Microbial FTases produced by filamentous fungi, its activity, and fermentation temperature of the maximum enzyme activity reached.
Fructooligosaccharides (FOS) are considered prebiotic compounds and are found in different vegetables and fruits but at low concentrations. FOS are produced by enzymatic transformation of sucrose using fructosyltransferase (FTase). Development of new production methods and search for FTase with high activity and stability for FOS production Is an actual research topic. In this article is discussed the most recent advances on FTase and its applications. Different microorganisms have been tested under various fermentation systems in order to identify and characterize new genes codifying for FTase. Some of these genes have been isolated from bacteria, fungi, and plants, with a wide range of percentages of identity but retaining the eight highly conserved motifs of the hydrolase family 32 glycoside. Therefore, this article presents an overview of the most recent advances on FTase and its applications.
- Enzyme production
- 1-β-fructofuranosyl nystose
At the present time, there is a growing consumer demand for healthier and calorie-controlled foods. For this reason, food industry has developed different alternatives for sweeteners, and among them is the fructooligosaccharides (FOS). Use of these compounds offers multiple health advantages. FOS are fructose oligomers with a terminal glucosyl unit and a general formula of GFn; where typical values of
FOS can be produced by the action of different types of enzymes with transfructosylation activity (i.e., fructosyltransferase—FTase [EC 184.108.40.206] and/or β-fructofuranosidase—FFases [EC 220.127.116.11]). These enzymes are obtained from different plants and microorganisms . The reaction mechanism of FTase depends on enzyme source . Most of these enzymes have been isolated from different fungal strains such as:
2. FTase: an overview
FOS can be synthesized in nature by the catalytic action of enzymes with transfructosylating activity. They are classified as 1F-FTases (E.C. 18.104.22.168, E.C. 2.4.1. 99, and E.C. 22.214.171.124), or β -FFases (, E.C. 126.96.36.199). FTase catalyzes the transfer of a fructosyl group to a molecule of sucrose or a FOS when a FOS with a chain longer by one fructosyl unit is formed . This enzyme also mediates polymerization reactions, where degree of polymerization (DP) decreases to the maximum by transferring fructosyl units from higher molecular mass fructans . The reaction mechanism of the FTases depends on the enzyme source. In plants and some microorganisms, a series of enzymes act together, whereas a single enzyme works in most of the microorganisms .
The FTase that converts sucrose to the shortest β (2–1) linked fructan 1-kestose is called sucrose: sucrose 1-FTase (1-SST) . It is reported that, FTase differs in molecular weight and properties depending on its origin . Properties of FTases can change according to the microorganism and culture medium composition, especially the carbon source, which can play a role as an inductor . FTase can be produced intra- and/or extracellular by different microorganisms, including bacteria and fungi. Despite the large number of microbial FTase producers, only some of them have the potential for industrial application and have been used in several studies about FOS production . The transfructosylating activity is responsible for FOS production from sucrose, although quantitative differences exist because of the microbial strains .
FTase has been produced using both solid and liquid fermentation, and FOS obtained by these fermentations have been reported. Factors affecting FOS by fermentation were mentioned, such as temperature, pH, and substrate concentration [14, 15]. FTase has a temperature optimum between 50 and 60°C, and the optimal pH is between 4.5 and 6.5 [1, 8].
