Stevia Glycosides

CHAPTER ONE

Stevia Glycosides: Chemical and Enzymatic Modifications of Their Carbohydrate Moieties to Improve the Sweet-Tasting Quality Gerrit J. Gerwig, Evelien M. te Poele, Lubbert Dijkhuizen, Johannis P. Kamerling Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction Steviol Glycoside Structures from S. rebaudiana Steviol Variants of Glycoside Structures from S. rebaudiana Stability of Steviol Glycosides Structure–Sweetness Relationship Chemical Modifications of Steviol Glycosides Enzymatic Modifications of Steviol Glycosides 7.1 Cyclodextrin Glycosyl Transferase Systems 7.2 α-Glucosidase Transglycosylation Systems 7.3 β-Glucosidase Transglycosylation and Deglycosylation Systems 7.4 α-Galactosidase Transglycosylation Systems 7.5 β-Galactosidase Transglycosylation Systems 7.6 β-Fructosidase Transglycosylation Systems 7.7 β-Glycosyltransferase Glycosylation Systems Using UDP-Sugars 8. Patents Regarding Enzymatic Modifications of Steviol Glycosides 9. Concluding Remarks Addendum Acknowledgments References

2 8 12 14 18 20 29 30 39 45 48 48 50 50 53 56 58 58 59

ABBREVIATIONS ADI acceptable daily intake Ara L-arabinose CAZymes carbohydrate-active enzymes CD cyclodextrin

Advances in Carbohydrate Chemistry and Biochemistry, Volume 73 ISSN 0065-2318 http://dx.doi.org/10.1016/bs.accb.2016.05.001

#

2016 Elsevier Inc. All rights reserved.

1

2

Gerrit J. Gerwig et al.

CGTase cyclodextrin glycosyltransferase DDase dextrin dextranase DIEA diisopropylethylethylamine DP degree of polymerization EFSA European Food Safety Authority FDA United States Food and Drug Administration FJGase β-glucosidase from Flavobacterium johnsonae Fruf D-fructofuranose Gal D-galactose Glc D-glucose Gtf glucosyltransferase Gtf180-ΔN N-terminal truncated glucosyltransferase from Lactobacillus reuteri 180 GRAS generally recognized as safe HPLC high-performance liquid chromatography IUPAC International Union of Pure and Applied Chemistry LC liquid chromatography Man D-mannose MD molecular dynamics MEP 2-C-methyl-D-erythritol-4-phosphate MS mass spectrometry NMR nuclear magnetic resonance PAM positive allosteric modulator PyBOP benzotriazol-1-yloxtri(pyrrolidinol)phosphonium hexafluorophosphate Qui D-quinovose (6-deoxy-D-glucose) Rha L-rhamnose (6-deoxy-L-mannose) SPGase steviol-producing β-glucosidase from Penicillium decumbens naringinase SSGase stevioside-specific β-glucosidase from Aspergillus aculeatus UDP uridine diphosphate UGT UDP-glucosyltransferase Xyl D-xylose

1. INTRODUCTION The herb plant Stevia rebaudiana Bertoni (Fig. 1), a rhizomatous perennial shrub of the sunflower family Asteraceae (Compositae; tribe Eupatorieae), native to Paraguay and Brazil, produces a host of natural sweet-tasting diterpene compounds as secondary metabolites in its leaves.1–7 The leaves have been used by local Guaranı´ Indian tribes as a natural sweetener and as traditional medicine for centuries.8 The Paraguayan kaa´-h^e-e or caa-he-e (meaning: sweet herb) plant was scientifically described for the first time in 1899 by the botanist M. S. Bertoni (1857–1929), and named S. rebaudiana Bertoni in 1905.9 In fact, the name “Stevia” originates from the surname of a Spanish botanist Pedro Jaime Esteve (
1500–1556), whereas the name “rebaudiana” honored the Paraguayan chemist Ovidio Rebaudi (1860–1931).

Native (Carbohydrate-Modified) Stevia Glycosides

3

Fig. 1 The Stevia rebaudiana Bertoni plant.

In view of the established intense sweetness of some of the leaf constituents, the so-called steviol glycosides, and the claims that they can be healthy and safe bio-alternatives for artificial (synthetic) sweeteners, nowadays the S. rebaudiana plants are commercially cultivated on a large scale in several countries in Europe, Asia, and America. Also cultivars of S. rebaudiana have been selected for enhanced production of specific steviol glycosides. The “generally recognized as safe” (GRAS) status of steviol glycosides has recently been approved by the United States Food and Drug Administration,10,11 as well as by the European Food Safety Authority12,13 (E960, European Index number). The global market for Stevia sweeteners is expected to grow to millions of metric tons in the coming years. The major steviol glycosides, stevioside (5) and rebaudioside A (11) (Table 111,14–52), taste about 250–350 times sweeter than the common table sugar, sucrose (0.4% aqueous solution). Both are currently in use as noncaloric (zero glycemic index) sugar substitutes in different kinds of drinks (fermented milk products, fruit nectars, flavored drinks) and food categories (ice cream, marmalades, chocolate products, candies, biscuits, soya sauce, processed potato-, cereal-, flour-, or starch-based snacks, chewing gum, dietary products for special medical purposes and weight control) in various countries. Most commercial table-top Stevia formulations consist of purified S. rebaudiana leaf extracts, having stevioside and rebaudioside A as the main components (purity must be >95%), but they can also contain smaller amounts of other steviol glycosides.53 For reviews on sweetness aspects, see Refs. 54–60.

Table 1 Structures of Naturally Occurring Steviol Glycosides in Stevia rebaudiana Leaves OR2

12 11

17

20

CH3

1 2

13 14

9

10

CH2 16

8

H 5

3 18

H3C R1O

No. Steviol Glycoside

7

4 6

H C 19

15

O

R1 (C-19/Carboxylic Acid)

R2 (C-13/Hydroxyl)

References

Glucosyl steviol family

1

Steviolmonosidea

H-

Glc(β1-

14

2

Steviol-19-O-glucoside

Glc(β1-

H-

15–17

3

Rubusoside

Glc(β1-

Glc(β1-

18–20

4

Steviolbiosidea

H-

Glc(β1-2)Glc(β1-

14,21–23

5

Stevioside

Glc(β1-

Glc(β1-2)Glc(β1-

14,19,21

6

Rebaudioside KA/Stevioside A Glc(β1-2)Glc(β1-

Glc(β1-

24,25

H-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

14,21,23,26

7

Rebaudioside B

8

Rebaudioside G

Glc(β1-

Glc(β1-3)Glc(β1-

17,26,27

9

Stevioside B

Glc(β1-3)Glc(β1-

Glc(β1-

28,29

10 Rebaudioside E

Glc(β1-2)Glc(β1-

Glc(β1-2)Glc(β1-

25,30

a

11 Rebaudioside A

Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

14,21,26, 31,32

12 Rebaudioside A2

Glc(β1-

Glc(β1-6)Glc(β1-2)Glc(β1-

33

13 Rebaudioside D

Glc(β1-2)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

30,34,35

14 Rebaudioside I

Glc(β1-3)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

26,36

15 Rebaudioside L

Glc(β1-

Glc(β1-6)Glc(β1-2)[Glc(β1-3)]Glc(β1- 26

16 Rebaudioside Q2

Glc(α1-2)Glc(α1-4)Glc(β1-

Glc(β1-2)Glc(β1-

17 Rebaudioside Qb

Glc(β1-

Glc(α1-4)Glc(β1-2)[Glc(β1-3)]Glc(β1- 11

18 Rebaudioside I2

Glc(β1-

Glc(α1-3)Glc(β1-2)[Glc(β1-3)]Glc(β1- 37

19 Rebaudioside Q3

Glc(β1-

Glc(α1-4)Glc(β1-3)[Glc(β1-2)]Glc(β1- 37

20 Rebaudioside I3b

Glc(β1-2)[Glc(β1-6)]Glc(β1-

Glc(β1-2)Glc(β1-

11

21 Rebaudioside M/ Rebaudioside X

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

25,26,35,38

Glc(β1-

Glc(β1-2)[Fru(β2-3)]Glc(β1-

33

Glc(β1-

Rha(α1-2)Glc(β1-

39,40

H-

Rha(α1-2)[Glc(β1-3)]Glc(β1-

26,39–41

Glc(β1-

Rha(α1-2)[Glc(β1-3)]Glc(β1-

26,41

26 Rebaudioside C (isomer)

Rha(α1-2)Glc(β1-

Glc(β1-3)Glc(β1-

11

27 Rebaudioside H

Glc(β1-

Glc(β1-3)Rha(α1-2)[Glc(β1-3)]Glc(β1- 26

27

Fructosyl steviol family

22 Rebaudioside A3 Rhamnosyl steviol family

23 Dulcoside A 24 Dulcoside Ba 25 Rebaudioside C

c b

Continued

Table 1 Structures of Naturally Occurring Steviol Glycosides in Stevia rebaudiana Leaves—cont'd R2 (C-13/Hydroxyl) No. Steviol Glycoside R1 (C-19/Carboxylic Acid)

References

28 Rebaudioside K

Glc(β1-2)Glc(β1-

Rha(α1-2)[Glc(β1-3)]Glc(β1-

26,42

29 Rebaudioside J

Rha(α1-2)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

26,43

30 Rebaudioside N

Rha(α1-2)[Glc(β1-3)]Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

25,26,44

31 Rebaudioside O

Glc(β1-3)Rha(α1-2)[Glc(β1-3)]Glc(β1- Glc(β1-2)[Glc(β1-3)]Glc(β1-

25,26,45

32 Stevioside D

Glc(β1-

Qui(β1-2)Glc(β1-

46

33 Stevioside E

Glc(β1-

Qui(β1-2)[Glc(β1-3)]Glc(β1-

46

34 Stevioside E2b

Qui(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

11

35

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Qui(β1-2)[Glc(β1-3)]Glc(β1-

47

36

Qui(β1-2)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

42

37 Stevioside F

Glc(β1-

Xyl(β1-2)Glc(β1-

48

38 Rebaudioside F

Glc(β1-

Xyl(β1-2)[Glc(β1-3)]Glc(β1-

49–51

39 Rebaudioside F2

Glc(β1-

Glc(β1-2)[Xyl(β1-3)]Glc(β1-

48

40 Rebaudioside F3

Xyl(β1-6)Glc(β1-

Glc(β1-2)Glc(β1-

52

41

Xyl(β1-2)[Glc(β1-3)]Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

47

Quinovosyl steviol family

Xylosyl steviol family

R1 and R2 represent carbohydrate moieties attached at the C-19 carboxylic acid and the C-13 tertiary hydroxy group of the common steviol aglycone, respectively. All monosaccharides, except rhamnose (L), have D configuration and, except fructose (furanose ring form), are in the pyranose ring form. The included references contain relevant chemical, NMR, MS, and/or HPLC analysis data. a Steviolmonoside (1), steviolbioside (4), rebaudioside B (7), and dulcoside B (24) can be artifacts formed from rubusoside (3), stevioside (5), rebaudioside A (11), and rebaudioside C (25), respectively, due to de-esterification during isolation protocols. b No analytical details available. c In literature, rebaudioside C (25) is sometimes named dulcoside B.

7

Native (Carbohydrate-Modified) Stevia Glycosides

Apart from their sweetness, steviol glycosides, in higher doses and regular consumption, have been claimed to display important pharmacological/ therapeutic activities, such as antioxidant, antibacterial, antifungal, antiviral, antitumor, diuretic, and gastroprotective (antidiarrheal) activities, and to have immunomodulatory effects and a positive influence on renal function, blood pressure, and blood glucose. They suppress neoplastic growth, improve cell regeneration, and strengthen blood vessels. For recent reviews on medical aspects, see Refs. 3,5,6,56,61–67. The use of steviol glycosides as sweeteners can be particularly beneficial to people suffering from obesity (metabolic syndrome), diabetes mellitus (type II), hypertension, cardiovascular disease, hypoglycemia, dental caries, and candidiasis, and the S. rebaudiana leaves are even used as dressings for healing wounds and skin abrasions (eczema and dermatitis).68,69 Moreover, Stevia leaf extracts seem to have a positive therapeutic effect in the treatments of neuralgia, anemia, lumbago, rheumatism, and amnesia. Steviol glycosides are considered as noncarcinogenic, nongenotoxic, and are not associated with any reproductive/developmental toxicity in humans. The general safety and noncaloric properties of steviol glycosides could be largely due to the fact that they are only minimally broken down and absorbed in the upper gastrointestinal tract due to their resistance to digestive enzymes present in saliva and the small intestine. Likely all steviol glycosides are metabolized in the colon to steviol (the aglycone part; Fig. 2) by glycosidases of the human intestinal microflora (eg, Bacteroides sp.).11,70–74 Consequently, steviol glycosides might have prebiotic properties. Steviol itself has been shown to be toxic only at very high concentrations, much higher than is formed from the acceptable daily intake (ADI 4 mg/kg body weight) of steviol glycosides for humans, and, therefore, does not pose a risk of genetic damage following human consumption of steviol glycosides.75,76 Although adverse effects of steviol glycosides have not been observed so 12

OH

20

2

13

CH3

1

H 5

18

HO

6

H C 19

CH2 16

9

2

18

H3C

10 5

4

15

HO

11

7

15

19

8 7

4

H3C

14

9

10

3

1

3 17

11

O

6

CH3

8

20

12 17

14 13

16

O OH

Fig. 2 Mills and three-dimensional depictions of the chemical structure of steviol.

CH2

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Gerrit J. Gerwig et al.

far, it has been recommended that pregnant women should avoid consuming Stevia extracts and that these extracts should not be added to infant formulas.77 Patients with Asteraceae plant allergies should be advised that cross reactions to Stevia-based sweeteners are unlikely, but cannot be completely ruled out.78 It has to be noted that, in addition to steviol glycosides, the leaves of the Stevia plant also contain other phytoconstituents, such as polyphenols, flavonoids, carotenoids, tannins, phenolic acids, chlorogenic acids, fatty acids, amino acids, proteins, and vitamins.79–82 A disadvantage of most steviol glycosides, in particular stevioside (5), rubusoside (3), and dulcoside A (23) (Table 1), is that they have a slight bitterness and astringency, giving a lingering, unpleasant metallic aftertaste, which partially restricts their use for human consumption, and thereby limits their application in food and pharmaceutical products. To minimize the bitter aftertaste of steviol glycosides, microencapsulation methods have been applied by using spray-drying techniques with maltodextrin and inulin as encapsulation agents.83 The use of flavor enhancers and taste modifiers is a further approach to improve taste profiles. In some commercially available Stevia extract products, attempts to mask the licorice-like aftertaste are made with additives such as other (artificial) sweeteners, rice/corn maltodextrin, erythritol, maltitol, xylitol, sorbitol, vegetable glycerin, inulin, fructooligosaccharides, dextrose, or even cane sugar (sucrose). Alternatively, to improve their edulcorating qualities, especially for food applications, chemical/enzymatic modifications (extra glycosylation and/or trimming) of the carbohydrate moieties of steviol glycosides might be an effective option.4,84,85 In this review, we present a summary of the chemical structures of all steviol glycosides found so far, and we give an overview of products that have resulted from recent chemical and enzymatic carbohydrate modifications of the main steviol glycosides. When possible, claimed specificity of enzymes and sweetness of generated new products are included.

