Khatun et al. compared wet and freeze-dried cells of Aureobasidium pullulans FRR 5284 [130]. They observed higher FOS yields with freeze-dried cells, possibly due to partial cell disruption or enzyme permeabilization during the drying process. The increased mass of freeze-dried cells required to match the wet cell mass may have also contributed to higher FTase levels, thus enhancing FOS synthesis. Further characterization of the dry cells identified the optimal conditions for FOS production as follows: 55 °C, pH 5.5, 500 g L⁻¹ of sucrose, and 5.0 g L⁻¹ cell loading. Under these conditions, the productivity and yield (based on initial sucrose) were 102 g L⁻¹ h⁻¹ and 61.2%, respectively.

Using whole cells of A. tamarii NKRC 1229, Choukade and Kango determined the optimal conditions of 20 °C, pH 7.0, 50% w v⁻¹ sucrose, 4.5% w v⁻¹ mycelial dosage, 48 h mycelial age, and 24 h incubation time, achieving 325 g L⁻¹ of FOS with a 0.55 yield [59]. A subsequent optimization study by the same authors [138] identified 28.4 °C, pH 7.0, and 50% (w v⁻¹) sucrose concentration as the optimal conditions.

Garcia et al. reported the optimal conditions for enzyme productivity by A. oryzae IPT-301 in batch culture to be 480.2 g L⁻¹ of sucrose, pH 5.5, and 50 °C [137]. Dias et al. subsequently evaluated the FTase activity of these whole cells in a packed bed reactor containing biocatalyst spheres with a diameter of 4.0 ± 0.2 mm [49]. Under comparable operational conditions to the batch process, enzyme productivity increased to 522 U g⁻¹ min⁻¹, significantly higher than the 12 U g⁻¹ min⁻¹ observed in batch mode [137]. This result demonstrates the advantages of continuous FOS production.

Metal ions and organic solvents can induce conformational modifications in enzyme active sites, thereby affecting catalytic activity. Choukade and Kango studied these effects on FTase activity of A. tamarii NKRC 1229 [58]. Among the metal ions tested, only the Cu²⁺ enhanced FTase activity, increasing it by 2.74%. In contrast, most solvents boosted catalytic activity, with chloroform, hexane, and ethyl acetate showing increases exceeding 10%.

To enhance FOS production from molasses, Khatun et al. pretreated the substrate with invertase-free Saccharomyces cerevisae cells to remove glucose, an FTase inhibitor [130]. Untreated molasses contained 23% (w w⁻¹) of sucrose, 3.6% (w w⁻¹) of glucose, and 3.4% (w w⁻¹) of fructose. After 12 h of treatment, glucose was completely consumed, with sucrose remaining stable. A slight decrease in fructose to 1.9% was observed. Consequently, the treated molasses improved FOS yield from 44.1% ± 0.1% to 56.5% ± 0.2% after 1 h and from 55.6% ± 0.3% to 60.0% ± 0.9% after 6 h.

6. Recovery and Purification of Fructooligosaccharides

6.1. Separation and Purification of Saccharides for FOS Production

Microbial fermentation is the primary method for large-scale FOS production. However, even under optimized conditions and using high-yield microbial strains, this process generates by-products or co-products, especially glucose and fructose, alongside non-hydrolyzed sucrose substrate.

Quantifiable amounts of other sugars have also been detected in commercial FOS products. For instance, one commercial mixture contained 5.25 g L⁻¹ of fructose and 28.7 g L⁻¹ of two unidentified hexoses, while another contained 12.23 g L⁻¹ of glucose, fructose, and sucrose [97]. This highlights the need for effective separation techniques to isolate FOS molecules and other medium components, particularly other sugars. Adsorption and tangential flow filtration (membrane filtration) are two prominent separation/purification methods described in the literature.