2.1. Mechanism of action
The reaction mechanism depends on the enzyme source and purity. The accepted mechanism is a type disproportionate reaction where FTase catalyzes the transfer of a fructosyl moiety of a sucrose or fructooligosacharide donor to another sucrose or FOS acceptor to provide a superior FOS . The reaction mechanism has been expressed as follows:
where GF is sucrose or FOS
3. Microbial and plant FTases
Several fungal strains, especially those from
Bacterial strains have been reported to produce different inulinases, but reports on enzymes able to produce FOS are scarce from bacterial strains. Someone bacteria mentioned to produce these enzymes are the following:
The FTases obtained from plants have other amino acid composition that is different from microbial FTases such as sucrose: sucrose 1-FTase (1-SST) and fructan: fructan 1-FTase (1-FFT). Plants such as
4. Structure of FTase
According to the Protein Data Bank (PDB) of Research Collaborators for Structural Bioinformatics (RCSB) data base, the crystal dimensional structure of FTase from
5. FTases properties
5.1. Factors affecting FTase activity
Fructosyltransferase (FTase) participates on FOS/fructan production by catalyzing the transfer of a fructose unit from one sucrose/fructan to another . This enzyme has been included in the glycoside hydrolase family 32 (GH32) and has been isolated from different sources, and the optimal conditions for the enzyme activity have been reported (Table 2). The optimal temperature reported for FTase enzyme activity ranges from 52 to 65°C, while the optimal pH varies widely from 4.5 to 8.0 [27, 28]. There are different reports mentioned about the chemical reagents and amino acids that positively affect FTase activity [29, 30]. On the other hand, there is a controversy in the use of detergents—some authors mention that these compounds enhance FTase activity , and in contrast there are others who mention that these compounds negatively affect FTase activity .
|52 °C||4.5||FeSO4, Fe2+, Fe2+ Ca2+||Intra- and extracellular|||
|55 °C||5.5||Leucine induced slightly|
|Intra- and extracellular||[30, 31]|
5.2. Carbon and nitrogen sources
Different reports mentioned that the preferred carbon source to produce FTase is sucrose. Patil and Butle  indicated that
5.3. FTase biochemical properties
Biochemical properties of FTase may change depending on its origin. Ghazi
6. FTases gene organization
Genome sequence of different microorganisms and vegetables has allowed identification of some enzymes, development of new products, improvement of strains, and increase of process efficiency. There are some reports of isolation and cloning of the FTase gene. The genes coding for FTase have been isolated from bacteria, fungi, and plants, with a wide range of percentages of identity but retaining the eight highly conserved motifs of hydrolyses family 32 glycoside . Fungal FTase genes have been isolated mainly from
The gene that encodes
FOS is a common name for fructose oligomers and corresponds to complex carbohydrates which are nondigestible oligosaccharide food ingredients and are fermentable by the gut microbiota. For this reason, they can be classified as prebiotics, and its commercial production has increased in response to a growing consumer demand for the so-called “health foods” [16, 39]. FOS are mainly composed of 1-kestose (GF2), 1-nystose (GF3), and 1- β-fructofuranosyl nystose (GF4), in which 1–3 fructosyl units (F) are bound at the β (2–1) status of sucrose molecule (GF) (Figure 1) .
FOS can be found in several vegetal sources such as tomato, onion, barley, garlic, Jerusalem artichoke, banana, rye, honey, sugar beet, to name a few; however, FOS concentration in these sources is low, and mass production are limited by seasonal conditions [3, 4]. At the industrial level, FOS are mainly produced from the disaccharide sucrose by action of different microbial enzymes with transfructosylating activity such as FTase (EC 188.8.131.52) and/or β-fructofuranosidase (EC 184.108.40.206), . Moreover, FOS compounds have received a generally recognized as safe status (GRAS) from the Food and Drug Administration (FDA) and has been consumed because of the several benefits of FOS to human health such as calorie-free and noncariogenic sweeteners, stimulate bifidobacteria growth, and activation of the immune system; have been claimed to contribute to the prevention of colon cancer and reduce the levels of serum cholesterol, phospholipids, and triglycerides; also promote calcium and magnesium absorption in animals and the human gut [14, 19, 22, 40, 41].