2. STEVIOL GLYCOSIDE STRUCTURES FROM S. REBAUDIANA Stevia glycoside preparations are commonly obtained by aqueous/alcoholic extraction from the leaves of the plant.6,86–89 The extract obtained initially is a dark particulate solution containing the steviol glycosides plus leaf pigments, soluble polysaccharides, and other impurities. Typically, the steviol glycosides are isolated further by different methods, eg, selective precipitation,

Native (Carbohydrate-Modified) Stevia Glycosides

9

ultrafiltration, or column chromatography, using adsorption or ion-exchange resins. The final product is commonly spray-dried. Chemical characterization studies of the major sweet components in the leaves have been carried out since 1908. A first preliminary structure of a steviol glycoside, called stevioside (5), appeared in 1955, followed by several additional structural studies14,90–95 and its organic synthesis starting from steviol in 1980.96 The stevioside structure was established to be a tetracyclic diterpene (ent-kaurene) glycoside built up from steviol, ent-13hydroxykaur-16-en-19-oic acid, as the aglycone (Fig. 2), esterified at the C-19 carboxylic acid function (R1) with a β-D-glucopyranosyl unit and substituted at the tertiary hydroxyl function at C-13 (R2) with a β-sophorosyl disaccharide, chemically formulated as 13-[(2-O-β-D-glucopyranosyl-β-Dglucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (5) (Fig. 3; Table 1). Although the carboxyl group in ent-kaurene-type diterpenoid structures is indicated as C-18 in IUPAC documents [IUPAC name for steviol: (5β,8α,9β,10α,13α)-13-hydroxykaur-16-en-18-oic acid; note that C-4 has the 4α configuration], the steviol glycoside literature nearly always uses C-19. To eliminate confusion, the latter system is followed in this review, whereby the C-19-linked glycan is generally denoted R1 and the C-13-linked glycan R2. At the present time, 43 different naturally occurring steviol glycosides from S. rebaudiana Bertoni have been identified, which are summarized in Table 1 (including references containing analytical details of the structural analyses) and the Addendum. In most cases, steviol (Fig. 2) is substituted at C-19 (R1) and C-13 (R2) with single β-D-glucopyranose units or relatively small oligosaccharides composed of D-glucopyranose units, whether or not containing additional single D-fructofuranose, L-rhamnopyranose, D-quinovopyranose (6-deoxy-D-glucopyranose), or D-xylopyranose units. For this reason, it has been proposed to classify the steviol glycosides, taking into account the type of monosaccharide residues present, into five groups as follows: glucosyl steviol family, fructosyl steviol family, rhamnosyl steviol family, quinovosyl steviol family, and xylosyl steviol family.11 In this context, the groups of Chaturvedula and Prakash must be mentioned for their extensive structural analyses of many steviol glycosides by NMR spectroscopy. Making use of tandem mass spectrometry, fragmentation schemes have been developed for steviol glycosides; besides the components proven by NMR spectroscopy, some additional components have been traced but not yet structurally characterized.97

1

3

9

2

18

H3C

H3C

11

7

15

O

O

6

CH3

8

11

7

15

19

12

20

10 5

4

19

9

2

18

10 5

4

1

3

O

O

6

CH3

8

17

14

O

16

13

12

20

17

14

CH2

O

OH

16

13

OH O

(5)

HO

O

(11)

HO O

OH

O

OH O

HO OH

O HO HO

OH OH

O HO OH

O HO

OH

HO

OH

HO HO

O HO

HO O

OH HO HO

Fig. 3 Chemical structures of stevioside (5) and rebaudioside A (11).

CH2

Native (Carbohydrate-Modified) Stevia Glycosides

11

Recently, conformational studies of rebaudioside A (11) (Fig. 3; Table 1) using NMR spectroscopy,31,98 molecular modeling,31,98 and X-ray diffraction,99,100 including its aglycone steviol,25,101 have been reported. The single-crystal X-ray structure of rebaudioside A4H2O1CH3OH showed a folding back on itself, with intramolecular hydrogen bonds between the OH-3 group (O97) of the C-19-ester-linked Glc(β1- residue and the OH-2 group (O41) of the Glc(β1-2) residue in the C-13 Glc(β1-2) [Glc(β1-3)]Glc(β1- trisaccharide moiety, and between the OH-2 groups of the Glc(β1-2) (O41) and Glc(β1-3) (O33) residues in the C-13 Glc(β1-2) [Glc(β1-3)]Glc(β1- trisaccharide moiety (Fig. 4). In addition, three other solvated crystal forms could be isolated.100 The conformational changes in rebaudioside A (conformational landscape) in water, established at a range of temperatures (5–60°C) using NMR and MD simulations, have been explored in a correlation with the parabolic change in sweet taste intensity with temperature (at moderate temperatures rebaudioside A is less sweet than at low and high temperatures). Interestingly, at 5°C the C-13-linked β-Glc residue is suggested to be in a 1,4B/1S3 conformation instead of in the usual 4C1 conformation.98 Aiming to alter the strength of the rebaudioside A–receptor interaction via controlled interactions with a binding protein, bovine serum albumin, thereby moderating its taste, a comprehensive saturation transfer difference NMR study has appeared.102

Fig. 4 Single-crystal X-ray structure of rebaudioside A (11). Reprinted with permission from Upreti, M.; Smit, J. P.; Hagen, E. J.; Smolenskaya, V. N.; Prakash, I. Single Crystal Growth and Structure Determination of the Natural “High Potency” Sweetener Rebaudioside A. Cryst. Growth Des. 2012, 12, 990–993. Copyright 2012 American Chemical Society.

12

Gerrit J. Gerwig et al.

It has been well established that both stevioside (5) [5–20% (w/w) of dried leaves] and rebaudioside A (11) [2–5% (w/w) of dried leaves] are the two major components in S. rebaudiana leaf extracts, followed in lower concentrations by rebaudioside B (7), C (25), D (13), E (10), F (38), steviolbioside (4), rubusoside (3), dulcoside A (23), and other steviol glycosides in very minor amounts (Table 1). The relative concentration of the different glycosides may vary depending on the Stevia leaf origin (genotype, phenological stage) and cultivation/climatic conditions (harvesting time of the plant). However, some studies have shown that increased amounts of steviolbioside (4) and rebaudioside B (7) might have been formed by partial hydrolysis during extraction processes of the S. rebaudiana leaves.103,104 Over the years, several reviews/studies have appeared on analytical methods3,6,7,105 employed for the characterization, distribution, and quantification of steviol glycosides, such as thin-layer chromatography/mass spectrometry,106 (HPLC) liquid chromatography,29,87,107–112 capillary electrophoresis,107,113 infrared spectroscopy,114 combined liquid chromatography–mass spectrometry,15,34,97,103,115,116 and NMR spectroscopy (Ref. 117 plus references in Table 1). Although, in general, LC protocols are applied for quantification purposes of steviol glycosides, a quantitative NMR (qNMR) approach has also been reported for determining the content of stevioside (5) and rebaudioside A (11) in standards.118 Also a quantification protocol for steviol at the picomole level using HPLC has been published.119 Several reviews/studies have paid attention to the biosynthesis of steviol glycosides.3,7,54,63,68,120–124 Steviol, generated via the 2-C-methyl-Derythritol-4-phosphate (MEP) pathway from pyruvate, is the key intermediate for the differential glycosylations to afford steviol glycosides. Scheme 1 shows the biosynthesis of rebaudioside A with part of the MEP pathway, starting with geranylgeranyl pyrophosphate. A review on the in vivo and in vitro metabolism of steviol glycosides and their relationship with overall plant physiology was published recently.7

3. STEVIOL VARIANTS OF GLYCOSIDE STRUCTURES FROM S. REBAUDIANA Besides the compounds presented in Table 1, a small number of glycosides containing variants of the steviol aglycone core structure have been characterized as being naturally present in very minor amounts in S. rebaudiana leaf extracts. However, it is possible that some of these compounds are

3−

CH2

CH3

3−

OP2O6

(1)

(2)

H3C

H3C

CH3

ent-Kaurene

Glc(β1 OH

11

(4)

2

13

CH3

5

H

18

HO

13

(5)

H

H C

HO

H3C

O

19

HO

Steviolmonoside

13

CH3

O 13

CH2

CH3

(8)

(7) H

H

Glc(β1

O

O

H

Glc(β1 Stevioside

H

H3C

C O

19

H C 19

O

Steviolbioside

Glc(β1-2)[Glc(β1-3)]Glc(β1 O

C

CH2

(6)

H3C

Glc(β1-2)Glc(β1

19

CH3

H

Steviol

H3C

ent-Kaurenoic acid

15

O

19

O

O 13

CH2

CH3

16

6

H C

C

Glc(β1-2)Glc(β1 O

CH2

8 7

4

H3C

14

9

10

3

HO

17

20 1

CH2

(3)

ent-Copalyl pyrophosphate (CPP)

Geranylgeranyl pyrophosphate (GGPP)

12

CH3

OP2O6

CH2

(1) ent-Copalyl pyrophosphate synthase (CPSent) (2) ent-Kaurene synthase (KSent) (3) ent-Kaurene 19-oxidase (KOent) (3 steps) (4) ent-Kaurenoic acid 13-hydroxylase (KAHent) (5) Glucosyl transferase UGT 85C2/UDP-Glc (6) Glucosyl transferase UGT?/UDP-Glc (7) Glucosyl transferase UGT 74G1/UDP-Glc (8) Glucosyl transferase UGT 76G1/UDP-Glc

O

Rebaudioside A

Scheme 1 Biosynthesis of steviol glycosides from geranylgeranyl pyrophosphate in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway.

14

Gerrit J. Gerwig et al.

artificial products of degradation and/or transformation events during extraction/isolation procedures under acidic or alkaline conditions (see Section 4). So far, only one naturally occurring compound built up from the isomeric aglycone of steviol, 16-methyl-13-oxo-17-nor-ent-kauran-19-oic acid, also formulated as 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid (8β,13β), called isosteviol (Fig. 5A), with a β-D-glucose residue at the C-19 carboxyl group (R1), has been reported.125 It should be noted that incubation of steviol with acid leads directly to isosteviol via a Wagner– Meerwein rearrangement (Scheme 2).126 Also diterpene glycosides containing the aglycone ent-13-hydroxykaur-15-en-19-oic acid, whereby the C-17 methyl group can be replaced by a hydroxymethyl (17-hydroxy) or an aldehyde (17-oxo) group (Fig. 5B), and decorated with the steviosidetype of glycosylation, have been isolated.52 Here, the exocyclic double bond between C-16/C-17 of the steviol core has been migrated to the endocyclic position between C-15/C-16 within the five-membered ring. This aglycone variant with R3 ¼ CH3 (Fig. 5B) occurs with rebaudioside B-type glycosylation.125 Also, the diterpene ent-13,16β-dihydroxykauran-19-oic acid (Fig. 5C), containing rebaudioside B-type carbohydrate moieties, was found.125 Another example is the isomeric diterpene ent-12αhydroxykaur-16-en-19-oic acid (Fig. 5D), which has been shown to contain stevioside-like carbohydrate moieties on C-19 (R1) and C-12 (R2).25 Most recently, a new steviol glycoside from S. rebaudiana with an extra hydroxy group at C-15 in the diterpene core, ie, ent-13,15α-dihydroxykaur-16en-19-oic acid (Fig. 5E), called 15α-hydroxy-rebaudioside M, containing a Glc(β1-2)[Glc(β1-3)]Glc(β1- moiety on C-13 as well as on C-19, was published.127

4. STABILITY OF STEVIOL GLYCOSIDES In view of the application of steviol glycosides as noncaloric sweeteners in beverages and in the food industry in general, the possible degradation of specific Stevia components has been studied. Acid-catalyzed hydrolysis of steviol glycosides gives isosteviol, together with released constituent monosaccharides. Incubation of steviol glycoside mixtures, containing mainly rebaudioside A (11), and minor amounts of rebaudioside B (7), rebaudioside F (38), rebaudioside Q3 (19) (Table 1), and the ent13-hydroxykaur-15-en-19-oic acid-based variants of stevioside and rebaudioside A with R3 ¼ CH3 and CH2OH, respectively (Fig. 5B), in

17 12

A

B

11 20 1

2

16

CH3

9

10

1

13 2

8

18

R1O

C 19

16 2

13

CH3

1

8

18

O R1O

10

C

OR2

18

20

CH3

1 2

13 14

9

10

E

2

5 4

18

6

H

H3C

C 1

R O

19

15

5

3

6

H

H3C

C

O

ent-12α-Hydroxykaur-16-en-19-oic acid R1 = R2 = H

7

4

18

1

R O

19

16

8

H 7

CH2

14

9

10

H 3

OR2 13

CH3

1

O

17

20

16

19

15

ent-13,16β-Dihydroxykauran-19-oic acid R1 = R2 = H

11

CH2

8

C

R1O

12 17

11

OH

6

H

O

12

D

7

4

H3C

ent-13-Hydroxykaur-15-en-19-oic acid R1 = R2 = H R3 = CH3, CH2OH, CHO

Isosteviol R1 = H

16

8 5

3

6

H 19

15

7

4

H3C

14

9

H

5

3

6

H

20

H 14

7

4

H3C

11

R3

14

9

10

H 5

3

C

17 13

CH3

OR2 17 CH3

12

11 20

O

15

OR2

12

CH3

OH

15

H

O

ent-13,15α-Dihydroxykaur-16-en-19-oic acid R1 = R2 = H

Fig. 5 (A–E) Chemical structures of five steviol variants found in glycosides from S. rebaudiana.