6.1.1. FOS Purification by Adsorption

Adsorption describes the concentration increase in compounds (adsorbate) from a fluid phase, onto the surfaces of a solid (adsorbent). This enrichment results from physical and/or chemical interactions with the adsorbent, which possesses specific electronic and steric properties. Notably, the internal porous surface of many adsorbents significantly contributes to adsorption, in addition to the external surface [139]. The three main adsorbents used in the separation and purification of FOS are activated carbon, cation exchange resins, and zeolites.

Sugar adsorption on activated carbon is a reversible process driven by van der Walls forces. Activated carbon is predominantly hydrophobic. The hydrophobicity of sugars correlates with the number of CH groups in their molecules, thus depending on carbon chain length, and consequently, molar mass. Due to their higher molar mass compared to monosaccharides and disaccharides such as glucose, fructose, and sucrose, FOSs exhibit a greater adsorption affinity [140,141].

Some studies have reported using activated carbon for FOS separation and purification. For example, one study investigated the separation of saccharides glucose, fructose, sucrose, and FOS from an enzymatically synthesized sugar mixture using activated carbon as the adsorbent and water–ethanol mixtures of varying concentrations as eluents. A fixed-bed column effectively separated FOS from glucose and FOS from fructose, obtaining optimal separation with an ethanol eluent at 50 °C. This process yielded approximately 80% FOS purification and 97.8% recovery [142].

A fixed-bed column with activated charcoal, using ethanol/water gradients as eluent, has been employed to separate FOS from mixtures with glucose, fructose, and sucrose derived from enzymatic synthesis. Using 15% (v v⁻¹) ethanol at 40 °C yielded a separation efficiency of 2.42, a FOS purity of 91%, and a recovery of 63.3%. Decreasing the ethanol concentration improved the separation between glucose and FOS, but this reduced FOS purity due to co-elution with sucrose [143].

In another study, a real fermentation broth was percolated through an activated charcoal fixed-bed column at 25 °C and a flow rate of 25 mL min⁻¹. Fructose and glucose exhibited the lowest adsorption, with 53.2% and 62.5% (w w⁻¹) of the initial amounts, respectively, remaining in the eluent. In contrast, 83.9%, 86.9%, 90.4%, and 95.1% of sucrose, ketose, nystose, and fructofuranosylnystose, respectively, were adsorbed. Sodium, potassium, and magnesium salts were also adsorbed, resulting in a salt-free sugar mixture with 92% FOS purity after ethanol desorption [144].

Although ion exchange between the adsorbate and solute is one of the main mechanisms in separation using ion exchange resins, sugars are electronically neutral. Therefore, their separation relies on complexation and size exclusion [145,146]. The cross-linking density of ion exchange resins affects the extent and type of sugar sorption and is particularly important for separating monosaccharides from oligosaccharides. At an optimal cross-linking density, oligosaccharides are excluded while monosaccharides are adsorbed, delaying their elution. On the other hand, excessive cross-linking can hinder monosaccharide adsorption due to steric hindrance, thus impairing separation [146,147]. Complexation between sugars and cations is facilitated by contiguous hydroxyl groups in an “a, e, a” configuration, where the near-equidistant oxygen atoms promote complex formation [147].

Batch adsorption studies at 60 °C investigated glucose, fructose, sucrose, and FOS adsorption from aqueous solutions onto four cation exchange resins with a poly(styrene-co-divinylbenzene) matrix functionalized with cationic groups. Adsorption equilibrium exhibited the linear behavior for both solid- and liquid-phase concentrations. Distribution coefficients indicated the following adsorption capacity order: fructose > glucose > sucrose > ketose > nystose > fructofuranosylnystose. The authors concluded that resins showed higher selectivity for FOS separation; individual FOS isotherms were unaffected by the presence of other saccharides; and size exclusion was the primary factor influencing saccharide partitioning [146].