7.1. FOS production
Production of FOS has received particular attention in recent years, so there is necessity for the development of new enzymatic systems . FOS represent one of the major classes of bifidogenic oligosaccharides in terms of production volume. Kestose and nystose are the main prebiotic compounds, which can be principally produced by hydrolysis of inulin or by transfructosylation of sucrose . The enzymes that are potentially useful for high production of FOS were reported about three decades ago. Hidaka
8. Use of FTAse for FOS production
FOS have demonstrated important properties for improving human health, thus they have attracted an increased interest mainly as ingredients for food applications. They contribute to 10% of the natural sweeteners, and their demand has risen rapidly (about 15% per year) in the last 15 years . Consequently, establishing sustainable and economically viable industrial process for the production of FOS with high yields and productivities is strongly desirable . These can be manufactured by three methods: (1) extraction from inulin-rich plant materials, (2) by enzymatic synthesis from sucrose, and (3) by enzymatic degradation of inulin [45, 47]. Most of the FOS marketed as food ingredients/nutritional supplements are synthesized either from sucrose by the action of FTases [48, 49] or by enzymatic degradation of inulin [50, 51]. In this section, we will discuss the production of FOS through FTase.
Commercial production of FOS was first developed using enzymatic fructosyl transfer on sucrose by Hidaka
Nowadays, to reduce cost, enzyme immobilization techniques have been applied. Fungal β-fructofuranosidase has been covalently immobilized onto inorganic supports such as porous glass or porous silica [54, 60].
However, it has been observed that enzymes immobilized on a porous support decrease apparently its enzymatic activity because of diffusion resistance. Instead, the use of magnetic nanoparticles is proposed, which offers (a) a higher specific surface area that permits binding of a larger amount of enzyme, (b) relatively low mass transfer resistance and (c) selective separation from a reaction mixture by application of a magnetic field [61, 62]. Chen
Another alternative for FOS production is the use of solid-state fermentation (SSF). Most investigations on FOS production are based on submerged fermentation systems, but SSF is attractive because of low capital cost and low demand of water, generating less wastewater as a consequence. Besides, higher productivities and yields could be obtained at industrial scale . Mussatto
9. Properties of FOS
General structure of FOS can be depicted as GFn, where “
FOSs contain several qualities that make its usage possible as an alternative sweetener in the food market. They are water soluble and one-third as sweet as sucrose . However, their viscosity and thermal stability is higher than sucrose. They are stable in a pH range from 4.0 to 7.0 and can be refrigerated for a period of one year. Their high moisture-retaining capacity provides prevention of excessive drying besides controlling microbial contamination owing their low water interacting activity .
They can be considered as noncariogenic sugar substitutes in confectionary, gums, and drinks since they cannot serve as a substrate of
10. FOS as prebiotic
The most widely used definition for prebiotic is “nondigestible food ingredient that beneficially affects host’s health by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon” . In 2004, the definition was updated, and it was defined as “selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” . According to this, prebiotics are a major part of the functional foods and among them, the FOS are in focus due to their functional properties and economical potential [65, 75].
A prebiotic must fulfill three criteria: (1) no hydrolysis or absorption in the upper part of the digestive system, (2) selective substrate for one or more desired bacteria species in the colon and stimulation of that species regarding growth and activation and (3) able to positively influence the numeric proportion of different bacteria species in the colon . FOS selectively stimulates the growth of
Many Bifidobacteria and
Antiinflammatory and antitumorigenic roles of SCFA have been reported [81, 82]. As a result of the prebiotic function, a decrease in inflammatory markers such as phagocytosis and interleukin (IL)-6 production by increasing CD3+, CD4+, and CD8+ populations has been observed . In case of antitumorigenic roles, especially in the context of colon cancer, the action mechanism is yet unclear . However, it is known that butyrate has an important role on DNA methylation thus modifying gene expression so it may directly enhance cell proliferation of normal cells, but suppress cell proliferation of transformed cells. Furthermore, in the presence of butyrate, apoptosis may be enhanced in transformed cells but inhibited in normal cells . Thus, the regular intake of FOS as a part of diet could help to improve health and over all well-being by providing resistance against the intestinal/extra intestinal pathogens, enhancing the growth of the colon microbiota which have metabolic activities and biochemical processes with a tremendous influence in human host .