OR2

12

12

11

17

20

CH3

1 2

13 14

9

10

CH3

1

16 2

H+

8

13

3

10

18

C 1

R O

19

15

16

18

7

C

O HO

15

18

6

H

H3C

C HO

Steviol glycoside

7

4

O

19

O

19

Steviol 17

17 12

12

CH3

11

2

16

CH3

15

9

+ 13

20

O

10

8 5

18

6

H

H3C

C HO

19

O 13

8

H 7

4

15

9

H 2

10

16

CH3

1

H 3

CH3

11

20 1

14

+

14

16

8 5

3

6

H

13

H

4

H3C

9

10

8 5

3

6

H

H3C

CH3

1

H 7

4

9

17

20

CH2

14

OH

11

2

H 5

17

20

CH2

12

H+

OH

11

3

5

7

4

18

6

H

H3C

C

O HO

19

14

O

Isosteviol

Scheme 2 Wagner–Meerwein rearrangement for the conversion of steviol into isosteviol under acidic conditions.126

15

CH3

Native (Carbohydrate-Modified) Stevia Glycosides

17

simulated formulations at different acidic pH values, temperatures, and times yielded six degradation products, depending on the applied conditions.17 Two steviol-based products were characterized, steviol-19-O-glucoside (2) and rebaudioside G (8) (Table 1), which can be seen as a partial and total hydrolysis of the Glc(β1-2)[Glc(β1-3)]Glc(β1- moiety at C-13 of rebaudioside A, respectively. Furthermore, besides de-esterification, the ent-13,16β-dihydroxykauran-19-oic acid-based variants of rebaudioside A and B (Fig. 5C), and an isosteviol-based compound (Fig. 5A), containing only a Glc(β1- residue at C-19, were identified, indicating that changes in the steviol aglycone structure indeed occur under acidic conditions. The finding of the ent-13,16β-dihydroxykauran-19-oic acid-based and isosteviol-based variants may raise the question whether such products, also reported as native products in S. rebaudiana leaves (see Section 3), are in fact degradation products formed during isolation. The same holds for the steviol glycosides 1, 4, 7, and 24 (Table 1), having a free carboxyl group (de-esterification).26,128 Incubation of rebaudioside M (21) under hydrolytic conditions at 80°C gave, besides deesterified rebaudioside M, also four minor degradation products, which turned out to be an ent-13-hydroxykaur-15-en-19-oic acidbased (Fig. 5B, R3 ¼ CH3) variant of rebaudioside M together with its deesterified form, an ent-13,16β-dihydroxykauran-19-oic acid-based (Fig. 5C) variant of rebaudioside M, and isosteviol (Fig. 5A) with a Glc (β1-2)[Glc(β1-3)]Glc(β1- moiety at C-19.129,130 Incubation of rebaudioside A under hydrolytic conditions gave rise to similar degradation products (except isosteviol variant), as presented for rebaudioside M.131 Although it has been reported that stevioside (5) was stable in buffer solutions in the range pH 2–10 for 4 h at 80°C132 and that rebaudioside A (11) appears to be more stable than stevioside, another study showed that long storage at pH < 2 at room temperature caused degradation and resulted in rare steviol variants.99 Strong acidic conditions for more than 2 days at about 50°C led to steviol glycoside isomers with the aglycone ent-13-hydroxykaur-15-en-17-methyl-19-oic acid (Fig. 5B).99 Furthermore, it was shown that stevioside and rebaudioside A were degraded when added to different carbonated soft drinks and stored for up to 72 h at 80°C.133 In an extensive study on the stability of steviol glycosides [stevioside (5), rebaudioside A (11), B (7), C (25), D (13), E (10), F (38), G (8), and dulcoside A (23)] in different food matrices, like soy drink, skimmed and fermented milk, and yogurt, no signs of decomposition were found.134

18

Gerrit J. Gerwig et al.

In a photostability study at pH
2.5, both rebaudioside A (11) and stevioside (5) showed only very minor degradation when subjected to sunlight. The observed degradation is mainly due to the applied acidic conditions. At first instance, rebaudioside A and stevioside gave rebaudioside B (7) and steviolbioside (4), respectively, due to partial de-esterification. The other formed products showed conversion of the steviol aglycone structure into the ent-13-hydroxykaur-15-en-19-oic acid-based (Fig. 5B; R3 ¼ CH3 or CH2OH) and ent-13,16β-dihydroxykauran-19-oic acid-based (Fig. 5C) aglycones.135 Also here, it is possible that the earlier reported minor native Stevia leaf products with aglycone structures different from steviol (Section 3) are degradation products, formed during the isolation process. In view of the reported contradicting results concerning the stability of steviol glycosides, further study in this area appears necessary. Finally, another aspect concerning stability is the fact that at high concentration, all steviol glycosides are likely to form micelles due to the hydrophobic core of these compounds, which also might influence the edulcorating properties of the steviol glycosides.

5. STRUCTURE–SWEETNESS RELATIONSHIP With respect to the structure–sweetness intensity relationship, it has been reported that the glycosyl residues at both C-19 (R1) and C-13 (R2) of the steviol core are essential for sweetness.26 Rebaudioside A (11) is considered as the sweetest steviol glycoside. Compared to stevioside (5) [R1 ¼ Glc(β1-; Table 1], steviolbioside (4), which is only missing glycosylation at the carboxyl group (R1 ¼ H; Table 1), had a lower sweetness.136 The number of glucose residues at the C-13 tertiary hydroxy group (R2) in case of R1 ¼ Glc(β1- also seems to influence the sweetness, as well as the quality of taste [rebaudioside A is less bitter than stevioside, and rubusoside (3) has the most bitter taste].84,137 Extending the C-19-ester-linked glycosyl unit led to an increase of sweetness.136 Rebaudioside D (13) seems to have an increased sweetness and the weakest bitter taste compared to other steviol glycosides. However, rebaudioside E (10) appears to be sweeter than rebaudioside D.86 On the other hand, it has been reported that the minor steviol glycoside rebaudioside M (21) is more potent, has higher sweetness intensity, and has a very slight licorice or bitter aftertaste compared to other steviol glycosides.130 So far, it seems that sweetness increases with the number of β-glucosyl residues and the bitterness perception also correlates with the total number

Native (Carbohydrate-Modified) Stevia Glycosides

19

of attached glucose units. Steviol glycosides bearing only few β-glucosyl residues have higher bitter intensities than steviol glycosides with more β-glucosyl residues.138 Replacement of the Glc(β1-2) unit in stevioside and rebaudioside A by a Rha(α1-2) unit, yielding dulcoside A (23) and rebaudioside C (25), respectively, led to a large decrease in sweetness.39 However, replacement of the Glc(β1-2) unit in rebaudioside A by a Xyl(β1-2) unit, yielding rebaudioside F (38), did not influence sweetness.50 Although it is still not exactly clear which parts of the steviol glycoside molecules are essential for the sweetness properties, it is obvious that the organoleptic properties are determined by both regioselectivity and linkage specificity of the glycosyl moieties. Notably, as mentioned in Section 2, rebaudioside A showed a change in sweet taste intensity with temperature due to molecular conformational changes. Rebaudioside A tasted significantly sweeter as the temperature dropped below ambient temperature.98 The sensations of bitter and sweet tastes are initiated by the interaction of molecules with the G protein-coupled receptors in the apical membranes of the taste receptor cells, which are typically clustered in groups within taste buds.139 It must be noted that still little is known about the human sweet taste receptor-binding/activating mechanisms.140–143 In a study on human psychometric and taste receptor responses to steviol glycosides, it was stated that one possible scenario to explain the decreased bitterness of steviol glycosides with increasing carbohydrate length is that the molecules become too bulky to fit into the taste receptor cell-binding cavity.138 Furthermore, it was found that hTAS2R4 and hTAS2R14 are the receptors that specifically mediate the bitter aftertaste of steviol glycosides. In fact, there is a genetic basis for steviol glycoside taste perception with various human populations, showing a differential ability to sense the bitter aftertaste.144 Recently, the binding patterns of steviol glycosides to the amino-terminal domain of the sweet taste receptor subunits T1R2 and T1R3, belonging to class C of the G protein-coupled receptor family, have been investigated to explore key binding interactions responsible for sweetness. Glide docking studies enabled prediction of the sweetness rank order of steviol glycosides by variation in docked poses with particular amino acids in T1R2 and T1R3. Rebaudioside A (11) was found to have maximum sweetness intensity, followed by rebaudioside E (10), rebaudioside D (13), rebaudioside B (7), stevioside (5), steviolbioside (4), and dulcoside A (23).145

20

Gerrit J. Gerwig et al.

6. CHEMICAL MODIFICATIONS OF STEVIOL GLYCOSIDES With the aim to understand what determines the quality of taste, searching for improvements, and also to investigate changes in cytotoxic affects, a diversity of modifications in native steviol glycosides using organic synthesis have been carried out over the years, including conversions between native steviol glycosides. In principle, aqueous alkaline hydrolysis (de-esterification at C-19 of the steviol core; Scheme 3) of stevioside (5) and rebaudioside E (10) gives steviolbioside (4), while rebaudioside C (25) gives dulcoside B (24), and rebaudioside A (11), D (13), I (14), M (21), J (29), N (30), and O (31) are all converted into rebaudioside B (7) (Table 1).23 In a similar way, steviolmonoside (1) can be obtained by alkaline treatment of rubusoside (3). For the de-esterification of stevioside, it has been established that the glucose residue at C-19 is released as 1,6-anhydro-β-D-glucopyranose (levoglucosan) due to its original β-linkage in combination with the highly sterically hindered carboxyl group of steviol.92 Treatment of stevioside and rebaudioside E (10) with a mixture of anhydrous LiI, 2,6-lutidine, and anhydrous methanol yielded the methyl glycosides of the C-19-ester-linked glucose and sophorose, respectively, together with steviolbioside (4).146 OR2 13

CH3

H

OR2 CH2

CH2

CH3

Alkaline conditions H

1 M NaOH, 80 °C, 2.5 h H3C R1O

H C 19

H3C O

R1 = saccharide R2 = saccharide

R1O

H C 19

O

R1 = H R2 = saccharide

Scheme 3 Alkaline treatment of steviol glycosides.

In an early study, chemical modification of steviol glycosides was carried out to improve their taste. Starting from steviolbioside (4), R1 ¼ H was converted into R1 ¼ (CH2)3SO3Na (sodiosulfopropyl ester), leading to a stevioside derivative with improved sensory properties over stevioside (5) (Scheme 4, track a). When the carboxyl function of steviol (Fig. 2) was converted into a sulfopropyl ester, the formed product, missing the

21

Native (Carbohydrate-Modified) Stevia Glycosides

OR2 13

CH3

CH2

H

H3C R1O

H C 19

O

1 (a) R = H R2 = Glc(β1-2)Glc(β1-

R1 = (CH2)3SO3Na R2 = Glc(β1-2)Glc(β1-

1 (b) R = H R2 = H

R1 = (CH2)3SO3Na R2 = H

1 (c) R = H R2 = H

R1 = (CH2)3SO3Na R2 = (CH2)3SO3Na

Scheme 4 Sulfopropyl ester/ether derivatives of stevioside and steviol.

acetal-linked β-sophorosyl fragment at C-13, exhibited only bitter taste or no sweet taste at all (Scheme 4, track b). However, when both the carboxyl and the hydroxyl functions of steviol were sodiosulfopropylated [R1 ¼ R2 ¼ (CH2)3SO3Na], the product that formed tasted mainly bitter but also exhibited a significant sweet taste (Scheme 4, track c).147 Further evaluation of a diversity of ester groups as replacement for the Glc(β1residue in stevioside (5) and rebaudioside A (11) demonstrated that the bitter-taste component in the natural glycosides may be eliminated by increasing the molecular hydrophilic character. Especially, no bitter-taste character was found when the Glc(β1- ester residues in stevioside and rebaudioside A were replaced by (CH2)2CH(SO3Na)2 and (CH2)3SO3Na groups, respectively.148 It has to be noted that, due to the uncontrolled reaction, multiple side products were also formed. In other studies, starting from steviolbioside (4), its C-19 carboxyl function has been glycosylated with different monosaccharides, ie, β-D-Xylp, α-L-Arap, α-D-Manp, β-L-Glcp, α-L-Rhap, and β-L-Quip, yielding a series of analogues of stevioside, not found in nature so far.149 In a similar way, the C-19 carboxyl function of steviolbioside was glycosylated with the disaccharide fragments L-Rhap-(α1-2)-D-Glcp-(β1-, L-Rhap-(α1-2)-DGalp-(β1-, and L-Quip-(α1-2)-D-Glcp-(β1-, respectively.136 Although

22

Gerrit J. Gerwig et al.

some differences in their levels of sweetness were recognized (90–300 times that of sucrose), an increase of sweetness compared to stevioside (5) was not seen; some of them were bitter-sweet. Note that D- and L-glucose played a similar role in the emerging of sweetness. Additionally, using steviol/ steviolbioside as precursors, the C-19 β-D-Galp-analogue of stevioside has been synthesized (Scheme 5).96 Using similar protocols, with deesterified rubusoside [steviolmonoside (1)] and stevioside [steviolbioside (4)] (alkaline saponification) as precursors, the C-19 β-D-Galp-analogues of rubusoside and stevioside were prepared, which were used as acceptors for the enzymatic synthesis of compounds 68–75 (see Section 7.5; Table 2).150 Starting from steviol, the chemical syntheses of steviol-19-O-glucoside (2) (Table 1) and steviol-19-O-glucuronide have been reported. The protocol comprised the acetylation of the C-13 hydroxyl function of steviol with acetic anhydride in pyridine, followed by glycosylation of the C-19 carboxyl group with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide or with methyl 2,3,4-tri-O-acetyl-α-D-glucopyranosyluronate bromide in the presence of K2CO3/tetrabutylammonium bromide/CH2Cl2/H2O, and subsequent deacetylation in Et3N/MeOH/H2O/hexane, and in case of the glucuronide, additional de-esterification with KOH/H2O.16 Starting from isosteviol, a similar protocol was followed for the synthesis of isosteviol-19O-glucoside. For the conversion of stevioside (5) into rebaudioside A (11), a chemoenzymatic route has been described (Scheme 6).128 Using Takadiastase® Y, a crude amylase preparation from Aspergillus oryzae, the terminal Glc(β1-2) residue at the C-13 site was removed (pH 4.0, 37°C, 80 h), yielding rubusoside (3), which was deesterified by alkaline treatment to give steviolmonoside (1). Then, steviolmonoside was converted into its 4,6-O-benzylidene derivative. The latter compound was a substrate for the chemical β-glucosylation at O-2 and O-3 of the C-13-attached β-D-Glcp residue and the free C-19 carboxyl group. After deprotection, rebaudioside A was finally obtained. In the early days, it had been shown via reduction experiments of the exocyclic C-16/C-17 methylene double bond of stevioside (C]CH2 ! CH–CH3; Pd–C/H2) that this bond was critical for sweetness.136 In more recent investigations, the double bonds in stevioside (5) and rebaudioside A (11) were converted into keto groups (C]CH2 ! C]O; OsO4–NaIO4), leading to products completely lacking the sweet taste, thereby confirming the earlier findings (Scheme 7).138,151 Additionally, chemical reduction using catalytic hydrogenation [Pd(OH)2/H2] of stevioside (5), rubusoside (3), and rebaudioside A (11), B (7), C (25), and D (13) (Table 1) yielded the corresponding compounds with CH–CH3 (C-17 αβ isomers) instead of

OR3

OR3 O

O

R3O

R3O

O

R3O

O

R3O O

OR3

O

O

OR3

Step 3

O

OR3

OR3

2,3,4,6-Tetra-O-acetylα-D-galactopyranosyl bromide 13

CH3

3

RO RO 3

CH2

13

Bu4NBr/toluene

CH3

3

RO RO 3

H

H OR4

H3C R1O

H C 19

CH2

OR4 O

H

H3 C

C

O R4 O

O

19

O

OR4

R1 = R3 = H (steviolbioside) Ac2O/pyridine

R1 = H R3 = Ac (heptaacetyl steviolbioside) Bis(tributyltin)oxide/ toluene

R3 = R4 = Ac

Step 1 NaOMe/MeOH

Step 4 R3 = R4 = H (β-D-Galp-analogue of stevioside)

Step 2 R1 = SnBu3 R3 = Ac

Scheme 5 Synthesis of the β-D-Galp-analogue of stevioside from steviolbioside (4). For the synthesis of stevioside itself, 2,3,4,6-tetra-O-acetylα-D-glucopyranosyl bromide was used.96

24

Gerrit J. Gerwig et al.

