Is Steviol Glycosides Healthy?

Stevia rebaudiana (Bertoni) Hemsl, also known as sweet leaf, sweet grass, dense chrysanthemum, sweet tea, etc., is a perennial herb in the Asteraceae family, native to the Amambay Mountains on the border of Paraguay and Brazil in South America [1]. Since its discovery in 1899, the plant has aroused the keen interest of many scholars. After its successful introduction in Japan in 1970, research into the cultivation, safety and chemistry of stevia quickly developed. In China, stevia was introduced in 1977 by scientific research institutions such as the Zhongshan Botanical Garden in Nanjing and the Chinese Academy of Agricultural Sciences. After successful trials, it developed rapidly, and to date it is cultivated in 23 provinces, municipalities and autonomous regions across the country. It has become the world's largest producer and exporter of stevia.

Stevioside is the sweet component in stevia, and is distributed in the leaves, stems, roots and other parts of stevia, but the content in the leaves is the highest [1], and its content changes with the growth process, reaching a maximum during the budding stage. Stevia is favored for its non-toxic, safe, and low-calorie properties. It is known as the “third sugar source in the world,” and research on its biological activity and product development has attracted the interest of many scholars at home and abroad.

This paper reviews the latest research progress on the chemical composition, extraction and purification process, pharmacological effects, and product development of stevia, combining the latest domestic and foreign literature.

1 Chemical composition and extraction process research

1. 1 Chemical composition

Wollwer-Rieck [2] reported in 2012 a comprehensive report on the chemical constituents of stevioside, flavonoids, phenols, etc. isolated from stevia. There are about 60 diterpenoid components, most of which are glycosides, but only two types of skeletons, kaurane and labdane. The type of squalane can be divided into four categories according to the different aglycones (as shown in Figure 1): I is 13-hydroxyl substitution, 16 and 17 double bonds, and 19 oxidation to a carboxyl group; II is 13-hydroxyl substitution, 15 and 16 are double bonds, and the 19 position is oxidized to a carboxyl group; III is 13 and 16 are substituted with 2 hydroxyl groups, and the 19 position is oxidized to a carboxyl group; IV is 16 is substituted with a carbonyl group, and the 19 position is oxidized to a carboxyl group.

Compounds in category I are the main chemical and sweetening components of stevia. Currently, 35 such compounds have been reported, of which 8 have a high sweetness and are the main components of commercial stevia (as shown in Table 1).

In addition, Markovic et al. [3] identified 88 compounds from Stevia leaves using GC-MS, including 17 monoterpenes, 32 sesquiterpenes, 2 diterpenes, and other mainly organic acid compounds.

1. 2 Extraction and purification process

1. 2. 1 Extraction process

All the glycosides in stevia are easily soluble in water. Water is an easily available, low-cost, and low-pollution solvent, and is therefore the extraction solvent commonly used in the industrial production of stevia.

Water decoction is still a traditional and economical extraction method commonly used in the industrial production of stevia. Scientific researchers have also been constantly improving and optimizing the decoction method. Chhaya et al. [4] optimized the decoction extraction process and concluded that the optimal extraction conditions were a liquid-to-material ratio of 1:14, extraction at 78°C for 56 minutes, and an extraction efficiency of 10.45%. This provides new extraction process conditions from an economically reasonable perspective.

With the progress of science and technology, some new extraction technologies have gradually come into the field of view of scientists. Among them, the continuous countercurrent extraction method for extracting stevia is an excellent modern industrial extraction method. Compared with the traditional process, its advantages are mainly simple operation, continuous production and short production time. Yu Jun et al. [5] extracted stevioside using three methods: three-stage countercurrent continuous extraction, three-tank tandem countercurrent extraction, and single-tank extraction. They found that the extraction rates of the three methods were similar, with the three-stage countercurrent continuous extraction having the shortest extraction cycle. The entire process only takes 20 min, and the extraction rate can still reach 10.1%.

In addition, Munish Puri et al. [6] and Feng Xue et al. [7] conducted a systematic study on the extraction process by adding enzymes such as cellulase, pectinase, and hemicellulase. It was found that when a single type of biological enzyme is used for assisted extraction, the hemicellulase has the highest stevia extract yield (about 14%) when the extraction time is 1 h, the extraction temperature is 60 °C, and the enzyme concentration is 3%. the highest stevia extraction rate (about 14%); if the enzymes are used in combination, the best extraction effect is obtained at an extraction time of 36-45 min, an extraction temperature of 51-60 °C, and a concentration of 2% for each of the three enzymes. Compared with the extraction method assisted by a single enzyme, the extraction rate and efficiency are both improved to a certain extent.