The separation of sugars from demineralized sucrose fermentation broth to produce FOS was evaluated using eight cation exchange resins in a fixed-bed preparative column. The highest retention factors were observed for resins in the K⁺ form, followed by the Na⁺ and H⁺ forms. The Li⁺ form presented the lowest selectivity for all sugars present. Resins in the Ca²⁺ and Mg²⁺ forms displayed selectivity dependent on the specific sugar pairs being separated. The Ca²⁺ form was more effective for separating sugar pairs with higher molar mass, such as sucrose–nystose and kestose–nystose, while the Mg²⁺ form was more suitable for fructose–glucose, glucose–sucrose, and glucose–kestose separation. Selectivity for the sucrose–kestose and glucose–nystose pairs was similar for both the Ca²⁺ and Mg²⁺ forms. The authors concluded that, in addition to size exclusion and complexation, the solvation of the cations plays a role in the separation mechanism [148].

Zeolites are materials with a primary structure based on interconnected silicon and aluminum tetrahedra, forming secondary polyhedral units linked by oxygen atoms. This three-dimensional crystalline network comprises these secondary units, forming cages that are connected by channels that permeate the structure. The channel size is determined by the number of interconnected silicon and aluminum atoms and the counterion associated with the negatively charged aluminum [139]. Component separation in zeolites is governed by both electrostatic interactions (ion-dipole and van der Waals forces) as mass transfer processes, including diffusion and size exclusion [149,150].

A study on the batch adsorption of model solutions (150 g L⁻¹) containing fructose, glucose, sucrose, and FOS in synthetic faujasite type zeolites with various cationic forms, Na⁺, Ca⁺², Ba⁺², Sr²⁺, K⁺, and Mg²⁺, reported the following adsorption sequences relative to glucose: NH⁴⁺ < Mg²⁺ < Ca²⁺ < Na⁺; fructose: NH⁴⁺ < Mg²⁺ < Na⁺ < Ca²⁺; and sucrose: K⁺ < Na⁺ < Sr²⁺ < Ba²⁺ < Ca²⁺ < Mg²⁺. FOS adsorption was generally low across most cationic forms. NaX zeolite exhibited the lowest mass transfer resistance, which resulted in higher adsorption rates [151].

Research on the separation and purification of enzymatically synthesized FOS using NaX zeolite fixed-bed columns (particle diameter 0.25–0.40 mm) and ethanol-water mixtures as eluents demonstrated that higher ethanol concentrations (around 60% (v v⁻¹)) and temperatures (40–45 °C) favored the separation of glucose and FOS, the most abundant sugars in the mixture, achieving separation efficiencies between 5.7 and 6.9 and FOS purities of 90%. On the other hand, the separation of sucrose and FOS was less effective, reaching a maximum separation efficiency of 3.69 with 83% FOS purity, and this was negatively impacted by increasing the ethanol concentration in the eluent [152].

A comparative study of fixed-bed column separation using Y-type zeolites and activated carbons revealed that the adsorption affinity order for saccharides was glucose > fructose > sucrose > FOS for both adsorbents. High ethanol concentrations favored sugar separation with zeolite. With activated carbon, ethanol concentration gradients from 8.0% to 20.0% (v v⁻¹) improved separation, with extraction yields following the order FOS > sucrose > fructose > glucose from low to high ethanol concentrations. High sugar concentrations hindered the separation of all saccharides when using activated carbon [153].

6.1.2. Membrane Technology and FOS Purification

Synthetic membranes used in tangential flow filtration, driven by transmembrane pressure, act as interfaces between two fluid streams, selectively permeating components (solutes) from the feed stream. The separation and purification achieved by the membranes depend on several factors, as follows: operating conditions; the physical and chemical properties of the solutes; the solvent; membrane characteristics (material, pore size, and structure, among others); and the interactions between solute, solvent, and membrane [154,155].

The properties of the saccharides are modulated by their constituent monosaccharides, intramolecular bonds, degree of polymerization, branching, and molar mass. These factors, in turn, determine the physical and chemical properties of the saccharide molecules and their solutions. The hydrophilicity/hydrophobicity of saccharides, determined by the number and spatial arrangement of OH groups, affects their solubility. This solubility, combined with molecular structural parameters, directly impacts membrane separation [156,157].