OR4

OR4 O

O

R5O R3O

R5O R3O

O

O OR3

O O

OR3

Takadiastaseâ Y (crude amylase)

OR3

13

CH3

3 R3O R O

13

CH2

CH2

CH3

H

H

OR3

OR3 O

H

H3C

R3O R3O

C O

19

O R3O R3O

O

H3C

C O

OR3

H O

19

OR3

R3 = R4 = R5 = H (stevioside)

R3 = R4 = R5 = H (rubusoside)

OR4

OR4

OR6 O

O

O

R5O R3O

R5O

R6O R6O

O OR3

O O

OR6

O OR6

O

3,4,6-Tri-O-acetyl-α-D-glucopyranose 1,2-(tert-Butyl orthoacetate)

5% NaOH/MeOH 13

CH3

CH2

OR6

chlorobenzene

13

CH3

6 R6O R O

H

CH2

H OR6

H3C

H C

19 R1O

O O

R6O R6O

H

H3C

C O

19

O

OR6

R1 = R3 = R4 = R5 = H (steviolmonoside) benzaldehyde/98% HCOOH R1 = R3 = H; R4,R5 = CHPh

R4,R5 = CHPh; R6 = Ac 1. 30% HOAc 2. 0.5 M BaO/MeOH R4 = R5 = R6 = H (rebaudioside A)

Scheme 6 Chemo-enzymatic conversion of stevioside (5) into rebaudioside A (11).

the exocyclic C-16/C-17 C]CH2 bond (Scheme 8). Also in this case, sensory evaluations of these derivatives indicated that the sweet taste was reduced by about 25–100%, depending on the formed product.152,153 Following the same protocol, hydro(deutero)genated forms (C]CH2 ! CH–CH3; C] CH2 ! CD–CDH2) of steviol glycosides were prepared for mass spectrometric analysis.34 In a search to study cytotoxic and antimicrobial activities, the carboxyl groups of steviol (Fig. 2), steviolbioside (4), and isosteviol (Fig. 5A) were converted into a series of different amides and/or amide dimers.154 To this end, the substrates were reacted with aliphatic alkylamines [NH2–(CH2)17– CH3; NH2–(CH2)8–CH]CH–(CH2)7–CH3] and alkyldiamines [NH2– (CH2)2–12–NH2] in the presence of benzotriazol-1-yloxtri(pyrrolidinol)

OH

OH O

O

HO

HO

O HO O OH

O HO O

1. Ac2O/pyridine 2. OsO4–NaIO4/THF/H2O 3. NaOMe/MeOH

O OH

OH

O OH

17 13

HO HO

13

CH2

CH3

HO HO

16

16

H

H

OH

OH O

HO

H

H3 C

C O

HO

19

O O

HO

OH

Scheme 7 Oxidation of the exocyclic C-16/C-17 methylene double bond of stevioside (5).

H

H 3C

C O

HO OH

O

CH3

19

O

OH

OH O

O

HO

HO

O HO

O HO

O OH

O

O

OH

Pd(OH)2/H2

OH 17 13

OH

EtOH.H2O

17 13

CH2

CH3

HO HO

O

16

H

H

OH

OH O

HO

H

H3C

C O

HO OH

19

O O

HO

H

H3 C

C O

HO

CH3

CH3

HO HO

16

OH

Scheme 8 Catalytic hydrogenation of the exocyclic C-16/C-17 methylene double bond of stevioside (5).

19

O

H

27

Native (Carbohydrate-Modified) Stevia Glycosides

OH O O

HO

O HO

OH

O

O O

OH O

CH2

OH

13

HO HO

O

13

OH CH3

CH2

HO HO

HO H3C

H

H HO OH

H C

H N

H

H3C

19

H3C

C N H

19

O

O

Fig. 6 Amide dimer of steviolbioside and NH2–(CH2)8–NH2.

phosphonium hexafluorophosphate (PyBOP) and diisopropylethylethylamine (DIEA) (Fig. 6 shows the dimer of steviolbioside). It was found that several of these compounds had cytotoxic effects on cancer and human embryonic lung cells and had enhanced activity against Gram-positive bacteria. In a more recent study, also to survey cytotoxic activities, a series of structural variants of steviol-19-O-(4,6-O-isopropylidene-β-D-glucopyranoside) and isosteviol-19-O-(4,6-O-isopropylidene-β-D-glucopyranoside) were synthesized.155 The chemical modifications that were introduced were directed to the C-15/C-17 part of steviol and the C-13/C-14 part of isosteviol and are presented in Fig. 7. It was concluded that some of these products were potential anticancer candidates. In a patent application,156 the chemical syntheses of rebaudioside D (13), I (14), M (21), N (30), and O (31) (Table 1) were described starting from rebaudioside B (7), which was generated from rebaudioside A (11). All these compounds have a Glc(β1-2)[Glc(β1-3)]Glc(β1- trisaccharide at the steviol C-13 site in common, but vary in the carbohydrate constituent at the steviol C-19 site. A great variety of carbohydrate-protecting groups and promoter systems/leaving groups for the oligosaccharide donors to be coupled to the free steviol C-19 site were proposed. It was stated that increasing the amount of rebaudioside D, usually present in concentrations <5% in Stevia leaf extracts, in a composition that includes rebaudioside A and/or other Stevia components can greatly overcome the bitter aftertaste. As a typical example, Scheme 9 shows the synthesis of rebaudioside D. First, rebaudioside A is converted into rebaudioside B by alkaline treatment, followed by acetylation of the free hydroxy groups of the trisaccharide at the steviol C-13 site. Then, the C-19 free carboxyl group is glycosylated with acetylated α-sophorosyl

OH 13

OH 13

CH2

CH3

15

R1O

H

O

15

O

CH2 H 3C

O

19

CH2 16

15

H

H C

CH3

16

O H3C

13

O

CH3

16

H

OH

R 1O

H C 19

16

C

R 1O

19

O

CH3

CH3 16

O

CH3

H

H 3C O

CH3

CH3

O

13 13

H

14

H

CH2 H3C R1O

H C 19

14

CH2 H 3C

O R 1O

O

H3C

O

R1 =

O O HO

H C 19

OH O

Fig. 7 A series of structural variants of steviol- and isosteviol-19-O-(4,6-O-isopropylidene-β-D-glucopyranoside).

29

Native (Carbohydrate-Modified) Stevia Glycosides

R3O

R3O R3O

R3O

R3O R3O

O O

OR3

O O

OR

O OR3

O

O

O

O

OR3

OR3

OR3

OR3

3

O OR3

O OR3

13

OR3

2. Neutralization

CH2

CH3

3 R3O R O

O

1. Aqueous KOH

13

CH3

3 R 3O R O

CH2

H

H OR3 O R3O R3O

H3C

C O

OR

19

H3C

H O

R1O

H C 19

O

3

R1 = R3 = H (rebaudioside B)

R3 = H (rebaudioside A)

Ac2O/4-dimethylaminopyridine/Et3N R1 = H; R3 = Ac

OR3

OR

OR3

3

R3O

R3O R3O

O

O O

OR3

O OR3

O

O

O R3O R3O

O OR3

Br

O OR3

OR3

R3 = Ac

13

CH3

R3O R3O

3 R3O R O

CH2

H

K2CO3/tetrabutylammonium bromide CH2Cl2/H2O

OR3 O R3O R 3O

H

H 3C

C O

19

O

O OR3

O OR3

R3 = Ac R3O R3O

1. NaOMe/MeOH 2. Neutralization R3 = H (rebaudioside D)

Scheme 9 Synthesis of rebaudioside D (13) from rebaudioside A (11).

bromide in CH2Cl2/K2CO3/tetrabutylammonium bromide, followed by deacetylation to afford rebaudioside D (13). Although of importance for academic studies on sweetness and taste of steviol glycosides, the industrial application of chemical methods to modify steviol glycosides is rather impractical, certainly in the case of multistep chemical reactions during the classical selective protection–deprotection oligosaccharide synthesis strategies. Furthermore, the use of toxic chemical reagents will cause problems for accepting these derivatives in the food industry. To overcome these problems, biocatalyst alternatives may be preferred and are more in line with the objectives of “green” chemistry.

7. ENZYMATIC MODIFICATIONS OF STEVIOL GLYCOSIDES Several types of carbohydrate-active enzymes (CAZymes)157 have been used in the glycosylation reactions of steviol glycosides. They comprise

30

Gerrit J. Gerwig et al.

glycosyltransferases using nucleotide sugar donor substrates, as well as glycoside hydrolases (glycosidases)/transglycosidases using oligosaccharides, polysaccharides, or monosaccharides as donor substrates. In the context of this review, these donor substrates are sucrose, maltose, cyclodextrins, starch, curdlan, pullulan, lactose, raffinose, and melibiose. Although glycosidases are in vivo mainly hydrolytic enzymes, many of them can be used in vitro for synthetic purposes as transglycosidases. They display robustness, stability, stereoselectivity, and broad substrate specificity. However, quite often, low regioselectivity and (minor) product hydrolysis (in contrast to glycosyltransferases) lead to mixtures of glycosylated products. Despite the limited specificity and moderate yields of these enzymes, the low cost of their glycosyl donor substrates (in contrast to the high costs of nucleotide sugars) is a major advantage for industrial applications. The structures of the enzymatically bioengineered steviol glycoside derivatives reported so far are summarized in Tables 2–8.

7.1 Cyclodextrin Glycosyl Transferase Systems Cyclodextrin glycosyltransferase (cyclodextrin glucanotransferase; cyclomaltodextrin glucosyltransferase; CGTase, EC 2.4.1.19) is a member of the α-amylase family 13 of glycoside hydrolases (GH 13). The enzyme is widely found in microorganisms, including mesophilic, thermophilic, alkaliphilic, and halophilic bacterial and fungal species. Although CGTase is predominantly used for the industrial production of cyclodextrins (CDs) [cyclomaltooligosaccharides; cyclic (α1-4)-linked oligosaccharides, mainly consisting of 6, 7, or 8 glucose residues (α-, β-, or γ-CDs)] from starch via a cyclization reaction (Scheme 10), more and more applications appear whereby CGTases are used to catalyze coupling and disproportionation reactions for the synthesis of modified oligosaccharides by using OH O HO HO OH OH

OR

OH

[(1 4)-α-D-Glcp]m O

O

CGTase

O HO OH

OR

CGTase

O HO OH

α-Cyclodextrin β-Cyclodextrin γ-Cyclodextrin

O

Starch

n

Scheme 10 CGTase-catalyzed formation of cyclodextrins and transglucosylated oligosaccharides from starch.

Table 2 α-Glucosylation of Stevioside, Rubusoside, Rebaudioside A, C-19-Gal-Stevioside, and C-19-Gal-Rubusoside Using Starch and Other Malto-Donor Substrates (See Text) in the Presence of CGTases as Biocatalysts (Glycosyl Extensions Are Marked in Italics) No. R1 (C-19/Carboxylic Acid) R2 (C-13/Hydroxyl) Acceptor References

42

Glc(β1-

Glc(α1-4)Glc(β1-2)Glc(β1-

Stevioside

162–165

43

Glc(β1-

[Glc(α1-4)]2Glc(β1-2)Glc(β1-

Stevioside

162–164

44

Glc(β1-

[Glc(α1-4)]3Glc(β1-2)Glc(β1-

Stevioside

162–164

45

Glc(α1-4)Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

162,164

46

[Glc(α1-4)]2Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

162,164,165

47

[Glc(α1-4)]3Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

162,164,166

48

Glc(α1-4)Glc(β1-

Glc(α1-4)Glc(β1-2)Glc(β1-

Stevioside

162,164

49

Glc(α1-4)Glc(β1-

[Glc(α1-4)]2Glc(β1-2)Glc(β1-

Stevioside

162,164

50

[Glc(α1-4)]2Glc(β1-

Glc(α1-4)Glc(β1-2)Glc(β1-

Stevioside

162,164

51

Glc(β1-

Glc(α1-4)Glc(β1-

Rubusoside

137,163,167

52

Glc(β1-

[Glc(α1-4)]2Glc(β1-

Rubusoside

137,163,167

53

Glc(α1-4)Glc(β1-

Glc(β1-

Rubusoside

137,167

54

[Glc(α1-4)]2Glc(β1-

Glc(β1-

Rubusoside

137,167

55

Glc(α1-4)Glc(β1-

Glc(α1-4)Glc(β1-

Rubusoside

137,167

56

Glc(β1-

[Glc(α1-4)]3Glc(β1-

Rubusoside

163,167

57

Glc(β1-

[Glc(α1-4)]4Glc(β1-

Rubusoside

163,167

58

Glc(α1-4)Glc(β1-

[Glc(α1-4)]2Glc(β1-

Rubusoside

167

59

[Glc(α1-4)]2Glc(β1-

Glc(α1-4)Glc(β1-

Rubusoside

167 Continued

Table 2 α-Glucosylation of Stevioside, Rubusoside, Rebaudioside A, C-19-Gal-Stevioside, and C-19-Gal-Rubusoside Using Starch and Other Malto-Donor Substrates (See Text) in the Presence of CGTases as Biocatalysts (Glycosyl Extensions Are Marked in Italics)—cont'd R2 (C-13/Hydroxyl) Acceptor References No. R1 (C-19/Carboxylic Acid)

60

[Glc(α1-4)]3Glc(β1-

Glc(β1-

Rubusoside

167

61

Glc(α1-4)Glc(β1-

[Glc(α1-4)]3Glc(β1-

Rubusoside

167

62

[Glc(α1-4)]2Glc(β1-

[Glc(α1-4)]2Glc(β1-

Rubusoside

167

63

[Glc(α1-4)]3Glc(β1-

Glc(α1-4)Glc(β1-

Rubusoside

167

64

[Glc(α1-4)]4Glc(β1-

Glc(β1-

Rubusoside

167

65

Glc(α1-4)Glc(β1-

Glc(α1-4)Glc(β1-3)[Glc(β1-2)]Glc(β1-

Rebaudioside A

166

66

Glc(β1-

[Glc(α1-4)]3Glc(β1-3)[Glc(β1-2)]Glc(β1-

Rebaudioside A

166

[Glc(α1-4)]3Glc(β1-3)[Glc(β1-2)]Glc(β1-

Rebaudioside A

166

67

Glc(α1-4)Glc(β1-

68

Gal(β1-

Glc(α1-4)Glc(β1-2)Glc(β1-

C-19-Gal-Stevioside

150

69

Gal(β1-

[Glc(α1-4)]2Glc(β1-2)Glc(β1-

C-19-Gal-Stevioside

150

70

Gal(β1-

[Glc(α1-4)]3Glc(β1-2)Glc(β1-

C-19-Gal-Stevioside

150

71

Gal(β1-

[Glc(α1-4)]4Glc(β1-2)Glc(β1-

C-19-Gal-Stevioside

150

72

Gal(β1-

Glc(α1-4)Glc(β1-

C-19-Gal-Rubusoside

150

73

Gal(β1-

[Glc(α1-4)]2Glc(β1-

C-19-Gal-Rubusoside

150

74

Gal(β1-

[Glc(α1-4)]3Glc(β1-

C-19-Gal-Rubusoside

150

75

Gal(β1-

[Glc(α1-4)]4Glc(β1-

C-19-Gal-Rubusoside

150

a

a

The authors mentioned both (α1-4) and (α1-6) for the same linkage.

