Enzymatic Conversion of Steviol Glycosides

From leaves of Stevia rebaudiunu (Compositae), several sweet glycosides of steviol (= 13-hydroxy-entkaur-16(17)-en-19-oic acid) have been isolated. Among all these glycosides, Stevioside and Rebaudioside A received most of the attention so far. 

 

The older breeds of Stevia plants contained higher proportion of Stevioside and Rebaudioside A was a minor component of the total glycosides. But, it was fond that Rebaudioside A has a more acceptable taste profile than the then predominant Stevioside. In the early years of adoption of steviol glycosides as sweeteners, the major glycoside, stevioside was used as a sweetener for seasonings, pickles and salted foods. Rebaudioside A was mainly used in more demanding applications like beverages as it has comparatively less aftertaste.

Structure of Steviol Glycosides

For Steviol R1 = R2 = H

Quality of taste -  Stevioside= 0, +ve better, -ve worse

Abbreviation of sugar units: Glc : b-D-glucoside, , Rha : a-l-rhamoside

In different sensory studies, Stevioside was found to be between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320, and rebaudioside C between 40 and 60. Dulcoside A was 30 times sweeter than sucrose. Rebaudioside A was the least astringent, the least bitter, had the least persistent aftertaste and was judged to have the most favourable sensory attributes of the four major steviol glycosides.

 

Stevioside tastes somewhat bitter, and show aftertaste. In order to improve of sweetness, modifications of sugar moieties of both the glycosides were conducted by enzymatic transglucosylation.

 

Cyclomaltodextrin-glucanotransferase (CGTase) efficiently catalyzes intermolecular glycosylation to transfer a-glucosyl units from starch to 4-OH of a glucosyl moiety (trans-a-l,4-glucosylation). CGTase (EC 2.4.1.19) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli were used for transglycosylating stevioside and rebaudiosides A with the use of starch or cyclo-dextrine as donor. CGTases produced by Bacillus stearothermophilus B-5076 B. macerans BIO-4m were the most effective biocatalysts. Optimum temperature and pH of these enzymes were 45°C and pH 6.5–7.5, respectively. The optimum stevioside-to-Cyclodextrine ratio and total concentration of dry matter for the synthesis of the best-tasting product were 1 : 1 (w/w) and 11.6%, respectivelyThis method can be used successfully for direct transglycosylation of Stevia extract without purification of its individual components.

 

Stevioside, when treated with CGTase, yields a complex mixture of products which were mono-, di-, tri and more glucosylated both at the 19-O-glucosyl unit and the terminal glucosyl unit of the 13-O-sophorosyl moiety.

 

Significant improvement in quality of taste was observed for most of the glucosylated products, especially for Sla and S2a which were mono- and di-glucosylated at the 13-O-sophorosyl moiety, respectively. Remarkable enhancement of intensity of sweetness was also observed for both the products, while glucosylation at the 19-0-glucosyl moiety resulted in decrease of intensity of sweetness. 

Products Sla, S2a and S2c were also obtained from stevioside by pullulan and crude pullulanase from Klebsiella sp though yields were rather low. Transglucosylation of stevioside by Pullulanase and pullulan exclusively afforded three products, 13-O-[b-maltotriosyl-(1----2)-b-D-glucosyl]-19-O-b-D-glucosyl- steviol (1), 13-O-[b-maltosyl-(1----2)-b-D-glucosyl]-19-O-b-D-glucosyl- steviol (2) and 13-O-b-sophorosyl-19-O-b-maltotriosyl-steviol (3). All of these products have already been obtained by trans-alpha-1,4-glucosylation of stevioside by the cyclodextrin glucanotransferase starch system, and 1 and 2 have been proven to be tasty and potent sweeteners.

 

Transglucosylation of stevioside by a amylase from Aspergillus niger and maltose afforded three new products, viz. 13-O-b-sophorosyl-19-O-b-isomaltosyl-steviol (4), 13-O-[b-isomaltosyl(1----2)-b -D-glucosyl]-19-O-b-D-glucosyl- steviol (5) and 13-O-[b-nigerosyl-(1----2)-b-D-glucosyl]-19-O-b-D- glucosyl-steviol (6). A significantly high quality of taste was evaluated for 4.

 

The enzyme system from Streptomyces sp. W19-1 forms several kinds of transfer products when incubated in the presence of both stevioside (ST) and curdlan. Three of the major were separated and purified and found to be (1) 13-O-b-sophorosyl-19-O-b-laminaribiosyl steviol; (2) 13-O-b-glucosylsophorosyl-19-O-b-glucosyl steviol, and (3) 13-O-b-sophorosyl-19-O-b-laminaritriosyl steviol.

 

Treatment of stevioside by l3-glucan and an enzyme from Streptomyces sp. DIC-108 yielded rebaudioside A, the better sweetener.