Membrane technology is a promising approach for oligosaccharide purification, offering several advantages over other separation processes. These advantages include the following: relatively low energy demand; ease of adjusting operating parameters (e.g., transmembrane pressure, temperature, and feed flow rate); scalability; a wide selection of membrane materials; compatibility with continuous and batch operation; removal of various impurities from fermentation broth to obtain high product purity; and adaptability based on differences in saccharide molecular structures. Consequently, numerous studies have investigated oligosaccharide purification, particularly FOS, using tangential flow filtration.

Studies have investigated membrane-based processes for FOS separation. One study examined diafiltration using zirconia ceramic membranes with molecular weight cutoffs of 600, 700, and 850 Da to separate FOS from a model sugar mixture (100 g L⁻¹ FOS). Optimal transmembrane pressure, linear flow, and temperature were determined to be 0.7 MPa, 1.2 m s⁻¹, and 60 °C, respectively. The 600 Da membrane exhibited the highest FOS rejection. Interestingly, the 850 Da membrane showed slightly higher FOS rejection than the 700 Da membrane. The authors concluded that this process effectively purifies FOS, yielding high concentrations, recovery, and purity, while maintaining permeability and separation [158].

Another study explored a serial nanofiltration sequence for concentrating FOS from a syrup containing residual fructose, glucose, and sucrose. Using membranes with molecular weight cutoffs of 1000, 2500, and 3500 Da, at 50 °C and a pressure of 8 or 12 bar, the process was modeled and validated. Optimized configurations increased the purity of monosaccharides, trisaccharides (containing three fructose units), and saccharides with five or more fructose units from 9%, 24%, and 34% to 47%, 34%, and 77%, respectively [159].

An integrated ultrafiltration–diafiltration–concentration process was developed for FOS production from enzymatically catalyzed sucrose. This approach achieved FOS purity exceeding 90%. Ultrafiltration retained and recovered 95.5% of the fructosyltransferase enzyme. Diafiltration with a PES10 membrane increased FOS purity from around 50% to 92.3%, while DL membranes recovered FOS and sucrose from the diafiltration permeate. This integrated approach reportedly reduced FOS purification costs by 68.2% compared to non-integrated methods [160].

In summary, several key factors influence FOS separation by membranes, as follows: nanofiltration membranes can retain FOS, while diafiltration removes ions, monosaccharides, and non-functional disaccharides. Separation efficiency depends on membrane material, pore size distribution, operating conditions, and feed composition and concentration. Membrane fouling, a complex phenomenon, requires further investigation for this application [161].

7. Global Market and Techno-Economic Analysis of FOSs

7.1. FOS Global Market

The global FOS market has been on a strong growth trajectory and is on track for further expansion. Valued at approximately USD 2.82 billion in 2023, the market is expected to reach USD 2.95 billion by 2024 [162] and USD 7.50 billion by 2032 [163]. The growth is attributed to the increasing demand for natural sweeteners and dietary fibers, driven by factors such as rising consumer interest in prebiotic ingredients, their usage in infant formulas and functional foods, and their associated digestive health benefits [162,163]. In 2023, Europe [164,165] and North America [165,166] held the highest share of the global FOS market.

The liquid form of FOS dominates the market, accounting for 54.2% of the revenue. Its widespread use stems from its syrupy consistency, which confers high water solubility, resulting in homogeneous solutions readily incorporated into diverse food and beverage products [163]. The FOS market is competitive, with several key players. Major companies operating in this market include AIDP; Beghin-Meiji SA (Marckolsheim, France); Baolingbao Biology Co. (Yucheng, China); Beneo GmbH (Mannheim, Germany); Cargill Incorporated (Wayzata, MN, USA); Cosucra Co. (Warcoing, Belgium); Galam (Kibbutz Maanit, Israel); GTC Nutrition (Golden, CO, USA); Ingredion Incorporated (Westchester, IL, USA); Jarrow Formulas (Los Angeles, CA, USA); Meiji Holdings Co., Ltd. (Tokyo, Japan); Nissin Sugar Co., Ltd. (Tokyo, Japan); Quantum Hi-Tech (Guangzhou, China); Biological Co., Ltd. (Guangzhou, China); Royal FrieslandCampina NV (Amersfoort, Netherlands); Sensus (Roosendaal, Netherlands); Tate & Lyle PLC Ltd. (London, United Kingdom); Tereos Group (Moussy-le-Vieux, France); and Yakult Pharmaceutical Industry Co., Ltd. (Tokyo, Japan) [162,163].