Native (Carbohydrate-Modified) Stevia Glycosides

33

alternative acceptor substrates. The intermolecular transglycosylation reaction (glucose or maltooligosaccharides) creates exclusively (α1-4)-glycosidic linkages.158–160 CGTase also catalyzes hydrolysis of these linkages, depending on incubation conditions.161 Using CDs or starch as the glucose donor substrate, especially CGTases from Bacillus spp. have been evaluated as biocatalysts for the transglycosylation of steviol glycosides, with the aim to improve the edulcorating quality of these compounds; most reports focus on stevioside (5) and rubusoside (3) as acceptor substrates (Scheme 11). Note that in examples where the structures of products are presented (Table 2137,150,162–167), characterizations have been carried out by NMR spectroscopy, quite often combined with mass spectrometry and microchemical analyses (eg, methylation analysis). Incubation of stevioside (5) with CGTase from B. macerans and soluble starch as donor substrate (pH 5.4, 40°C, 8 h, followed by 28°C, 10 h) yielded a mixture of mono- to over deca-(α1-4)-glucosylated products. Nine products were purified and subjected to structural analysis.162 As presented in Table 2 (compounds 42–50), they reflect elongations of the C-13-β-sophorosyl unit and the C-19-ester-linked Glc(β1- residue with one to three Glc(α1-4) residues (major products were 42 and 43). When compared with stevioside, an improvement in both the intensity and quality of sweetness was observed for the C-13 mono- and di-(α1-4)-glucosylated products 42 and 43, but sweetness then decreased for the C-13 tri-(α1-4)glucosylated product 44. An (α1-4)-glucosylation at the C-19 glucosyl moiety led to a decrease in sweetness and an increase in bitterness, as experienced for products 45 and 46. It should be noted that the glycoside mixture obtained may contain large amounts of maltooligosaccharides, whereas the steviol glycoside products yields were low (
2–6%). In a much broader study, the (α1-4)-glucosylation of stevioside (5) was carried out with α- and β-CD as donor substrates and CGTases from various groups of microorganisms (mesophilic B. macerans, mesophilic B. circulans, thermophilic B. stearothermophilus, alkalophilic B. alcalophilus, and halophilic B. halophilus) as biocatalysts.164 The same set of products (42–50; Table 2) was isolated as reported for the incubation of stevioside with CGTase and starch.162 Compounds 42 and 45 were acknowledged as the most sweet in this investigation, which is in contrast with an earlier finding that (α1-4)glucosylation at the C-19 glucosyl moiety leads to a decrease in sweetness.162 Maximal yields of 42 plus 45 (30–40%, depending on the bacterial species) were obtained, using a ratio between stevioside and β-CD of 1:1 at pH 7 and 40–50°C.

OH

OH O

O

HO HO O OH

OH

O

13

R

CH2

13

O HO OH

OH C O

19

CH3

O HO

R H

OH

1

2

O HC 3

O

OH

CGTase Starch

CH3

HO HO

O O

O OH

OH

HO HO

HO HO

n

O

O

CH2

H

O HO

OH OH

H

O HO

O

OH

O HC 3

C O

19

H O

OH

m Stevioside

n,m = 0; Stevioside n and/or m = 1; R1 and/or R2 = H n and/or m >1; R1 and/or R2 = [(1 4)-α-D-Glcp]p

Scheme 11 CGTase-catalyzed (α1-4)-transglucosylation reaction with starch as donor substrate and stevioside as acceptor substrate (see Table 2 for more details of formed products).

Native (Carbohydrate-Modified) Stevia Glycosides

35

Making use of traditional, ultrasound-assisted or microwave-assisted conditions, the glycosylation of stevioside (5), incubated with CGTase from an alkalophilic B. firmus strain in the presence of β-CD, was evaluated.165 Best results were obtained by the microwave-assisted reaction; when using stevioside/β-CD in a molar ratio of 1:2, after 1 min at 50°C and pH 7, an optimal production of two products was observed, ie, compounds 42 (66% yield) and 46 (24% yield) (Table 2). Removal of aftertaste bitterness and an improvement in the sweetness index were claimed for both derivatives. To avoid rapid multi-substitution, it must be noted that the microwaveaccelerated transglycosylation conditions are very critical. For additional glycosylation experiments with stevioside (5), a wide range of different glucose donor substrates were tested in a study using a CGTase with a high transglycosylation activity, excreted by a newly characterized alkalophilic Bacillus sp. BL-12 strain, isolated from the soil of a hot water spring.168 The donor substrates evaluated were glucose, maltose, maltotriose, α-CD, β-CD, γ-CD, water-soluble dextrin (DP 4–6), maltodextrin (DP 4–7), corn starch, potato starch, and soluble starch, and the optimal reaction conditions were determined by varying the amounts of CGTase, stevioside, and donor substrates. The highest conversion yields (70–76%) were obtained with maltodextrin and soluble starch (pH 8.5, 40°C, 12 h). HPLC analysis of the reaction mixture showed putative mono-, di-, and oligo-glucosylated stevioside; however, the positions of the glucosyl residues were not determined. Most of the glucosyl residues could be cleaved from the products by incubation with amyloglucosidase, indicating the presence of terminal Glc(α1-4) units. More recently, transglycosylations directed to stevioside (5) with CGTases from Thermoanaerobacter (Toruzyme® 3.0 L), B. macerans, and Paenibacillus macerans, using α-amylase (B. subtilis)-treated cornstarch as donor substrate under different conditions (temperature, mass ratio of reactants, pH, and enzyme units), were investigated to improve the edulcorating quality.169 The aim was to identify conditions that allow synthesis of mainly mono- and di-glucosylated stevioside at a high conversion rate. The Toruzyme® 3.0 L preparation, showing the highest activity, gave rise under optimal conditions to a mixture of 14 stevioside derivatives (60°C, 10 U CGTase/g stevioside, pH 5–6; conversion of 77%), including mono- up to penta-glucosylated products, with the mono- and di-glucosylated compounds (
30%) as the main components (HPLC–MS analysis). Very high enzyme loading enhanced the formation of tri-glucosylated steviosides. Hydrolytic activity probably reduced the presence of higher substituted glucosylation products. However, structural data and edulcorating quality tests were not reported.

36

Gerrit J. Gerwig et al.

In additional studies using Toruzyme® 3.0 L, different glucosyl donors were compared in the transglycosylation of stevioside (5), and it was found that α/β-CDs and starches provided a transglucosylation yield up to 80% within 5 h, while mono- and disaccharides (glucose, maltose, sucrose) were not effective glucosyl donors.170 CGTase first converts α- and β-CDs into linear oligosaccharides (hydrolysis), followed by their use as donor substrates in a disproportionation reaction with stevioside. As mentioned earlier, CGTase cleaves mono- or disaccharides from starches, resulting in the formation of a covalent enzyme intermediate, followed by transglycosylation of stevioside. To get more information on the reaction pathway, testing different reaction parameters (power, temperature, time, ratio of substrates, and pH), the microwave-assisted transglycosylation of stevioside (5), catalyzed by Toruzyme® 3.0 L, using α- and β-CDs and gelatinized/hydrolyzed cornstarch as the glucosyl donor, was further investigated.171 Isomeric monoto penta-glucosylated steviosides (structures not mentioned) were quickly formed, using gelatinized starch, at 60°C in 3 min under low-power microwave irradiation, whereby mono- and di-glucosylated steviosides are the main products. Higher microwave power did not help the formation of higherglucosylated stevioside, but only increased the stevioside conversion and the yield of mono-glucosylated stevioside. Furthermore, a transglucosylation pathway of stevioside with CDs under microwave irradiation was proposed, showing no transformation between α-CD and β-CD, and that the ringopened intermediate of reducing saccharide (disproportionation) instead of glucose produced from CDs donated the glucosyl residue to stevioside. Also enzymatic modifications of other steviol glycosides using CGTases have been exploited. In an early study, it was shown that, next to stevioside (5) and steviolbioside (4), incubation of rubusoside (3) with CGTase produced by B. megaterium strain No. 5, using soluble starch as the donor substrate, produced a mixture of mono- to hexa-glucosylated products in a total yield of 52% (pH 5.4, 40°C, 8 h, followed by 28°C, 10 h).137 Both the C-13-acetal- and C-19-ester-linked Glc(β1- units were found to be elongated with Glc(α1-4) residues. The mono- and di-glucosylated rubusoside products were characterized in detail (compounds 51–55; Table 2). An improvement of the quality of taste, compared to stevioside and rebaudioside A, was observed for the C-13-elongated products, while a change of sweetness and taste for the worse was observed for the C-19-elongated products, demonstrating the influence on taste of the number and ratio of glucose units at these positions. The same modification of rubusoside was also carried out with the CGTase from B. circulans as biocatalyst.167 The 14 isolated and

Native (Carbohydrate-Modified) Stevia Glycosides

37

characterized products 51–64 (up to tetra-glucosylated products) are presented in Table 2. To achieve steviol glycosides, which are exclusively (α1-4)-glucosylated at C-13, stevioside (5) and rubusoside (3) were first deesterified at C-19 with alkali, affording steviolbioside (4) and steviolmonoside (1), respectively.163 Subsequently, (α1-4)-glucosylation of steviolbioside and steviolmonoside was performed by incubation with B. circulans CGTase in the presence of soluble starch as donor substrate (pH 5.4, 40°C, 8 h); γ-CD was added to increase the solubility of the glycosides by formation of inclusion complexes.172 Then, the glycans created at the C-13 tertiary hydroxyl function of the obtained mixture were acetylated, the C-19 carboxyl group was chemically β-glucosylated, followed by deacetylation, affording the mixture of (α1-4)-glucosylated stevioside and rubusoside derivatives, specifically elongated with one to four Glc(α1-4) residues at the C-13 site. The characterized compounds were stevioside derivatives 42, 43, and 44 and rubusoside derivatives 51, 52, 56, and 57 (Table 2). The derivatives were reported to have improved sweetness compared to stevioside.163 In additional experiments, after protection of the C-19-ester-linked Glc(β1- residue of rubusoside with a Gal(β1-4) residue (a B. circulans β-galactosidase-catalyzed transgalactosylation with lactose as the donor; see Section 7.5173), incubation with CGTase from B. stearothermophilus was performed, followed by enzymatic de-galactosylation with B. circulans β-galactosidase.167 The products obtained were 51, 52, 56, and 57 (Table 2). In a different approach, the C-19-ester-linked Glc(β1- residue in stevioside (5) and rubusoside (3) was chemically replaced by a C-19-ester-linked Gal(β1residue as a blocker for (α1-4)-glucosylation at the C-19 site.150 This replacement changed the taste somewhat for the worse in intensity and character of sweetness. Using CGTase from B. macerans and soluble starch as the donor substrate, the C-19-Gal modification of stevioside showed only elongation at the C-13-β-sophorosyl part with Glc(α1-4) residues (pH 5.4, 40°C, 8 h, followed by 26°C, 16 h). Using CGTase from B. stearothermophilus and soluble starch as the donor substrate, the C-19-Gal modification of rubusoside showed only elongation at the C-13-β-glucosyl unit with Glc(α1-4) residues (pH 5.4, 40°C, 4 h, followed by 45°C, 12 h). The isolated mono- up to tetraglucosylated products 68–75 were characterized (Table 2). Improved sweetness was observed for compounds 68, 69, and 72–74, while 70, 71, and 75 showed a reduction in intensity of sweetness. Testing CGTases from different bacteria (see above, Ref. 164) with 1:1 mixtures of stevioside (5) and rebaudioside A (11) as acceptor substrates and

38

Gerrit J. Gerwig et al.

soluble starch as donor substrate under various conditions (concentration, pH, and temperature) showed that the CGTases of B. stearothermophilus and B. macerans gave the best results in forming (α1-4)-glucosylated derivatives of the steviol glycosides (HPLC analyses), up to penta-(α1-4)glucosylated products. The generated products were transformed into mono- and di-(α1-4)-glucosylated forms by treatment with α-amylase, resulting in greater sweetness and more delicate taste than that of the high-molecular-weight forms. However, the resultant products were not structurally analyzed.174 Characterization of some products from a purified Stevia extract, incubated with the CGTase of B. stearothermophilus and starch, revealed the stevioside-related compound 47 and the rebaudioside A-related compounds 65, 66, and 67 (Table 2).166 In compound 65, the C-19 site was extended with a Glc(α1-4) residue, whereas the C-13 site was specifically elongated with a Glc(α1-4) residue at the Glc(β1-3) residue, not at the Glc(β1-2) residue. Compound 66 showed an extension of the Glc(β1-3) residue at the C-13 site with a maltotriose sequence. For compound 67, the authors showed an additional extension with one Glc(α1-6) residue at the C-19 site, as was drawn in figs. 1 and 5 of their paper,166 but in their abstract and conclusions, they indicate the linkage as (α1-4). In view of the substrate specificity of CGTases, we have chosen to depict (α1-4) in Table 2. In a recent transglycosylation study, stevioside (5) was exclusively transformed into a mono-glucosylated product in high yield. A CGTase from a Paenibacillus strain, isolated from Stevia planting soil, with soluble starch as the glucosyl donor substrate was used. The structure of the product, having a Glc residue attached to the β-sophorosyl moiety at the C-13 site of stevioside, was given as a possibility (42), but not proven. The sweetness and taste quality were significantly improved compared to stevioside.175 In the context of all studies focused on modification of steviol glycosides by (α1-4)-glucosylation with CGTases, it is interesting to note that the leaves of S. rebaudiana already naturally contain, in minor amounts, two rebaudioside A derivatives with a terminal Glc(α1-4) residue linked to the Glc(β1-2) residue (rebaudioside Q; 17)11 or to the Glc(β1-3) residue (rebaudioside Q3; 19)37 of the C-13 glycosyl moiety (Table 1). A complicating factor of the enzymatically obtained (α1-4)-glucosylated steviol glycoside derivatives with improved taste is that they are sensitive to breakdown in the human mouth by the amylolytic enzymes present in saliva,

Native (Carbohydrate-Modified) Stevia Glycosides

39

which could imply an increase of the caloric content of these compounds. No study on this feature was found in the literature.