Xu Zhongwei et al. [8] also conducted a systematic study of the combined enzyme-assisted extraction method and determined that the optimal process for extracting stevia is as follows: enzyme dosage 0.20%, extraction temperature 50°C, extraction time 30 min, liquid-to-material ratio 1:11, pH 5.0, enzyme added in four equal portions, extraction for 7 times, and the final extraction rate can be as high as 13.93%. This method greatly improves the extraction rate, but the operation is the extraction method commonly used in the industrial production of stevia today. Scientific researchers are also constantly improving and optimizing the water decoction method. Chhaya et al. [4] optimized the decoction extraction process and concluded that the optimal extraction conditions were a liquid-to-material ratio of 1:14, extraction at 78 °C for 56 minutes, and an extraction efficiency of 10.45%. This provides new extraction process conditions from an economically reasonable perspective.

With the progress of science and technology, some new extraction technologies have gradually come into the field of view of scientists. Among them, the continuous countercurrent extraction method for extracting stevia is an excellent modern industrial extraction method. Compared with the traditional process, its advantages are mainly simple operation, continuous production and short production time. Yu Jun et al. [5] extracted stevioside using three methods: three-stage countercurrent continuous extraction, three-tank tandem countercurrent extraction, and single-tank extraction. They found that the extraction rates of the three methods were similar, with the three-stage countercurrent continuous extraction having the shortest extraction cycle. The entire process only takes 20 min, and the extraction rate can still reach 10.1%.

In addition, Munish Puri et al. [6] and Feng Xue et al. [7] conducted a systematic study on the extraction process by adding enzymes such as cellulase, pectinase, and hemicellulase. It was found that when a single type of biological enzyme is used for assisted extraction, the hemicellulase has the highest stevia extract yield (about 14%) when the extraction time is 1 h, the extraction temperature is 60 °C, and the enzyme concentration is 3%. the highest stevia extraction rate (about 14%); if the enzymes are used in combination, the best extraction effect is obtained at an extraction time of 36-45 min, an extraction temperature of 51-60 °C, and a concentration of 2% for each of the three enzymes.

Compared with the extraction method assisted by a single enzyme, the extraction rate and efficiency are both improved to a certain extent. Xu Zhongwei et al. [8] also conducted a systematic study of the combined enzyme-assisted extraction method and determined that the optimal process for extracting stevia is as follows: enzyme dosage 0.20%, extraction temperature 50°C, extraction time 30 min, liquid-to-solid ratio 1:11, pH 5.0, enzyme added in four equal portions, extraction for 7 times, and the final extraction rate can be as high as 13.93%. This method greatly improves the extraction rate, but the operation is complicated and the extraction cycle is long.

Ultrasonic extraction is an emerging extraction method, but there are few reports on its use for stevia extraction. Liu Jie et al. [9] systematically optimized the ultrasonic-assisted extraction method and obtained the optimal extraction conditions: an extraction temperature of 68 °C, an extraction power of 60 W, and an extraction time of 32 min, with an extraction rate of up to 12.2%. This method greatly reduces the extraction time compared with the traditional extraction method, while significantly improving the extraction rate. It is of great research value in the extraction process of stevia.

Vikas Jaitak et al. [10] concluded that the optimal extraction conditions for microwave extraction are an extraction temperature of 50 °C, an extraction time of 1 min, and an extraction power of 80 W. The extraction time is greatly reduced, but there is no significant improvement in the extraction rate compared with the traditional method. In addition, emerging extraction methods such as ultra-high pressure technology [11] and supercritical fluid extraction [12] have also been used in the research of improving the extraction process of stevioside.

The above new extraction methods all have significant advantages over traditional extraction methods, but there are certain obstacles to industrialization due to key factors such as cost issues or poor continuous operability. In view of this, the author believes that a variety of method combinations can be designed to extract stevioside according to the characteristics of each method. For example, ultrasonic extraction is inexpensive, has a short cycle, a high extraction rate, and can be operated continuously. Continuous countercurrent extraction of stevioside has the advantages of greatly improving the extraction rate and shortening the extraction cycle. Combining the ultrasonic principle with continuous countercurrent extraction to extract stevioside may be a feasible research direction.