FOS pricing varies depending on production costs, purity levels (due to the additional processing required for higher purity), raw material availability, and market demand [162,165]. Retail prices for consumer products containing FOSs may also vary by brand, grade, and packaging.

Table 2. Price of FOS in the market.

Value	Observation/Information	Reference
USD 3.0/kg to USD 4.0/kg	Standard Purity FOS (50–70% Purity) Applications: Used in general food and beverage products as fiber or low-calorie sweetener where ultrapure is not critical. Characteristics: Contains a blend of FOS with other sugars such as glucose and sucrose.	[165]
USD 4.5/kg to USD 6.0/kg	High Purity FOS (70–90% Purity) Applications: Dietary supplements, functional foods, and products requiring higher concentrations of prebiotic activity. Characteristics: Enhanced prebiotic benefits due to reduced levels of monosaccharides and disaccharides.	[165]
USD 6.5/kg to USD 8.0/kg	Ultra-High Purity FOS (Above 90% Purity) Applications: Essential for infant formulas, pharmaceutical products, and specialty nutraceuticals where maximum efficacy and minimum contamination are critical. Characteristics: High concentration of FOS with minimal (or no) presence of other sugars, ensuring optimal prebiotic functionality.	[165]
USD 10.90/kg	Brand Tiens, Packet size of 1 kg.	[166]
USD 15/kg	Sample price of the supplier Kap (xiamen) Bio-Tech Co., Ltd.	[167]
USD 44/kg	FOS powder, brand SaporePuro, packet size of 250 g.	[168]
USD 2.61/kg	Selling price considered for FOS powder, 90% purity.	[169]
USD 3.20/kg	Selling price considered for FOS powder, 95% purity.	[170]
USD 0.47/kg	Selling price considered for 75% Brix FOS syrup.	[170]
USD 5.00/kg	Selling price considered for FOS powder, 95% purity.	[171]
USD 5.00/kg	Selling price considered for FOS powder, 97% purity.	[172]
USD 1057.00/kg a	Selling price considered for FOS ≥ 97% purity.	[173]
Marketplace value	There are several suppliers, each offering a different specified product.	[174]
USD 3.50/kg to USD 30.00/kg

^a Adopted exchange rate of USD 1.08 per euro.

shows some examples of FOS prices.

Prices of FOS vary considerably (Table 2), primarily due to product purity, which directly impacts production costs. Higher purity requires more complex purification processes and adherence to stricter quality standards. This translates to higher selling prices for several reasons as follows: enhanced efficacy (a higher concentration of active compounds provides more effective prebiotic benefits for intestinal health), improved safety (removing impurities reduces the risk of adverse reactions, particularly in sensitive applications such as infant formulas), and compliance with stringent regulatory requirements for ingredient purity in certain industries [175]. Furthermore, a positive correlation between the price and quality is generally observed in the market. Consequently, consumers often perceive higher-priced FOS as superior in quality and are willing to pay a premium [176]. Manufacturers, therefore, balance production costs, selling prices, and functional and regulatory requirements to determine the appropriate purity level based on specific product needs and market demand [177]. In addition to variations in purity, market price fluctuations often reflect variations in the source material, production methods, and additional quality parameters such as moisture content, degree of polymerization, and the presence of residual mono- and disaccharides. As shown in Table 2, FOS can also be found in the marketplace from multiple sources with a wide range of prices and qualities. Higher-priced FOS formulations tend to offer greater purity, enhanced functional properties and stricter quality control measures, while lower-cost options may contain a wider range of saccharide compositions and fewer processing refinements.