7.2 α-Glucosidase Transglycosylation Systems Among the native steviol glycosides, found in the leaves of S. rebaudiana, in fact, four compounds with α-glucosylated units have been identified (Table 1). Rebaudioside Q (17) and Q3 (19), being extensions of rebaudioside A (11) with an extra Glc(α1-4) residue at the Glc(β1-2) or Glc(β1-3) residue of the C-13 glycosyl moiety, have already been mentioned in Section 7.1.11,37 Additionally, the presence of rebaudioside A with a Glc(α1-3) residue linked to the Glc(β1-2) residue of the C-13 glycosyl moiety (rebaudioside I2; 18)37 and stevioside with a Glc(α1-2)Glc(α1-4) extension of the Glc(β1- unit at the C-19 site (rebaudioside Q2; 16)27 have been established. In a study focused on the suitability of commercially available α-glucosidases to glycosylate stevioside (5), namely, α-glucosidases from baker’s yeast (type I) and yeast (type II), β-glucanases from A. niger (Finzym) and B. subtilis (Cereflo), pullulanase from Klebsiella sp., and β-amylases (Biozyme® A, C, M, L) from Aspergillus sp. or malt, only pullulanase and Biozyme® L led to positive results.22 Incubation of stevioside with pullulan, a polysaccharide built up of maltotriose units connected via (α1-6)-glycosidic linkages, in the presence of a pullulanase (α-1,6-glucosidase) preparation (pH 6.05, 50°C, 96 h) resulted in transglucosylation, yielding three major products 42, 43, and 46 (Table 322,176–181), that were also found in studies with CGTases (Table 2). It has remained unclear why Glc(α1-4) residues were incorporated by this preparation (either a new function or a contamination). The relative sweetness to sucrose of compounds 42 and 43 was somewhat better than stevioside; that of compound 46 a little bit worse with some bitter aftertaste. Incubation of stevioside with maltose in the presence of Biozyme® L (pH 5.45, 50°C, 32 h) yielded mainly three products, reflecting the attachment of a Glc(α1-6) residue at the C-19-ester-linked Glc(β1- residue (compound 76) or a Glc(α1-6) (compound 77) or Glc(α13) (compound 78) residue at the terminal Glc(β1-2) residue of the β-sophorosyl disaccharide at the C-13 site (Table 3). As Biozyme® L (β-amylase) is known to produce maltose from amylose hydrolysis, the unexpected transglucosylation may have been caused by a contaminating α-glucosidase in the preparation. The relative sweetness to sucrose of compound 77 is much lower than stevioside, whereas compound 78 has a bitter taste. Compound 76 showed a decrease in sweetness, but a remarkable improvement in the quality of taste (less bitter).

Table 3 α-Glucosylation of Stevioside and Rebaudioside A, Using Different Suitable α-Gluco-Donor Substrates (See Text) in the Presence of α-Glucosidases or Glucansucrases as Biocatalysts (Glycosyl Extensions Are Marked in Italics) R2 (C-13/Hydroxyl) Acceptor References No. R1 (C-19/Carboxylic Acid)

42

Glc(β1-

Glc(α1-4)Glc(β1-2)Glc(β1-

Stevioside

22

43

Glc(β1-

[Glc(α1-4)]2Glc(β1-2)Glc(β1-

Stevioside

22

46

[Glc(α1-4)]2Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

22

76

Glc(α1-6)Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

22

77

Glc(β1-

Glc(α1-6)Glc(β1-2)Glc(β1-

Stevioside

22,176

78

Glc(β1-

Glc(α1-3)Glc(β1-2)Glc(β1-

Stevioside

22

79

Glc(β1-

Glc(β1-2)[Glc(α1-6)]Glc(β1-

Stevioside

176

80

Glc(β1-

Glc(α1-6)Glc(β1-2)[Glc(α1-6)]Glc(β1-

Stevioside

176

81

Glc(α1-2)Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

177

82

Glc(α1-4)Glc(α1-2)Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

177

83

Glc(β1-

Glc(α1-3)Glc(α1-6)Glc(α1-3)Glc(β1-2)Glc(β1-

Stevioside

178

84

Glc(β1-

Glc(α1-6)Glc(β1-2)Glc(β1-

Stevioside

179

85

Glc(α1-6)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

180,181

86

Glc(α1-3)Glc(α1-6)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

180,181

87

Glc(α1-6)Glc(α1-6)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

180,181

88

Glc(α1-6)Glc(α1-3)Glc(α1-6)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

180,181

Native (Carbohydrate-Modified) Stevia Glycosides

41

In another study directed to the α-glucosylation of stevioside (5), incubation was performed (40°C, 4 h) with a starch hydrolysate in the presence of isoamylase (for debranching, creating linear dextrins) and dextrin dextranase (DDase) from Acetobacter capulatus ATCC 11894.176 The DDase enzyme is known to transfer (α1-4)-linked nonreducing terminal Glc residues of dextrins predominantly to nonreducing terminal Glc residues of various saccharides, forming (α1-6) linkages. Three major products 77, 79, and 80 (Table 3) were obtained, showing mono- and di-(α1-6)-glucosylation of the β-sophorosyl disaccharide at C-13; additionally minor tri- and higher-glucosylated stevioside products were detected in the HPLC analysis. Interestingly, both Glc residues of the β-sophorosyl moiety are acceptors for (α1-6)-glucosylation. No glucosylation of the C-19-ester-linked Glc(β1residue was observed. The relative intensity of sweetness of the compounds was lower than that of stevioside. More recently, a commercially available α-amylase preparation from B. amyloliquefaciens (1,4-α-D-glucan glucanohydrolase; EC 3.2.1.1; BAN® 480L) has been applied as biocatalyst for the α-glucosylation of stevioside (5), using soluble starch as the glucosyl donor.177 Under optimal conditions (pH 6.5, 70°C, 12 h, selected stevioside and starch concentrations) a conversion yield of 38% could be realized for stevioside, and five transglucosylated products were detected. Of the five modified stevioside products, the two major ones (96%) showed elongations of the C-19-ester-linked Glc(β1- residue with Glc(α1-2) (compound 81) and with Glc(α1-4)Glc(α1-2) (compound 82) units (Table 3). The three minor products corresponded with isomeric tri-glucosylated steviosides (LC–MS analysis). Sensory data showed that, compared with stevioside, the bitter aftertaste of the modified steviosides was significantly reduced. Surprisingly, rebaudioside A (11) turned out to be a poor substrate (only 1% conversion). In a related study, the α-amylase BAN® 480L was replaced by a commercial α-amylase preparation from A. oryzae (TAKA), giving rise to a mixture of one mono-, one di-, three tri-, and one tetra-glucosylated stevioside derivatives based on HPLC–MS analysis, with the mono-glucosylated one as a major peak.182 Under optimal conditions of substrates (stevioside/starch ratio 1:10), a conversion yield of 48% was obtained within 0.5 h at pH 6.4 and 70°C. Again, rebaudioside A was a relatively poor transglycosylation acceptor, yielding three minor peaks (13%), identified by MS as mono-, di-, and tri-glucosylated rebaudioside A. The modified steviol glycosides (exact positions of the extra glucose residues are unclear) were sweeter and exhibited less bitter aftertaste than stevioside at the same concentration. It should be

42

Gerrit J. Gerwig et al.

noted that α-amylase enzymes are known as hydrolases, which suggests that the transglycosylation activities observed are most likely due to the presence of contaminants. Glucansucrase enzymes from lactic acid bacteria also provide an interesting option for glycosyl modification of steviol glycosides. The different glucansucrases characterized have been shown to synthesize glucans with (α1-2), (α1-3), (α1-4), and/or (α1-6) linkages, using sucrose as cheap donor/acceptor substrate.183 Recently, the α-glucosylation of stevioside (5) has been reported, using sucrose as glucosyl donor substrate and alternansucrase (EC 2.4.1.140) from Leuconostoc citreum SK24.002 as biocatalyst.184 When incubated with sucrose, alternansucrases are known to catalyze the synthesis of glucans with alternating (α1-3) and (α1-6) linkages, affording the polysaccharide alternan (Scheme 12).185 Under optimal concentrations of substrates and catalysts, the highest transglucosylation yield of about 44% was achieved at pH 5.4, 20°C, 24 h. LC–MS analysis showed the formation of a mixture of nine products, including three mono-, three di-, and three tri-glucosylated stevioside derivatives. The major products comprised the mono- and di-glucosylated stevioside derivatives. One of the tri-glucosylated stevioside derivatives was characterized in detail (Scheme 12).178 It comprised a specific elongation at the C-13 site with an alternating Glc(α1-3)Glc(α1-6)Glc(α1-3) sequence attached to the terminal Glc(β1-2) residue (compound 83 in Table 3). The yield of the mono-glucosylated stevioside after 5 h significantly increased with increased reaction shaking velocity. This product (84) was characterized as stevioside with an additional Glc(α1-6) residue at the terminal Glc(β1-2) residue of the β-sophorosyl disaccharide at the C-13 site (Table 3; Scheme 12).179 Edulcorating quality tests were not reported. Very recently, the α-glucosylation of rebaudioside A (11) has been investigated, using the glucansucrase/glucosyltransferase Gtf180 enzyme from Lactobacillus reuteri 180 and sucrose as donor substrate.180,181 This enzyme was selected from a screening of more than 80 wild-type and mutant glucansucrase enzymes with different product specificity from several L. reuteri strains. When incubated with sucrose as the glucosyl donor/acceptor substrate, the Gtf180 enzyme produces a (1-3,1-6)-α-D-glucan polymer, built up from different lengths of isomaltooligosaccharides, interconnected by single (α1-3) bridges (Scheme 13). Truncation of the N-terminal variable domain of Gtf180, yielding the recombinant Gtf180-ΔN enzyme, had no significant effect on the product spectrum. However, mutagenesis of specific amino acid residues in the active site region of the Gtf180-ΔN enzyme yielded several mutants that produced modified α-glucans from sucrose.186–189

OH O HO HO

[3)-α-D-Glcp-(1 6)-α-D-Glcp-(1 ] n Alternan OH

O O

O OH O

O OH

OH O

13

HO HO

O

HO

HO

O OH

H

O

OH OH 13

CH3

HO HO

CH2

O HC 3

HO HO

Alternansucrase Sucrose

H

C O

O

19

OH OH

H

O

OH HO HO

CH2

CH3

HO HO

O HC 3

C O

HO HO

H

19

O O

O OH

OH

OH

O

O OH

OH

O

OH HO OH O

O

13

O

CH3

HO OH

HO HO HO HO

O

CH2

H

OH HO HO

O HC 3

C O

H

19

O

OH

Scheme 12 Structure of the alternan polysaccharide and major reaction products obtained from the incubation of stevioside (5) with sucrose in the presence of alternansucrase.

OH

OH

O

HO

HO HO

O

OH

O

O

O

OH

O

O

HO

HO HO

OH

OH O

O

O

OH

O

OH

Gtf180-ΔN Sucrose

OH

O OH

OH 13

CH3

HO HO

CH2

CH3

HO HO OH

H

CH2

H

O

OH HO HO

13

O HO HO

O HC 3

C O

OH

19

H O

HO HO

O HC 3

C O

19

H O

OH

Scheme 13 Composite structure of the Gtf180 polysaccharide (EPS180) and the major reaction product (85) obtained from the incubation of rebaudioside A (11) with sucrose in the presence of the Gtf180-ΔN enzyme.

Native (Carbohydrate-Modified) Stevia Glycosides

45

The glucansucrase Gtf180-ΔN enzyme was able to α-glucosylate rebaudioside A (11) specifically at the C-19 site (Scheme 13). Several mutants of Gtf180-ΔN displayed higher transglucosylating activity than Gtf180-ΔN. One mutant, Q1140E, even showed a conversion of 96% rebaudioside A into α-glucosylated products, in contrast to 55% conversion with nonmutated Gtf180-ΔN. Optimal incubation reactions were performed in 25 mM sodium acetate (pH 4.7), containing 1 mM CaCl2, 50 mM steviol glycoside, and 10 U enzyme/mL, whereby 1 M sucrose was added at t ¼ 0 and t ¼ 3 h (37°C, 24 h). Detailed structural analysis by NMR spectroscopy of the products obtained from rebaudioside A revealed an initial extension of the β-linked Glc residue at the C-19 carboxyl group with a Glc(α1-6) residue (major compound 85; Scheme 13; Table 3). Higher-glucosylated products showed elongation on this site with Glc residues mainly in alternating (α1-3)/(α1-6) and to a lesser extent in a successive (α1-6) way (eg, compounds 86, 87, and 88; Table 3). A comparative taste evaluation of the mono-glucosylated derivative (85) showed an increased and more natural sweetness and reduced bitterness compared to rebaudioside A.181 It should be noted that the presented examples, wherein enzymes were used that yield steviol glycoside derivatives with extensions of Glc(α1-3) and/or Glc(α1-6) residues, avoid the problems of product breakdown by saliva amylolytic enzymes, as mentioned in Section 7.1.

7.3 β-Glucosidase Transglycosylation and Deglycosylation Systems Incubation of stevioside (5) with curdlan, a (β1-3)-glucan, as glycosyl donor in the presence of an enzyme system consisting of β-1,3-glucanase and β-glucosidase from Streptomyces sp. W19-1 (pH 6.0, 55°C, 30 min) afforded several transfer products containing (β1-3)-linked Glc residues, in a total yield of 30%.190 The major mono- and di-glucosylated compounds 89, 90, and 91 (Table 4190–193) were isolated and characterized. It was shown that Glc(β1-3)-elongations are present at both the C-13 and C-19 sites of stevioside with some preference for the C-19 site. The bitter taste of the mixture was reduced compared to stevioside; however, their relative sweetness was lower than that of stevioside. In a similar approach, with the actinomycete strain K-128 cultured in a medium containing both stevioside and curdlan (35°C, 148 h), a major product (yield 20%) was isolated that revealed a Glc(β1-6)-elongation at the terminal Glc residue of the β-sophorosyl disaccharide at the C-13 site of stevioside (compound 92; Table 4).191 Interestingly, it was mentioned that when using the enzyme

46

Gerrit J. Gerwig et al.