1. 2. 2 Purification process

As water is currently the solvent of choice in industrial production, there are a large number of water-soluble impurities such as polysaccharides, proteins and tannins in the extraction solution, which are 3 to 6 times more concentrated than stevioside. If these impurities are not removed, they will greatly interfere with the next purification step. Therefore, it is particularly important to pretreat the extraction solution. At present, the main purification methods for stevioside in industrial production are alcohol precipitation, macroporous adsorption resin, chemical flocculation and membrane separation.

The alcohol precipitation method is simple, ethanol is readily available, it is less toxic, easy to recycle and reuse, and low in cost, and is a commonly used method for purifying stevioside. Fu Junfang et al. [13] found through experimental comparison that when the alcohol concentration reaches 80%, the impurity removal rate is 16.47%, which can almost completely remove impurities such as starch, polysaccharides, proteins, and inorganic salts.

In addition to the alcohol precipitation method, in traditional production, the common adsorption method is also often used to purify stevioside, that is, chemical flocculants and activated carbon are used to adsorb impurities, and then anionic and cationic resins are used for desalination and decolorization to achieve the purpose of purification. However, the selectivity of chemical flocculants [13] and modified attapulgite methods [13] is poor, and the loss rate of stevioside is relatively high. As the impurity removal rate increases, the loss rate also increases significantly. For example, in chemical flocculation impurity removal, , when the impurity removal rate is 24.12%, the loss rate is as high as 12.29%; when the impurity removal rate is 31.44%, the loss rate is 17.98%.

The macroporous adsorption resin separation technology has been increasingly widely used in the purification and separation of stevioside due to its stable physical and chemical properties, good selectivity, fast adsorption and exchange speed, and convenient regeneration. Zhang Qianghua et al. [14] screened eight macroporous resins, including 001 × 16 cation exchange resin, D941, AB-8, DM130, HPD-100, NKA-9, D392, and D3520, The resin with the best decolorization effect was selected as D941, and it was found that the optimal static decolorization conditions were an adsorption time of 90 min, a temperature of 45 °C, a pH of 8.5, and a resin dosage of 60 g/L.

In recent years, the research and application of membrane technology has developed rapidly. Membrane separation technologies such as electrodialysis, microporous filtration, ultrafiltration, and reverse osmosis are all of significance to the purification process of stevia. Chen Shaopan et al. [15] first used microporous filtration and ultrafiltration to remove macromolecular impurities, and then used reverse osmosis to concentrate the filtrate. They believe that this technology has the advantages of high flux, good effect, fast speed, and energy saving, but the complicated membrane cleaning process prolongs the entire production cycle and increases costs.

Zhao Yongliang et al. [16] passed the stevia leaf infusion through a primary membrane to remove impurities, then concentrated it through a secondary membrane, passed it through a macroporous resin, and spray-dried it to obtain the product stevioside. This process uses membrane separation technology to replace the flocculation process in the traditional production process. Since the membrane separation process does not allow the infiltration of colorless ions, it can simplify the ion exchange resin process in the traditional production process. Improving the application of the process has improved the purity of stevioside as well as production efficiency, and the membrane regeneration rate has been greatly improved. Yao Guoxin et al. [17] first used a two-stage membrane consisting of a microfiltration membrane and a ultrafiltration membrane to remove impurities from the stevioside water extract, such as pectin, pigments, and water-soluble proteins. The results showed that the liquid was concentrated 15 times after membrane treatment, and the purity reached about 87%.

Compared with traditional industrial purification processes for stevioside such as alcohol precipitation, flocculation, and macroporous resin column chromatography, membrane technology for purifying stevioside has the advantages of better purification results and faster speed. With the development of membrane technology and the progress of membrane regeneration technology, it is gradually replacing traditional processes in the industrial production of stevioside, and is the future trend of industrial production.

1. 2. 3 Purification and separation of rebaudioside A

Stevia extract after impurity removal and decolorization is a mixture of components, the main ones being stevioside, rebaudioside A and rebaudioside C. Stevioside and rebaudioside A account for a relatively large proportion of the extract. Of these components, rebaudioside A has the highest sweetness and best taste, and its mouthfeel is closest to that of glucose. Therefore, many researchers and manufacturers have used increasing the content of Rebaudioside A as a starting point for improving the quality of stevia products.