FOS production faces challenges due to the high costs associated with its extraction, processing, and purification. To maintain market growth, research and development efforts continue to focus on improving yields and enhancing cost-effectiveness [163,165].

7.2. Techno-Economic Analysis of FOS Production

FOS can be produced from various plant materials, such as sugarcane [172], sugar beet [174], bagasse, molasses [13], and fruit peels [13,178]. These feedstocks are also commonly used in large-scale biofuel production. Consequently, integrating FOS production into existing biomass biofuel facilities has been frequently proposed in the literature [13,170,171,179]. Although FOS is produced in smaller quantities compared to biofuels, its higher market price (Table 2) presents profitable opportunities for integrated biorefineries. Co-producing FOS with high-volume, low-cost products like ethanol and energy can optimize resource utilization, reducing waste, and improve overall economic performance [13,170,171]. This approach promotes sustainability by maximizing value extraction from agricultural feedstocks while addressing the growing demand for both bioenergy and health-oriented ingredients [13].

Amanful et al. assessed the feasibility of producing low-calorie sweeteners including short-chain fructooligosaccharides (scFOSs) in a sugarcane-mill-annexed biorefinery utilizing A-molasses as feedstock [170]. Aspen Plus^® simulations indicated positive economic performance, with an internal rate of return (IRR) of 46% in the African context and a minimum selling price of USD 1540/ton of scFOS in the single-product biorefinery. This minimum selling price was lower than the market price assumed in the study (USD 2610/ton). The authors considered a 90% purity, powdered scFOS with a production capacity of 39,000 tons/year, using A-molasses at USD 194/ton. However, this production capacity would theoretically meet 95% of global demand, suggesting potential market oversupply. This scenario may not be realistic due to economies of scale and the market’s limited absorption capacity. In addition, such a large-scale production could significantly impact FOS prices due to supply and demand dynamics.

Vacharanukrauh et al. [180] performed a techno-economic comparison of different reactor types for FOS production from sucrose. The processes included chitosan bead production, enzyme immobilization, and the production of 2000 m³/year of levan-type FOS (L-FOS). The final product was sugar syrup containing 15.2% (w v⁻¹) L-FOS, which exhibits a higher bifidogenic effect than commercially available inulin-type FOS. The estimated minimum selling price ranged from USD 0.847/L to USD 1.234/L. Reactors using immobilized enzymes demonstrated greater economic advantages than those using free enzymes, as the cost savings from reduced enzyme purchases offset the additional equipment and chemical costs associated with enzyme immobilization. However, comparing the minimum selling price of the FOS produced in this study with the market selling price of commercial FOS is potentially misleading. Vacharanukrauh et al. considered a 15.2% (w v⁻¹) FOS syrup, thus omitting the capital and operating costs associated with FOS purification, which may underestimate the overall production costs [180].

Klaver et al. compared the economic viability of small-scale, decentralized biorefineries producing scFOS and/or ethanol with that of large-scale, centralized scFOS production integrated with existing sugarcane mills [179]. Using a 20% discount rate, they estimated the minimum selling prices for scFOSs of USD 1.0/kg and USD 0.42/kg for small- and large-scale scenarios, respectively. These values are lower than the assumed market price of USD 5.0/kg, indicating potential economic feasibility for scFOS production.

Dogbe et al. developed Aspen Plus simulations and conducted an economic assessment of various biorefinery scenarios within South African sugar mills, focusing on the production of succinic acid and scFOS from A-molasses [171]. The authors assessed the integration of these processes into existing sugarcane biorefineries. All evaluated scenarios demonstrated positive economic performance, with IRR ranging from 24.1% to 61.1% exceeding the required 9.7% discount rate. The authors determined the average selling prices of USD 226/ton for scFOS syrup and USD 1283/ton for scFOS powder, both lower than the market prices they used for comparison (USD 471/ton for scFOS syrup and USD 2610/ton for scFOS powder). The positive economic performance can be partly attributed to economies of scale, as the proposed plant capacity (31,000 tons/year) could supply 75% of the current global market. Furthermore, shared infrastructure with the sugar mill (including a shared cogeneration system and the use of A-molasses instead of crystalline sucrose) contributes to reduced capital investment and operating costs.