 

An Actinomycete strain K- 128 isolated from soil was cultured in a medium containing stevioside and curdlan to give a trans-p-1,6-glucosylated product, 13-[p-glucosyl-( 1-6)-p-glucosyl-( 1-2)-p-glucosyl]- 19-0-p-glucosyl-steviol.

 

Incubation of stevioside with sucrose and p-fructofuranosidase from Arthrobactor sp. K- 1 afforded a product trans-O-2,6-fructofuranosylated at the 19-0-glucosyl moiety in a high yield. Relative intensity of sweetness was not enhanced, however, significant improvement of quality of taste was observed for both the compounds. It is noteworthy that the fructofuranosyl linkage is rather unstable, being hydrolyzed on standing in foods.

 

These results strongly suggested that for enhancement of intensity of sweetness of steviol glycosides, the elongation of the 13-0-glucosyl moiety up to a total of four glucosyl units under suppression of glucosylation at 19-0-glucosyl moiety, may be desirable.

 

CGTases from B. stearothermophillus, B. Macerans and B. halophillus are commercially available from many suppliers. These enzymes are shipped in as liquid stebilised preparations in 25 litre HDPE containers. The activity of the commercial preparations is within 1200-1600 IU/g, and their temperature optima is 500 -650C. The suppliers claim that the active shelf life of these preparations is about 8 months when preserved in 20-40C.

 

Enzymatic transglycosylation of stevioside usually takes long reaction time and concerns thermo-deactivation of the enzyme. Using a thermophilic enzyme and employing low power microwave irradiation, transglycosylation of stevioside was found to be speeded up over 10-folds. 

 

It has been observed that, to achieve better sweetness with less astringency, suppression of glycosylation at 19-O-glucosyl moiety is necessary.  b amylase may be employed to shorten long oligo-glucosyl chain in 19 position generated after transglycosylation of stevioside. b Amylase hydrolyzes a- 1,4- glucosyl chain from the non-reducing end to release maltose. By treatment with this enzyme, tri- and more a-1,4-glucosyl chains are converted into a mono- or di-a-1,4-glucosyl chain. Since decrease of sweetness was observed for products with a poly-a-1,4-glucosyl chain, treatment of glucosylated products with b-amylase resulted in the further improvement of sweetness.

 

After transglycosylation, the stevioside derivatives are to be separated from the reaction mixture consisting water, dissolved donor starch or cyclo-dextrin and residual enzyme formulation. This may be a very complicated and cumbersome step. Extraction and purification of stevioside from crude Stevia extract is a multi-step and cost intensive process. If transglycosylation is performed with purified stevioside, preparation of modified steviosides will involve another costly post-transglycosylation recovery and purification stage. The total economics of this two stage purification may not be commercially feasible. Thus, to achieve commercial success, a CGTase system effective on crude Stevia extract is to be identified, so that, modified steviosides can be directly recovered from the crude reaction mixture in a single stage purification process.

 

Direct fermentation of stevioside by specific organisms may also not be commercially feasible at present. After fermentation, the modified steviosides are to be separated from the residual medium components and microbial metabolites – all of which may not be entirely non toxic. Industrial Fermentors with efficient process control along with all its peripherals are very expensive. Moreover, to ensure freedom from fermenting organisms, a very capital intensive biomass separation system comprising cross flow micro-filtration system is to be employed. Total cost of ownership of this setup, its maintenance cost and the cost of manpower to run this setup will be really prohibitive. The maximum stevioside concentration usable in the medium may not be very high – thus the setup may not have high throughput.

 

Hence, I feel that the most cost efficient setup for transglycosylation process will be immobilized enzyme column array. Specific CGTase enzyme may be immobilized on a proper matrix either by gel entrapment or by adsorption or by covalent linking and a percolation column is to be filled with that matrix. Crude Stevia extract with dissolved donor starch can be recirculated through these columns until desired extent of transglycosylation is achieved.

 

After transglycosylation, the solution may be passed through amylase columns for 19-O- chain shortening. Then modified steviosides can be recovered from the reaction mixture by conventional method.

 

 

Development of commercially feasible technology for the process should involve the following steps –

 

  • Identification of a CGTase and amylase system effective in crude Stevia extract

  • Determination of most effective immobilization process.

  • Working out of process parameters like optimal temperature, pH, enzyme cofactors, donor substrate ratio, reaction kinetics etc. in lab scale.

  • Construction of a pilot facility and further standardization of process parameters.

  • Scaling up of the process and determination of immobilized enzyme column dimensions, design of specific liquid handling and process control systems and further standardization of process parameters.

  • Development of cost effective post extraction and transglycosylation purification technology.

  • Development of “in-process” and finished product rapid QC protocol.      

 

Keeping in view the recently discovered anti-diabetic, anti-hypertensive and anti-neoplastic activity of absolutely natural sweetener steviosides, I feel, it has tremendous market potential in future. Since there is wide apprehension of shortening of market life expectancy of sucralose for it’s recently discovered immunosuppressive and thymo-toxic effectes, stevioside may win further importance in very near future.