Li Pei et al. [18] studied the technology of separating rebaudioside A by recrystallization, and on this basis, Zhao Hao et al. [19] used the method of solvent extraction and crystallization to separate rebaudioside A. When 50% ethyl acetate was added to methanol, the solution concentration was 20 g/L, and the crystallization rate at a crystallization temperature of 18°C was 0.34, and the separation factor could reach 3.8. Liu Jie et al. [20] used methanol-isopropanol (99:1, v/v) to induce crystallization to separate lobetyolin A. The effects of the crystallization solvent, solid-liquid ratio, temperature, time, and addition of seed crystals were optimized were optimized. The results showed that the relative purity of a single recrystallization can reach 92.7%, the crystallization rate 0.61, and the purity of a second recrystallization can reach 95.8%, the crystallization rate 0.35. This method has the significant advantages of being simple to operate and low-cost, but the crystal lattice is prone to encapsulate the organic solvent used for crystallization (such as methanol, ethyl acetate, etc.) during the crystallization process, and it is difficult to remove the organic solvent encapsulated by the crystal lattice by ordinary means such as heating and drying, which can easily cause harmful organic solvent residues.

The macroporous resin adsorption method is one of the most commonly used methods for refining and separating stevioside. Hu Jing et al. [21] optimized several types of macroporous resins and found that D107 and D108 macroporous resins have strong separation ability for rebaudioside A and stevioside in stevia. D107 has a large adsorption capacity and can increase the content of rebaudioside A in stevia to more than 80%, while the adsorption capacity of D108 is less than that of D107, but it can achieve a content of 90% of stevioside A in stevia. Li et al. [22] used mixed-bed ion exchange resins to separate and purify stevioside A in stevia, which can increase its purity to 97%.

In addition, there are reports on advanced separation methods such as high-speed countercurrent chromatography [23], high-performance liquid chromatography [24], and capillary electrophoresis [24] for the separation and enrichment of rebaudioside A. However, these methods have low throughput and are generally only used for laboratory separation and identification.

2 Pharmacological activity and safety

As a new source of sugar, the pharmacological activity and safety of stevia have always been the focus of attention of many scholars. At present, the biological activities of stevia are reported to be very extensive, among which the main ones that have been studied in depth are anti-diabetes, blood pressure lowering, antibacterial and antiviral, anti-inflammatory activities, etc.

2. 1 Pharmacological activity

2. 1. 1 Anti-diabetic activity

Stevia leaves have been used as an anti-diabetic drug in the Americas for many years, and modern pharmacological studies have also shown that stevia leaves [25] have a good anti-diabetic effect and can effectively prevent the increase in blood glucose caused by alloxan [26]. Stevia has three main ways of exerting its anti-diabetic activity: (1) it prevents blood glucose from rising by inhibiting gluconeogenesis in the liver; (2) stevioside and rebaudioside A can improve the sensitivity of the islets of Langerhans, stimulating the secretion of insulin by islet cells, and has the effect of safely treating type 2 diabetes; (3) Stevioside can also lower blood sugar by increasing the utilization of insulin in mice.

2. 1. 2 Blood pressure and blood fat lowering effects

Studies have found that stevia leaf extract has blood fat lowering effects [27] and significant blood pressure lowering effects [28]. It has been found that the main mechanism of action is to inhibit Ca2+ influx into vascular cells, promote vasodilation, and thereby lower blood pressure.

2. 1. 3 Antibacterial and antiviral effects

The diameter of the bacteriostatic circles of Stevia rebaudiana Bertoni leaf ethanol extract at a concentration of 1 000 μg/mL against Bacillus cereus, Bacillus subtilis and Staphylococcus xylosus was 6, 6 and 6 mm, respectively; the diameter of the bacteriostatic circles of acetone extract at a concentration of 1 000 μg/mL against Bacillus cereus, Bacillus subtilis, Staphylococcus xylosus, Alcaligenes acidoterrestris, Pseudomonas aeruginosa, the diameter of the inhibition zone was 7, 5, 6, 7, 9 mm, respectively; the diameter of the inhibition zone of stevioside at at a concentration of 100 μg/mL, the diameter of the bacteriostatic circles for Bacillus cereus, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa is 12, 10, 10 and 10 mm, respectively [29-30]. In addition, Kataev et al. found that stevia extract also has anti-tuberculosis activity [31], and the active ingredients are stevioside and rebaudioside A, with anti-tuberculosis MIC values of 7.5 μg/mL and 3.75 μg/mL, respectively [32 ]. Takahashi et al. [33] found that a water extract of Stevia rebaudiana Bertoni can bind to the outer capsid glycoprotein VP7 of human rotavirus, increasing the steric hindrance when VP7 binds to the cell receptor, thereby preventing the virus from attaching to normal cells and thus exhibiting anti-human rotavirus activity.

