Complete Hydrolysis Fructose Syrups

Fructose syrups are widely utilized in the food industry in that they are sweeter than sucrose, thus allowing less sugar to be used to achieve a given level of sweetness (i.e., fructose is 1.2 times sweeter than sucrose on a weight basis (Shallenberger, 1993)). In addition, fructose metabolism in humans is not insulin dependent, and it produces less tooth decay than other sugars (Roch-Norlund et al., 1972). Currently, much of the fructose used by the food industry is produced from corn starch, a glucose polymer, via hydrolysis followed by isomerization. The fructose content is around 42% but can be increased to 95% by chromatographic separation of the residual glucose and further isomerization.

In contrast, as a polymer of fructose, inulin is an excellent candidate for producing a high-fructose syrup. It can be readily hydrolyzed enzymatically or chemically (GrootWassink and Fleming, 1980). Chemical hydrolysis can be achieved, for example, by acidifying to pH 2 to 3 with a strong acid cation exchanger and heating at 70 to 100°C (Yamazaki and Matsumoto, 1986). However, undesirable contaminants are produced and must be removed.

Enzymatic hydrolysis requires a single enzyme, inulinase, which yields a high-purity product (Barwald and Flother, 1988; Zittan, 1981). The percentage of fructose varies with the degree of polymerization of the inulin, a condition that is influenced by species, cultivar (Chabbert et al., 1985a), time of harvest (Chabbert et al., 1983), and other factors (Modler et al., 1993b). The average degree of polymerization for early harvested Jerusalem artichoke inulin was 10 to 15, while for late harvest it was only 3 to 5. Shorter chain lengths yield syrups with progressively higher glucose and lower fructose contents (e.g., 96% fructose for early harvest vs. 65% for late harvest). Therefore, the production of high-fructose syrups from inulin involves two processes: hydrolysis and, when required, fructose enrichment.

Several classes of enzymes are capable of hydrolyzing the fructosidic linkages of inulin. The endo-inulinases cleave linkages within the chain, yielding fructans with reduced degrees of polymerization (e.g., p-D-fructofuransoidase (EC 3.2.1.26)). Exo-inulinases (2,1-p-D-fructan fructano-hydrolase (EC 3.2.1.7)), in contrast, cleave single D-fructose molecules from the terminal end. Exo-inulinases are preferred and can be produced by a host of microorganisms, including fungi, yeast, and bacteria (Khanamukherjee and Sengupta, 1989; Pandey et al., 1999); typically, the enzyme is isolated from the organism for use. Microorganisms used for the production of inulinases include (Pandey et al., 1999):

Fungi:

Aspergillus sp.: A. aureus, A. awamori, A. ficuum, A. fischeri, A. flavus, A. nidulans, A. niger, A. phoenicis Cladosporium sp.

Chrysosporium pannorum Fusarium sp.: F. oxysporum

Penicillium sp.: P. purpurogenum var. rubisclerotium, P. rugulosum, P. trzebinskii Streptomyces sp.: S. rochei

Bacteria:

Acetobacter sp. Achromobacter sp. Arthrobacter sp. Bacillus sp.: B. subtilis

Clostridium sp.: C. acetobutylicum, C. pasteurianum, C. thermoautotrophicum, C. ther-

mosuccinogenes Escherichia coli Flavobacterium multivorum Pseudomonas sp. Staphylococcus sp.

Yeast:

Candida sp.: C. kefyr, C. pseudotropicalis Kluyveromyces sp.: K. fragilis, K. lactis, K. marxianus Pichia sp.

Isolated inulinase is utilized for either batch hydrolysis of inulin or immobilization for flow-through column hydrolysis systems. For example, purified inulinase from K. fragilis has been immobilized on 2-aminoethyl cellulose (Kim et al., 1982).

To achieve high-purity fructose syrup, it is necessary to remove either inulin with a lower degree of polymerization before or the glucose after hydrolysis. Removing shorter-chain-length inulin can be achieved by chromatography, enzymatic removal, precipitation of higher molecular weight fractions using ethanol or low temperatures (Chabbert et al., 1985b), or ultrafiltration (Kamada et al., 2002). Enzymatic removal generally utilizes yeast strains whose fermentation of inulin is restricted to lower molecular weight fractions (e.g., Saccharomyces cerevisiae, Saccharo-myces diastaticus) (Schorr-Galindo et al., 1995). The raw inulin is initially fermented, producing ethanol from the low molecular weight fraction. The residual high-dp inulin is then hydrolyzed using an exo-inulinase. Using this technique, both early- and late-harvest Jerusalem artichoke inulin can produce syrup of up to 95 and 90% fructose, respectively.

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