A FOS market analysis was performed by our group with accurate numbers being difficult to come by. FOS are grouped in the prebiotic market which includes a range of oligosaccharides and sugars. Based on a market price of USD 10/kg and projections of published figures, Shedlock (2014) places the global FOS market between 50 000 and 100 000 tonnes.
2.6.1 Industrial FOS preparations derived from inulin
The compositions of industrial inulin-type fructan preparations are quite varied and contain low DP FOS, high DP inulin or combinations of both. Table 2.1 details the compositions of some commercial fructan preparations. Inulin is derived from chicory roots and the DP varies from 2 to ~60 units with an average DP = 12. The partial hydrolysis of inulin using endoinulinases yields oligofructose in which the DP varies from 2 to 7 with an average DP = 4. It is composed mostly of lower-DP oligosaccharides, namely, 1-kestose (GF2), nystose (GF3) and
β-fructofuranosylnystose (GF4), as well as inulobiose (F2), inulotriose (F3), and inulotetraose
(F4). Oligomers with DP < 10 may be removed by applying chromatographic separation
techniques to produce a high molecular weight inulin-type fructan or inulin HP (Raftiline HP in Table 2.1). A mixture of 2 distinct populations of the low molecular weight oligofructose and the high molecular weight inulin is known as oligofructose-enriched inulin or Synergy (Roberfroid 2007). The preparations produced by Orafti are made from chicory inulin (BENEO GmbH 2013).
2.6.2 Industrial FOS preparations from sucrose
The synthesis of FOS from sucrose is the method used by the remaining companies listed in Table 2.1. The products contain FOS with a DP ranging from 2 to 4 and average DP = 3.6 (similar to oligofructose) (Roberfroid 2007).
Although bacteria, plants and fungi produce enzymes for FOS synthesis, fungal enzymes are preferred for industrial FOS production. Typical reaction conditions are ~50-70% sucrose as substrate, pH5-6.5 and reaction temperatures at 50-60 °C (Yun 1996, Nishizawa et al. 2001). The use of plant enzymes is limited by the requirement of two enzymes for FOS synthesis; the production of two enzymes being uneconomical compared to a single enzyme. Microbial enzymes also display better thermal stabilities than plant enzymes (Yun 1996). The FOS yields of fungal enzymes range between 50-60% of total sugars (Nishizawa et al. 2001) whereas bacterial inulosucrases and levansucrases produce significantly lower yields. Furthermore, levansucrases generally yield high DP levan which has many industrial uses but is not the target fructan of industrial FOS synthesis (Bittencourt et al. 2014; Ozimek et al. 2006). Thus fungal β-fructofuranosidases/fructosyltransferases possess a number of traits that render them more suitable to industrial conditions than their bacterial- and plant-derived counterparts.
There are two main industrial processes for FOS synthesis – a batch system using soluble enzyme or a continuous process using immobilised enzyme or whole cells (Yun 1996). The complete FOS synthesis process is a two-stage operation with initial production of enzyme or microbial biomass under specific conditions followed by the synthesis of FOS in a batch or continuous process using the produced biocatalyst (Fig. 2.4). The high substrate concentrations are used to promote fructosyltransferase enzyme activity and limit downstream processing costs, but together with the relatively high temperatures also limit unwanted microbial contamination of reaction vessels. Immobilisation increases the stability of the enzyme or cells and facilitates easy removal of the catalyst from the final reaction mixture. However, it also limits substrate and product diffusion capacities in column reactors. A batch process requires an additional step to remove enzyme which is not a factor in a continuous process but the latter is limited by lower flow rates required to maximise diffusion of substrates and products in gel matrices (Yun 1996). The productivities of continuous processes using immobilised enzyme are superior to immobilised cells, with values of 1190 g/l h reported for the former (Hayashi et al. 1991) and 180 g/l h for the latter (Yun et al. 1992). The design of a FOS production process therefore must consider catalyst stability, dosage, yields and productivity factors.