Bedzo et al. performed a comparative techno-economic analysis of three methods for producing scFOSs (powder and syrup) at a 2000 tons/year scale via enzymatic sucrose synthesis [172]. The objective was to assess the economic potential of immobilized enzyme systems relative to a free enzyme system. The systems compared were free enzyme (FE), calcium alginate immobilized enzyme (CAIE), and amberlite IRA 900 immobilized enzyme (AIE) systems. The economic analysis showed that all three systems were economically viable, with the minimum selling prices ranging from USD 1.82/kg to USD 3.10/kg, well below the target scFOS price of USD 5/kg. The FE system proved the most profitable, exhibiting the lowest minimum selling price (1.82/kg). This was attributed to the fact that cost savings from enzyme immobilization could not offset the additional immobilization-related expenses.

Mussatto et al. evaluated and compared three different fermentation processes for FOS production, considering both economic and environmental aspects [122]. The processes were as follows: submerged fermentation of sucrose solution by Aspergillus japonicus using free cells (FCF); submerged fermentation using cells immobilized in corn cobs (ICF); and solid-state fermentation (SSF) using coffee silverskin as a support material and nutrient source. The authors reported positive economic performance for the three scenarios, with IRRs of 9.61%, 11.64%, and 33.36% for FCF, ICF, and SSF, respectively. SSF was identified as the most economically attractive process due to its higher annual FOS productivity (232.6 tons) and purity (98.6%) compared to the other processes, resulting in the highest annual profit (EUR 6.55 million) and the shortest payback time (2.27 years).

Economic analyses frequently explore co-production within biorefineries, aiming to enhance overall profitability by producing FOS alongside other biobased products. The market price of FOS offers substantial economic potential for such facilities. Furthermore, incorporating this sweetener into product portfolios can contribute to the long-term sustainability and profitability of biobased industries. These analyses also serve to identify challenges in FOS production, highlighting the key process variables and guiding R&D efforts.

Balancing the production of high-value, low-volume products like FOS with low-cost, high-volume commodities such as biofuels, ethanol, and bulk sugars is crucial for economic viability. This strategy allows biorefineries to capitalize on both premium markets and large-scale industrial demand. Therefore, the integration of FOS production within biorefineries exemplifies the evolving role of biotechnology in creating diverse value-added products. Consequently, these integrated biorefineries represent a potentially viable strategy for revitalizing the sugar industry.

8. Conclusions

Research on FOS emerged in the 1980s, with ninety-three countries/regions contributing to FOS publications between 1983 and 2024. Publication output concerning FOSs’ functional properties, production, purification, and applications increased by over 87% from 2012 to 2022. Brazil leads in contributions, followed by China and the United States. Fermentation studies for FOS production employ solid-state or submerged methods, with solid-state fermentation demonstrating superior performance, exhibiting higher productivity and product stability. Aureobasidium sp. and Aspergillus sp. are the most extensively studied microorganisms for FOS production, achieving higher transfructosylation activities and FOS conversion. Activated carbon has shown better results than ion exchange resins and zeolites for FOS separation and purification via adsorption. However, membrane filtration is a more promising method due to its relatively low energy demand, ease of operating parameter adjustment, scalability, compatibility with continuous and batch operations, wide variety of membrane materials, and simultaneous removal of various impurities from the fermentation broth, yielding high product purity. Large-scale FOS production studies have demonstrated positive economic performance. Integrating FOS production with sugarcane biorefineries, utilizing free enzymes compared to calcium alginate and amberlite immobilized enzymes, and employing solid-state fermentation using coffee silverskin compared to submerged fermentation using cells immobilized in corn cobs or sucrose solution, have been indicated to be more economically attractive processes.