Table 4 β-Glucosylation of Stevioside and Rubusoside, Using Different Suitable β-Gluco-Donor Substrates (See Text) in the Presence of β-Glucosidases as Biocatalysts (Glycosyl Extensions Are Marked in Italics) R1 (C-19/Carboxylic Acceptor References No. Acid) R2 (C-13/Hydroxyl)

89 Glc(β1-3)Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

190

90 Glc(β1-

Glc(β1-3)Glc(β1-2)Glc(β1-

Stevioside

190

91 [Glc(β1-3)]2Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

190

92 Glc(β1-

Glc(β1-6)Glc(β1-2)Glc(β1-

Stevioside

191

11 Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1- Stevioside

7

H-

93 Glc(β1-4)Glc(β1a

Glc(β1-2)[Glc(β1-3)]Glc(β1- Stevioside Glc(β1-

191,192 a

192

Rubusoside 193

Stevioside is partly converted into steviolbioside (enzymatic deglucosylation at C-19).

system of Streptomyces sp. DIC-108, a Glc(β1-3)-elongation occurred at the internal Glc residue of the β-sophorosyl disaccharide at the C-13 site of stevioside, yielding rebaudioside A (11) (Table 4).191 A similar feature was observed when stevioside (5) as donor/acceptor substrate was incubated with a cell-free extract from the fungus Gibberella fujikuroi ATCC12616 for 15 days at pH 5 and 28°C.192 HPLC–MS/MS analysis showed a stepwise hydrolysis of stevioside, giving rise to steviolbioside (4), steviolmonoside (1), and steviol, and the generation of nearly equal amounts of rebaudioside A (11) and rebaudioside B (7) (Table 4) in a total yield of 80%, whereas stevioside itself had disappeared nearly completely. It was stated that this protocol is highly suitable for taste improvement of Stevia extracts due to the increase of rebaudioside A, which has better organoleptic properties than stevioside. Note that steviol should be removed before use. The specific G. fujikuroi ATCC12616 enzymes involved in these conversions remain unknown. During a screening for β-glucosidases able to release the C-19-ester-βlinked glycosyl moiety in steviol glycosides, the β-glucosidase (CMGase) of the plant pathogenic actinomycete, Clavibacter michiganense, was found to have this hydrolytic activity, as shown for stevioside (5) [! steviolbioside (4)], rubusoside (3) [! steviolmonoside (1)], rebaudioside A (11) [! rebaudioside B (7)], and steviol-19-O-glucoside (2) (! steviol) (Table 1).193 Furthermore, a slow hydrolysis was seen for the oligosaccharide at the C-13 site: rebaudioside B ! steviolbioside ! steviolmonoside; the glycosyl linkage of the remaining Glc residue in the latter compound was

Native (Carbohydrate-Modified) Stevia Glycosides

47

found to be stable, as was the same linkage in rubusoside. Interestingly, in case of rubusoside as donor/acceptor substrate, a transglucosylation product was also found displaying a Glc(β1-4)Glc(β1- unit at the C-19 site (compound 93; Table 4). This compound was seen at an early stage of the reaction, but then its level decreased due to hydrolytic activity. In another study, incubation of stevioside (5) and rebaudioside A (11) with a β-glucosidase (FJGase) from Flavobacterium johnsonae demonstrated de-esterification at C-19 and no hydrolysis in the saccharide chain at C-13, yielding steviolbioside (4) and rebaudioside B (7), respectively.194 In contrast, incubation of rubusoside (3) with FJGase yielded both steviolmonoside (1) and steviol-19-O-glucoside (2) (Table 1), affording finally steviol, thereby demonstrating that both the single ester- and ether-β-linked Glc residue can be released. Furthermore, rubusoside and steviol-19-O-glucoside could be produced in high yields (12–24 h) from stevioside by the hydrolytic activity of a commercial β-glucosidase (Viscozyme® L, SSGase) from Aspergillus aculeatus.195 A similar conversion of stevioside into rubusoside by enzymatic hydrolysis was reported earlier for Takadiastase® Y, a crude amylase preparation from A. oryzae (Scheme 6).128 A high-yield preparation of rubusoside from stevioside was also reported for a β-galactosidase preparation from an Aspergillus sp. (see Section 7.5).20 In search for optimal production of steviol from steviol glycosides, a novel β-glucosidase (SPGase) was purified from Penicillium decumbens naringinase, which was able to hydrolyze stevioside (5) via rubusoside (3) and steviolmonoside (1) into steviol.196 More recently, 30 commercial hydrolyzing enzymes (having the mixed activities of pectinase, cellulases, hemicellulases, α-galactosidase, β-galactosidase, and/or β-glucanase) and a purified recombinant lactase (β-galactosidase) from Thermus thermophilus (expressed in Escherichia coli) were screened for conversion of stevioside (5) into rubusoside (3) by selective cleavage of the (β1-2) glycosidic linkage of the β-sophorosyl moiety at the C-13 site of stevioside. Only crude pectinases from A. niger and naringinase from Penicillium spp. could convert stevioside to rubusoside as the main product; the recombinant lactase showed the highest rubusoside productivity.197 A recombinant β-glucosidase (BGL1) from Streptomyces sp. GXT6 was found to produce rubusoside from stevioside at a yield of 78.8% in 6 h (pH 8.5, 50°C). The enzyme was also active on steviolbioside (4) but not on rebaudioside A (11).198 The use of different commercially available lipases (eg, Novozyme® 435, Lipozyme® TL100I, Lipozyme® TL IM, Lipozyme® RM IM, Lipase® AF-15, Amano® AY 30G) to release the Glc moiety from the C-19 site of stevioside has not been successful.199

48

Gerrit J. Gerwig et al.

7.4 α-Galactosidase Transglycosylation Systems In a comprehensive study, the α-galactosylation of rubusoside (3) has been investigated for a series of commercial α-galactosidases of various microbial origins, ie, from the fungi Mortierella vinacea and Absidia reflexa, the bacterium E. coli, or green coffee beans, with raffinose or melibiose as donor substrates.200 When using M. vinacea α-galactosidase and raffinose under optimal conditions (pH 6.0, 50°C, 15 h, 3 g rubusoside, 20 g raffinose), a major product with a Gal(α1-6) extension at the C-13-acetal-linked Glc(β1- unit (94, 12%) and a minor product with a Gal(α1-6)Gal(α1-6) extension at the same site (95, 3%) were formed (Table 5200). These results revealed that the enzyme preferred elongation at the C-13 Glc moiety. In case of the α-galactosidase from A. reflexa, the products 94 and 96 formed in equal amounts (
8%), reflecting a Gal(α1-6) elongation at the C-13 or the C-19 Glc residue, respectively. Just minor activities were found for the two other α-galactosidases from E. coli and coffee beans, yielding mainly compound 94. No taste studies were mentioned in the report.

Table 5 α-Galactosylation of Rubusoside, Using Different Suitable α-Galacto-Donor Substrates (See Text) in the Presence of α-Galactosidases as Biocatalysts (Glycosyl Extensions Are Marked in Italics) No. R1 (C-19/Carboxylic Acid) R2 (C-13/Hydroxyl) Acceptor Reference

94

Glc(β1-

Gal(α1-6)Glc(β1-

Rubusoside

200

95

Glc(β1-

[Gal(α1-6)]2Glc(β1-

Rubusoside

200

96

Gal(α1-6)Glc(β1-

Glc(β1-

Rubusoside

200

7.5 β-Galactosidase Transglycosylation Systems The β-galactosylation of rubusoside (3) has been investigated for a series of commercial β-galactosidases from various microbial origins, ie, the bacteria B. circulans and E. coli, the fungi A. oryzae and Penicillium multicolor, and the yeast Kluyveromyces lactis, with lactose as the donor substrate.173 Following incubation with B. circulans β-galactosidase (pH 6.0, 40°C, 20–160 min), four products 97–100 could be isolated (Table 6173). In the course of the reaction under different conditions (ratio of substrates), it was found that in the early stage of the incubation mainly the C-19-ester-linked

49

Native (Carbohydrate-Modified) Stevia Glycosides

Table 6 β-Galactosylation of Rubusoside, Using Different Suitable β-Galacto-Donor Substrates (See Text) in the Presence of β-Galactosidases as Biocatalysts (Glycosyl Extensions Are Marked in Italics) No. R1 (C-19/Carboxylic Acid) R2 (C-13/Hydroxyl) Acceptor Reference

97

Gal(β1-4)Glc(β1-

Glc(β1-

Rubusoside

173

98

Glc(β1-

Gal(β1-4)Glc(β1-

Rubusoside

173

99

Gal(β1-6)Glc(β1-

Glc(β1-

Rubusoside

173

100

Glc(β1-

Gal(β1-6)Glc(β1-

Rubusoside

173

Glc(β1- residue was elongated with a Gal(β1-4) residue (97), followed by the formation of minor amounts of a product with Gal(β1-4) elongation of the C-13-linked Glc(β1- residue (98). After reaching maximum levels of these products, a rapid decrease was observed together with the formation of the C-19 and C-13 Gal(β1-6) analogues 99 (major) and 100 (very minor), respectively. After 15 h of incubation, the amounts of transfer products were 97+ 98, 13.6%; 99, 12.3%; and 100, 0.9%. When using E. coli β-galactosidase, these amounts were 2.3%, 12.5%, and 0.5%, respectively. The β-galactosidases from A. oryzae and P. multicolor produced only low amounts of these isomers, whereas the β-galactosidase of K. lactis did not yield transgalactosylated products. From this study, it is clear that for the β-galactosidase enzymes studied, the C-19 site is the attachment site of choice. Surprisingly, during the evaluation of a β-galactosidase preparation from Aspergillus sp. CICIM F0620 for the transgalactosylation of stevioside (5), mainly hydrolytic β-glucosidase activity was observed.20 The enzyme preparation cleaved the (β1-2) linkage of the β-sophorosyl unit, thereby converting stevioside into rubusoside (3). Only minor transglycosylating activity was observed, yielding, tentatively, a mono- and a di-galactosylated product. The enzyme preparation showed no activity on rebaudioside A and rebaudioside C. In later studies, the action of a β-galactosidase from Sulfolobus sp. (S. solfataricus) on stevioside was investigated.201,202 In the absence of a galactose donor, total hydrolysis to steviol was observed. However, in the presence of lactose, three (galactosylated) products were formed, ie, mono-, di-, and tri-glycosylated stevioside (structures not determined) in a total yield of 87%. Using a β-galactosidase preparation from K. lactis in a packed bed reactor, stevioside could be converted into steviolbioside (4) in a yield of 90% within 6 h.199 It was mentioned that steviolbioside inhibits human breast cancer cells.

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7.6 β-Fructosidase Transglycosylation Systems Using the system stevioside (5) (acceptor substrate)/sucrose (donor substrate)/β-fructofuranosidase enzyme from Arthrobacter sp. K-1 (pH 6.5, 40°C, 30 min), the C-19-ester-linked Glc(β1- residue was efficiently (β2-6)fructosylated (furanose form) at a low acceptor concentration in a yield of 80%.19 When compared with stevioside, the isolated product, containing a Fruf(β2-6) residue linked to the β-Glc-C-19 unit (101; Table 719,203,204), showed an improvement in the quality of taste, even superior to that of rebaudioside A (11). After depletion of sucrose, hydrolysis of the fructosyl linkage was observed.205 Replacing stevioside by rubusoside (3) as acceptor substrate and using similar conditions (low acceptor concentration, pH 6.5, 40°C, 1 h) also showed that the C-19-ester-linked Glc(β1- residue was (β2-6)-fructosylated in high yield (88%).19 The quality of taste of the isolated product 102 (Table 7) was comparable with that of rebaudioside A. When replacing stevioside by rebaudioside A (11) and using Microbacterium sp. H-1 as source for the β-fructofuranosidase enzyme, under similar conditions (low acceptor concentration, pH 6.5, 40°C, 2 h), again only the C-19-ester-linked Glc(β1- residue was (β2-6)-fructosylated in high yield (80%), giving compound 103 (Table 7).203 Longer incubation times led to lower yields, due to enzymatic hydrolysis of the fructosyl linkage. When compared with rebaudioside A, some improvement in the quality of taste was noted. Using crude β-fructofuranosidase from Arthrobacter sp. 101137, a mixture of stevioside (55%) and rebaudioside A (28%) was (β2-6)-fructosylated at pH 6.5 and 30°C, yielding compounds 101 plus 103 (Table 7), as suggested from HPLC analysis (transfructosylating activity 65% after 15 h).204

7.7 β-Glycosyltransferase Glycosylation Systems Using UDP-Sugars Only a few studies concerning the modification of steviol glycosides, using β-glycosyltransferases in combination with nucleotide sugars as donor substrates, have been reported. Incubation of acceptor substrate rebaudioside A (11) with UDP-Glc as glucose donor and a β-glucosyltransferase UGTSL2 (produced in yeast) as biocatalyst showed a partial bioconversion (Scheme 14), yielding three products, namely, rebaudioside D (13) and two other components, denoted rebaudioside M2 (104) and rebaudioside D2 (105) (Table 835,36,206,207) (ratio A:D:M2:D2 ¼ 49:24:6:21).35,206 Clearly, the C-19-ester-linked Glc(β1residue is an acceptor for Glc(β1-2)- and Glc(β1-6)-elongations, including

Table 7 β-Fructosylation of Stevioside, Rubusoside, and Rebaudioside A, Using Sucrose as β-Fructo-Donor Substrate in the Presence of β-Fructofuranosidases as Biocatalysts (Glycosyl Extensions Are Marked in Italics) R2 (C-13/Hydroxyl) Acceptor References No. R1 (C-19/Carboxylic Acid)

101

Fruf(β2-6)Glc(β1-

Glc(β1-2)Glc(β1-

Stevioside

19,203,204

102

Fruf(β2-6)Glc(β1-

Glc(β1-

Rubusoside

19

103

Fruf(β2-6)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

203,204

Table 8 β-Glycosylation of Stevioside, Steviolbioside, or Rebaudioside A, Using UDP-Glc or UDP-Gal as Donor Substrate in the Presence of β-Glucosyltransferase or β-Galactosyltransferase as Biocatalysts (Glycosyl Extensions Are Marked in Italics) R2 (C-13/Hydroxyl) Acceptor References No. R1 (C-19/Carboxylic Acid)

13

Glc(β1-2)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

35

104

Glc(β1-2)[Glc(β1-6)]Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

35

105

Glc(β1-6)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

206

14

Glc(β1-3)Glc(β1-

Glc(β1-2)[Glc(β1-3)]Glc(β1-

Rebaudioside A

36

106

Glc(β1-

Gal(β1-4)Glc(β1-2)Glc(β1-

Stevioside

207

107

H-

Gal(β1-4)Glc(β1-2)Glc(β1-

Steviolbioside

207

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Gerrit J. Gerwig et al.