11. Applications of FOS
FOS are components of functional food that are becoming popular in the society because they have a potential for enhancing flavor quality and physicochemical properties of food products, besides FOS offer various benefits for human health and are also of industrial interest . FOS are used in different food applications and other areas because of its positive impact on human health, physical performance, or state of mind , and the most relevant uses in food formulations are the following: beverages (fruit drinks, coffee, cocoa, tea, soda, health drinks, and alcoholic beverages), dairy products (fermented milk, instant powders, powdered milk, and ice cream), also in light jam products and confectionary .
12. Functional foods
The growing interest of consumers in the relationship between nutrition and health has increased demand among the population for food products that improve or benefit their health beyond basic nutrition [87, 88]. Because of this demand, both the academic community and the food industry have focused on developing products that meet these characteristics, which are now called functional foods.
The term “functional foods” was first coined in Japan after a group of scientists and nutritionists conducted numerous studies and defined them as “Food for specified health uses” (FoSHU) . Though there is a great number of definitions, Doyon and Labrecque  identified four key concepts after reviewing more than 20 definitions: (a) health benefits, (b) the nature of the food, (c) level of function, and (d) consumption pattern. The definition of functional food has evolved, and the latest is that proposed by the European Commission for concerted action Functional Food Science in Europe (FUFOSE) that mentions that “a food can be regarded as ‘functional’ if it is satisfactorily demonstrated to beneficially effect one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease. Functional foods must remain as food and they must demonstrate their effects in amounts that can normally be expected to be consumed in the diet: they are not pills or capsules, but part of a normal food pattern” [91, 92].
The challenges for the food industry are great so that the biological value of the functional ingredient is not disturbed and sensory characteristics of the food are acceptable . According to various investigations, the success and acceptance of these foods is influenced by factors such as clarity and understanding of the information about nutrition and health benefits that are provided to consumers, especially in elderly consumers .
Experts recognize that there are scientific evidence on the effectiveness of various functional foods, which can be useful to balance a poor diet or assist in avoiding health problems in some cases . In general, the consumers’ attitude towards functional foods is positive and have great potential in the food industry.
13. Applications of FOS in food formulations
FOS are ingredients that have been applied in a variety of food matrices, their prebiotic potential has been proven, and its technological properties allow easy incorporation into foods, mainly those that are probiotics . The FOS have comparable glucose syrups and sugar properties and proved approximately a 30–50% sweetness compared with sugar table. Therefore, their application has a dual benefit: (1) as a substitute for sugar and (2) for their prebiotic properties .
Akalin and Erisir  evaluated the effect of supplementation of oligofructose or inulin in symbiotic ice cream, in the rheological properties and probiotics survival. They found that the survival rate of the probiotics during storage at 30, 40, and 90 days was better with oligofructose. The FOS were evaluated in cookies and Quicks bread and found that consumers had preference equal to or greater for products supplemented with FOS [96, 98]. Although the FOS are easy to incorporate into foods such as yogurt, processing conditions such as acidity and temperature should be considered since they have reported low prebiotic activities under acidic conditions and high thermal processing times .
New applications in different food matrices are being evaluated. Valencia
There is an innovative trend in the FOS application in different types of food and, undoubtedly, to maximize the benefits that can confer the FOS, factors as type of food matrix, processing conditions, and added amount should be considered.
14. Future trends
Because of the importance of this enzyme in the modern industry, it is important to relate a set of FTases from different organisms to allow the identification of features that could be used for the identification and classification of new FTases, and also it is necessary to improve the conditions and costs of FTases production process. Further studies of gene sequencing will allow distinguishing among the set of FTase and β FFase enzymes.
The studies on production and application of FOS are of high interest for food industry because of several health benefits and biofunctional properties that these compounds provide. FOS can be synthesizing from precursors such as sucrose using FTase enzymes. These enzymes can be obtained from different microorganisms (bacteria and fungi) and plants. The main disadvantage of this production is the low yields of enzymatic activity and FOS. Thus, search for new microbial sources of FTase enzymes is a very important research topic as well as studies about the evolution of FTase genes from different sources, and relate their function with the nucleotide sequence using functional genomics studies.
This project was financially supported by the Universidad Autónoma de Coahuila. MRMM would like to thank the financial support received from CONACYT during her master’s degree.