Table 2.1 Commercial preparations of inulin-type fructans (Andersen et al. 1999; Nishizawa et al. 2001)
Trade name FOS content DP Range DP average Free glucose content Free fructose content Free sucrose content Producer
Raftilose L30 28-32% oligofructose, syrup 2-8 NR 6-10% 57-61% 1-5% Orafti
Raftilose L60 58-62% oligofructose, syrup 2-8 NR 8-12% 28-32% Orafti
Raftilose L85 83-87% oligofructose, syrup 2-8 NR 6-10% 5-9% Orafti
Raftilose L95 93.2-95% oligofructose, syrup 2-8 NR 4.2-6.8% Orafti
Raftilose P95 93-97% oligofructose, powder 2-8 NR 3-7% Orafti
Raftiline ST 90-94% chicory inulin, powder 2-60 10-12 0-4% 4-8% Orafti
Raftiline GR 90-94% chicory inulin,granulated powder 2-60 10-12 0-4% 4-8% Orafti
Raftiline LS 99% chicory inulin 2-60 10-12 1% Orafti
Raftiline HP 99% chicory inulin 2-60 22-25 0.5% Orafti
Neosugar G 27% GF2 + 27% GF3 + 6% GF4 3-5 NR 30% - 10% Meiji Seika Kaisha
Neosugar P 36% GF2 + 50% GF3 + 9% GF4 3-5 NR 2% - 3% Meiji Seika Kaisha
Meioligo P 42% GF2 + 46% GF3 +9% GF4 NR NR NR NR NR Meiji Seika Kaisha
Actilight 550S 45+3% GF2 + 45+3% GF3 + 10+3%
GF4, FOS 55%, syrup 3-5 NR 45% Beghin-Meiji
Actilight 950S 37+6% GF2 + 53+6% GF3 + 10+4%
GF4, FOS 95%, syrup 3-5 NR 5% Beghin-Meiji
Actilight 950P 37+6% GF2 + 53+6% GF3 + 10+4%
GF4, FOS 95%, powder 3-5 NR 5% Beghin-Meiji
NutraFlora 95% FOS NR NR NR NR NR Golden Technologies
Oligo-Sugar 60% FOS NR NR NR NR NR Cheil Foods and Chemicals
Fructafit HD 90% inulin NR NR 1.5% 4.0% 4.5% Fructall
Fibruline NR 2-50 9 NR NR NR Cosucra
NR – not reported Beghin-Meiji, France Orafti, Belgium Cosucra, Belgium
Meiji Seika Kaisha, Japan
Cheil Foods and Chemicals, Korea
Fig. 2.4 Schematic of the industrial processes for FOS production. Redrawn from Yun et al. (1996).
2.6.3 Opportunities to improve enzymatic FOS synthesis from sucrose
A major limitation of the enzymatic synthesis of FOS from sucrose is enzyme inhibition by the glucose product which limits maximum conversion yields to 55-60% (Dominguez et al. 2013; Jong and Seung 1993; Jung et al. 1989; Sangeetha et al. 2005). The enzyme from
A. pullulans is competitively inhibited from glucose while that of A. japonicus displays non-
competitive inhibition (Jung et al. 1989; Nishizawa et al. 2001). The residual sucrose, glucose and fructose (if any) pose problems for certain applications of FOS preparations, for example in products tailored for diabetics. Methods to remove the unwanted sugars to yield high-content FOS preparations include: simulated moving-bed chromatographic separation for the removal of sugars from the final reaction mixture; a mixed enzyme system for FOS synthesis in which glucose oxidase is included to convert glucose to gluconic acid; application of a membrane reactor system using a nano-filtration membrane through which glucose permeates but sucrose and FOS do not (Jong and Seung 1993; Nishizawa et al. 2001; Yun 1996). Only the latter two methods can potentially relieve product inhibition by glucose but they will increase process costs. Additionally, in the mixed enzyme system
Cell culture
Enzyme extraction Cell immobilisation
Enzyme reaction Column reactor
Sucrose solution Purification Concentration
Sterilisation Product
optimal temperatures for the two enzymes differ. Also, glucose oxidase requires oxygen, the solubility of which is decreased at the higher temperatures required for optimal β-fructofuranosidase activity. Thus operational parameters have to be adjusted to suit both enzymes, often resulting in a compromise at the expense of productivity (Yun and Song 1999). Engineering the enzyme to relieve product inhibition offers a way to increase FOS yields yet limit additional input costs.
The stability of the enzyme used in the process is affected by relatively high incubation temperatures, typically between 50 and 60 °C. Immobilised cells remain stable for approximately 100 days while immobilised enzymes only remain stable for 35 days, however at lower operational temperatures (Yun and Song 1999). To be able to exploit the higher productivities of immobilised enzyme systems as opposed to cells, improved thermal stability of the enzymes would be advantageous. A batch process would also benefit from higher operational temperatures, permitted by a thermostable enzyme as follows. An increase in temperature is known to affect enzyme reactions on two levels, namely the catalysed reaction itself and the thermal inactivation of the enzyme. At lower temperatures enzyme inactivation is very slow and has no noticeable effect on the rate of the catalysed reaction. Thus the overall reaction rate therefore rises with increases in temperature, as do all chemical reactions. At higher temperatures enzyme inactivation becomes more prominent, causing the effective concentration of active enzyme to decrease during the course of the reaction, accompanied by a reduced reaction rate (Laidler and Peterman 2009). An enzyme displaying higher thermal stability would delay the effect of enzyme inactivation and allow for reactions to be conducted at higher temperatures, reducing the time to completion of the reaction. Again, protein engineering offers a means to achieve such improvements.
The cost of enzymes remains an important factor in the economics of enzyme-catalysed bioprocesses. The higher the specific activity of the enzyme the less is required to complete a given reaction. This impacts the scale of the required enzyme production stage as well as the required enzyme dosage in the FOS synthesis stage. Alternatively, the turnaround time of the FOS synthesis stage can be reduced by an enzyme with higher specific activity, while maintaining set enzyme dosages. Enzyme activity has an impact on the amount of enzyme required per gram of FOS produced and ultimately dictates the design and scale of the facility as well as the operational procedures, all with cost implications (Newman et al. 2013).