OH

OH

O

O HO

HO HO

O

O O

OH O

OH

OH

Rebaudioside A

OH

OH

O

O 13

CH3

HO HO

CH2

UGTSL2 UDP-Glc

H

HO

HO HO

O

O O

OH O

OH

OH

OH O H C 3

HO HO

C O

Rebaudioside D

H O

19

UGT76G1 UDP-Glc

HO

O

O OH

O H C 3

HO HO

O

OH O

OH

H O

O

O OH

13

CH3

HO HO

C 19

O

OH

Rebaudioside I

CH2

OH O

O

13

H

OH

OH

HO HO

CH3

HO HO

OH

CH2 HO HO

H OH

OH

O H C 3

O HO

HO HO

C O

O

H

19

O

OH

OH

OH

OH

OH

O

OH

HO

13

CH3

HO

H

OH 13

CH2

O

OH

CH2

H O

O H C 3

C O

19

H O

HO HO

O H C 3

C O

H

19

O

OH

O

O

CH3

HO HO

HO

O

HO HO

O OH

HO

OH HO HO

O

OH

OH

O

O O

O

HO HO

HO

HO HO

O O

OH

O

O

HO

HO HO

OH

OH O

O

Rebaudioside D2

OH

Rebaudioside M2 HO HO

Scheme 14 Bioconversion of rebaudioside A (11) into rebaudioside I (14), rebaudioside D (13), rebaudioside M2 (104), and rebaudioside D2 (105).

branching, affording linkage isomers of rebaudioside M (21) and D (13) (Table 1). The rare Glc(β1-6) residue has been found in rebaudioside L (15)26 and in rebaudioside A2 (12),33 although present in the C-13acetal-linked oligosaccharides (Table 1). Incubation of acceptor substrate rebaudioside A with UDP-Glc as glucose donor and β-glucosyltransferase UGT76G1-R11-F12 (produced in E. coli) as catalyst showed a partial

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bioconversion by introduction of a Glc(β1-3) residue at the Glc(β1-C19 site, affording rebaudioside I (14) (Tables 1 and 8) (A:I ¼ 77:23).26,36 Interestingly, with both β-glucosyltransferases only the C-19-ester-linked Glc(β1- residue functioned as an acceptor, not the C-13-acetal-linked trisaccharide Glc(β1-2)[Glc(β1-3)]Glc(β1- moiety. Bovine colostrum β-1,4-galactosyltransferase has been tested as a biocatalyst for the β-galactosylation of stevioside (5) and steviolbioside (4) using UDP-Gal as galactose donor.207 In both cases, the terminal Glc residue of the C-13-β-sophorosyl disaccharide could be elongated with a Gal(β1-4) residue in high yields of 81% (106) and 95% (107) (Table 8), respectively. The synthesized galactosylated products showed improved sweetness and decreased aftertaste. Recently, to improve the organoleptic properties of steviol glycoside products, an efficient (78%) enzymatic conversion of stevioside (5) into rebaudioside A (11) was reported, using recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana, together with the sucrose synthase AtSUS1 from Arabidopsis thaliana.208 The latter enzyme was used to catalyze the generation of the glucose donor, UDP-glucose, from sucrose and UDP. In a similar way, stevioside was converted to rebaudioside A using recombinant Saccharomyces cerevisiae, in which UGT76G1 was overexpressed as the whole-cell biocatalyst in a glucose-containing medium. The addition of citrate regulated the yeast metabolism toward enhanced synthesis of UDP-glucose.209

8. PATENTS REGARDING ENZYMATIC MODIFICATIONS OF STEVIOL GLYCOSIDES During the last two decades, several patents regarding modifications, including enzymatic transglycosylations, of steviol glycosides to improve the quality of taste have been honored. Some recent patents of interest for this review will be highlighted below. Unfortunately, in many cases, definite chemical structures were not presented. The patents focused on isolation protocols of steviol glycosides from S. rebaudiana plants and addition of Stevia sweeteners to food products are outside the scope of this review. As already mentioned in Section 6, the chemical synthesis of rebaudioside D (13), from rebaudioside A (11) via rebaudioside B (7), was described in a patent application, claiming the improved quality of taste of this product.156 Also, the patent describing novel isomers of steviol glycosides with modified steviol cores obtained by heating aqueous solutions under strongly acidic conditions for long time has already been mentioned (Section 4).99

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Concerning enzymatic modifications, an early patent claimed the process of producing a sweetener, containing α-glucosyl steviosides, by allowing an α-glucosyltransferase (eg, α-glucosidase, α-amylase, CGTase, dextransucrase) to react on an aqueous solution of stevioside (5) as the acceptor substrate and an α-glucosyl carbohydrate compound [eg, maltooligosaccharides, starch (hydrolysate), sucrose] as the glucose donor substrate.210 Nearly all stevioside was converted into products, containing one to three α-Glc residues, but structures of the products were not mentioned. The resultant reaction mixture possessed a pleasant sweetness, superior to stevioside. Instead of α-glucosylation, β-glucosylation of stevioside (5) or Stevia leaf extract was performed in another early patent,211 using enzymes having β-1,3-glucosyltransferase activity (eg, laminarinase) or microorganisms (eg, Streptomyces sp., Bacillus coagulans, Arthrobacter luteus) capable of producing these enzymes, together with curdlan [(β1-3)-glucan] or laminarin [(β1-3) (β1-6)-glucan] as the glucosyl donor. In a similar way, the β-glucosylation of stevioside was achieved using the β-1,4-glucosyltransferase enzyme from the yeasts Rhodotorula minuta or R. marina with cellobiose as the glucosyl donor substrate. The quality of (β1-4)-glucosylated stevioside was superior to that of stevioside, missing the bitterness and astringency. Furthermore, (β1-4)-galactosylation of stevioside by a transferase enzyme from the yeasts R. marina, R. minuta, or R. lactosa with lactose as galactosyl donor was investigated, and an improvement of the quality compared to stevioside was observed. However, structures of products were not mentioned. In a more recent patent, CGTase from B. stearothermophilus in the presence of starch was used for the transglucosylation of a steviol glycoside mixture of S. rebaudiana.212 A nonbitter, nonlingering sweet preparation was obtained, consisting of mainly mono-, di-, and tri-(α1-4)-glucosylated stevioside and mono-, di-, and tri-(α1-4)-glucosylated rebaudioside A, together with higher transglucosylated derivatives (DP up to 10) in much lower amounts. The attachment sites of the extra α-Glc residues were not mentioned. Transglucosylation of stevioside (5), rebaudioside A (11), and purified Stevia leaf extract, using starch as donor substrate and CGTases produced by B. stearothermophilus,213 Thermoactinomyces vulgaris,214 or B. halophilus214 as biocatalysts, gave a reaction mixture with a superior sweetness quality. Glucosylation of stevioside and rebaudioside A with 1 to over 10 α-Glc residues was observed on HPLC, but the positions of the extra α-Glc residues were not mentioned. In a later patent, the same authors again described the transglucosylation of a commercial Stevia extract, containing mainly stevioside, rebaudioside A, and rebaudioside C (25), using CGTase from

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B. stearothermophilus and starch as donor.215 Mono-, di-, and tri-α-glucosylated derivatives of stevioside and rebaudioside were detected as major products. In a series of patent applications,216–220 CGTase produced by B. stearothermophilus in the presence of liquefied starch was used for the transglucosylation of purified Stevia leaf extract, and in particular steviolbioside (4) and rebaudioside B (7). With respect to the Stevia leaf extract, HPLC analysis revealed products of stevioside (5) and rebaudioside A (11), which were mono- up to deca-(α1-4)-glucosylated. Eventually, the derivatives that formed were subjected to a β-amylase treatment to produce monoto tetra-(α1-4)-glucosylated derivatives. In another procedure, after removal of maltooligosaccharides, the mixture was subjected to a second enzymatic transglucosylation by the same CGTase/starch system, yielding a mixture of (α1-4)-glucosylated derivatives up to DP 20. The increase in the number of (α1-4)-glucosyl residues improved the taste quality, but at the same time reduced the sweetness level, although it was concluded that overall the samples with short-chain glycans (DP < 5) possessed better taste profiles compared to samples with long-chain glycans and derivatives with only one or two (α1-4)-glucosyl residues. For rebaudioside B, the sweetness and taste quality of derivatives with mono- up to deca-(α1-4)-glucosyl residues at the C-13 site were improved. The positions of the extra α-glucosyl units were not mentioned. By preparation of blends containing different ratios of steviol glycosides and α-glucosylated steviol glycosides, obtained by the CGTase/starch transglucosylation system, variations of sweet and bitter tastes were observed.221 Recently, the biocatalytic conversion of stevioside (5) to rubusoside (3) (Table 1) by enzymes with glycoside hydrolase activity (eg, β-glucosidase, cellulase, or hesperidinase) has been claimed, showing a yield of 98% within 24 h at 37°C for the enzyme hesperidinase from Aspergillus niger.222,223 This was directly followed by another patent application224 claiming methods of converting certain steviol glycosides particularly into rebaudioside A (11), D (13), and M (21), by using free or immobilized host-expressed UDP-glucosyltransferases (eg, UGT76G1 and UGT91D2) from S. rebaudiana Bertoni. A similar approach was also reported in Ref. 208. Finally, a few patent applications are available, dealing with the aim to produce commercially viable sweeteners that are molecularly identical to Stevia leaf extract components (Table 1), making use of recombinant host cells, such as yeast species, comprising the relevant biosynthetic genes (Scheme 1) that lead to the production of specific steviol glycosides [eg, rebaudioside A (11), D (13), and M (21)].225,226

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In a very recent patent, starting from rubusoside (3), the enzymatic preparation of a series of natural and nonnatural steviol glycosides has been reported.227 To this end, three recombinant β-glucosyltransferases, coded UGT76G1, EUGT11, and HV1, were explored. It was found that UGT76G1 has a β-1,3-glucosyltransferase activity not only for the C-13 site but also for the C-19 site. EUGT11 has a β-1,2-glucosyltransferase activity for both the C-13 and C-19 site, and in addition a β-1,6-glucosyltransferase activity for the C-13 site. HV1 has a β-1,2-glucosyltransferase activity for both the C-13 and C-19 site. UDP-Glc was used as glucosyl donor, eventually generated via a sucrose synthase system, whereby UDP-Glc can be continuously regenerated from UDP and sucrose. The naturally occurring steviol glycosides thus prepared comprised stevioside (5), rebaudioside KA (6), rebaudioside G (8), rebaudioside E (10), rebaudioside A (11), rebaudioside D (13), and rebaudioside M (21) (Table 1). The prepared nonnatural steviol glycosides are coded rebaudioside V [R1 (C-19 site) ¼ Glc(β1-2)Glc(β1-; R2 (C-13 site)¼ Glc(β1-3)Glc(β1-], rebaudioside W [R1 ¼ Glc(β1-2)[Glc(β1-3)] Glc(β1-; R2 ¼ Glc(β1-3)Glc(β1-], rebaudioside Z1 [R1 ¼ Glc(β1-2)Glc(β1-; R2 ¼ Glc(β1-2)Glc(β1-2)Glc(β1-], rebaudioside Z2 [R1 ¼ Glc(β1-2)Glc(β12)Glc(β1-; R2 ¼ Glc(β1-2)Glc(β1-], and rebaudioside D2 [R1 ¼ Glc(β1-2)Glc(β1-; R2 ¼ Glc(β1-2)[Glc(β1-6)]Glc(β1-]. Unfortunately, the name rebaudioside D2 was earlier used for the enzymatically prepared steviol glycoside with R1 ¼ Glc(β1-6)Glc(β1- and R2 ¼ Glc(β1-2)[Glc(β1-3)]Glc(β1(105) (Table 8).206

9. CONCLUDING REMARKS The demand for natural sweeteners as alternatives for sucrose is constantly increasing due to the worldwide ever-growing incidence of obesity, diabetes, hyperlipidemia, and dental caries in children. On the other hand, the use of artificial (synthetic) sweeteners, like aspartame, acesulfame, saccharin, sucralose, and neotame, is strongly discouraged, due to several toxic side effects.228 The many publications reviewed clearly show that the Stevia plant is likely to remain a major source of noncaloric high-potency sweeteners for the growing natural food market in the future.57,229 In Japan, for instance, Stevia already makes up more than 40% of the sweetener market. Many studies have also reported the health-promoting properties of Stevia. An increased number of S. rebaudiana plantation farms is found in subtropical regions, in countries such as Tunisia, China, India, Malaysia, Thailand,

Native (Carbohydrate-Modified) Stevia Glycosides

57

and Indonesia.230 But, although the sales of products that contain Stevia compounds as the sole alternative sweetener source have more than doubled in the last 4 years, the bitter aftertaste of steviol glycosides, perceived by almost half of the human population, is still a troublesome feature. During the last 10 years, a great deal of attention has been paid to improve the quality of taste and simultaneously to increase the sweetness of steviol glycosides. One approach to obtain steviol glycosides with good organoleptic properties is the development of new varieties of the S. rebaudiana plant.4,105,231,232 For instance, plants with a higher content of rebaudioside A and a reduced content of stevioside can be obtained through selection and intercrossing among various desirable genotypes. By selective breeding of S. rebaudiana Bertoni, a new cultivar S. rebaudiana Morita, and varieties thereof, were developed, producing a higher ratio of rebaudioside A relative to stevioside.26,233 Genetic engineering could provide a complementary tool for Stevia plant breeders to enhance the steviol glycosides content. Since many details of the biosynthetic pathway of steviol glycosides are well elucidated, the glycoside profile of the steviol glycosides could be biotechnologically manipulated by up- and/or downregulations of genes.123,234–236 Furthermore, recombinant production of desired steviol glycosides via engineered microbial host cells is a challenging option. In view of the worldwide increasing demand for Stevia-derived medicines, studies for rapid and large-scale production of Stevia plants and efficient extraction procedures are in full progress,230,237–246 as well as studies for the development of rapid analytical methods for quantitative profiling of Stevia glycosides in large numbers of Stevia leaf extracts.87,108,112,115 The main topic of this chapter is an overview of chemical and enzymatic modifications that have been performed with the aim of improving the organoleptic properties of steviol glycosides. In this respect, the modification of the carbohydrate moieties via in vitro carbohydrate–enzyme bioengineering is of special interest. From the foregoing survey, which reviews research on several different enzyme–glycosyl donor combinations by the application of CGTases, α- and β-glucosidases, glucansucrases, β-glucosyland β-galactosyl-transferases, α- and β-galactosidases, and β-fructosidases, it will be apparent that this is not as straightforward as it looks. A major problem is that there is a lack of knowledge in exactly understanding structure– sweetness and structure–taste relationships, which makes it difficult to develop straightforward protocols for improvements. The results obtained so far are not always in line with each other. Trans-α-glucosylation was

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studied the most. Several enzyme systems have been tested that introduce extra glucose residues by transglucosylation. Most of the early studies revealed that for enhancement of the intensity of sweetness of stevioside (5), rubusoside (3), and rebaudioside A (11), the elongation of the carbohydrate moiety at the C-13 site from one up to four α-glucose residues, under suppression of the α-glucosylation of the β-glucose residue at the C-19 site, might be desirable. In contrast, recent publications emphasize a more important role for the carbohydrate moiety at the C-19 site. Interestingly, in a recent study it was shown that a clean sweetness taste without bitterness could be obtained by replacing the C-19 glucose residue of stevioside by amino acids, eg, glycine or L-alanine.247 Also, studies to use positive allosteric modulators in Stevia formulations to inhibit the bitterness and enhance the sweet sensory are in progress.59 In conclusion, it can be stated that many more systematic studies are needed, for instance, in making use of organic chemical approaches to investigate large series of related variants. In case of positive results, biotechnological approaches should be worked out. There is also room for more explorative research, investigating the glycosylation specificity of new or engineered carbohydrate enzymes. Another aspect is that, since some steviol glycosides exhibit strong biological/pharmacological activities, they may be of importance for the development of novel drug substances.

ADDENDUM In addition to the 41 native steviol glycosides reported in Table 1, two new minor compounds have been identified in the leaves of S. rebaudiana, denoted rebaudioside R, belonging to the xylosyl family, and rebaudioside S, belonging to the rhamnosyl steviol family. In case of rebaudioside R, the C-19 saccharide is Glc(β1- (R1) and the C-13 saccharide is Glc(β1-2) [Glc(β1-3)]Xyl(β1- (R2). In case of rebaudioside S, the C-19 saccharide is Rha(α1-2)Glc(β1- (R1) and the C-13 saccharide is Glc(α1-2)Glc(β1- (R2).248

ACKNOWLEDGMENTS The authors acknowledge the financial support by the EU project NOVOSIDES FP7KBBE-2010-4-265854. This work was financially supported by the European Union/ European Regional Development Fund and by the Dutch Ministry of Economic Affairs, Agriculture and Innovation, the Dutch innovation program “Peaks in the Delta,” the Municipality of Groningen, the provinces of Groningen, Frysl^an, and Drenthe, and the Dutch Carbohydrate Competence Center.

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