2. 1. 4 Anti-inflammatory effect

The chloroform and methanol extracts of Stevia rebaudiana Bertoni leaves have significant anti-inflammatory effects and can prevent the swelling of rat toes induced by carrageenan [34]. Some scholars have speculated through experiments that stevioside may be the active ingredient that exerts anti-inflammatory effects, and its mechanism of action is mainly to stimulate innate immunity and thereby reduce the occurrence of pro-inflammatory responses [35]. Further studies have confirmed that stevioside can inhibit NF-κB activity and protein kinase inhibitory signal transduction, thereby exhibiting anti-inflammatory properties [36]. Bunprajun et al. [37] also found that stevioside can promote satellite cell activity by regulating NF-κB pathway signals, thereby promoting the recovery of injured muscles.

2. 1. 5 Immune regulation

Stevia leaf extract and steviol have immunomodulatory activity [38], and their mechanism of action is to interfere with the NF-κB pathway, which exhibits anti-inflammatory and strong immunomodulatory effects [39].

2. 1. 6 Anti-cancer effect

Bhattacharyya et al. [40] found that the ethyl acetate, acetone, chloroform and water extracts of Stevia rebaudiana leaves all showed anti-cancer potential. Takahashi et al. [41] recently reported that stevioside can promote the expression of Bax and cytochrome C, which are then released into the cytoplasm to induce apoptosis in cancer cells. In addition, the results of experiments by Konoshima et al. [42] show that stevia can also be used to prevent chemical carcinogenesis.

2. 1. 7 Other effects

Stevia has the effects of anti-amnesia, preventing obesity, preventing heart disease, preventing tooth decay, killing larvae, improving the quality of pork as feed, and treating metabolic syndrome.

2. 2 Safety studies

The safety of stevia as a food additive is particularly important. Modern pharmacological studies have shown that stevia and stevioside are safe to consume and have no toxic side effects. Andrey et al. [43] randomly divided laboratory rats into three groups and fed them 500, 1,000 and 2,000 mg/(kg·d) of three different doses of rebaudioside A for 90 days. lebaudioside A for 90 days, and the results showed no toxic side effects. Geuns et al. [44] injected each fertilised egg of broiler chickens with 0.08, 0.8 or 4 mg of stevioside, or 0.02 5, 0. 25, 1. 25 mg of steviol, and found that the embryos developed normally during incubation. Williams et al. [45] used the Ames test, chromosomal aberration test, bone marrow micronucleus test and DNA synthesis assay to demonstrate that rebaudioside A is non-genotoxic.

3 Product development

Currently, there are three main categories of stevia products: (1) sweeteners, which have the advantages of high sweetness, low calorie, safety and non-toxicity, and stable physical properties; (2) adjuvant drugs, which are used as adjuvant products for the treatment of diabetes, hypertension, poultry mastitis inflammation, and cattle infertility; (3) feed and fertilizer, mainly the industrial waste from the production of stevioside added to animal feed and crop fertilizers, which has the effect of regulating the digestive function of poultry, increasing egg production, promoting the early maturity of fruits and vegetables, and sweetening fruits and vegetables.

Stevioside is a natural sweetener with a high sweetness and low calorie content. It has advantages over common sweeteners such as sucrose and glucose, but it has a serious aftertaste of bitterness. With the improvement of people's quality requirements for stevia, the extraction, purification and taste improvement of stevioside will become the focus of future research, and the improvement of the sweetness of stevioside will become the key to its further development as a sweetener.

3.1 Factors affecting the sweetness, sweetness and aftertaste of stevia

Wang Deji [46] started from the perspective of molecular chemical structure and combined the sweetness and sweetness of the components of stevioside to draw the following conclusions: (1) The C-13 sugar group is the main functional group for sweetness, and compounds with 3 to 4 sugar groups generally have the highest sweetness and better sweetness , and the order of the types of linked sugar groups that are beneficial to sweetness and sweetness is fructose > glucose > rhamnose or other galactose groups; (2) the C-19 ester group of the glycoside is a taste-enhancing group. If it is not linked to a glucose group such as H, it will greatly affect the sweetness and sweet taste quality; (3) the strong bitter aftertaste of the glycoside is the root cause of the bitter aftertaste of stevioside, and impurities such as tannins, flavonoids, and sesquiterpene lactones can increase the bitter taste of stevioside.

3. 2 Stevia quality improvement technology

3. 2. 1 Blending method

When stevia is used together with other high-calorie sweeteners (such as sucrose, glucose, etc., with cyclodextrin sugar being the most commonly used) or inorganic salts, the sweetness of stevia is greatly increased, while mixing with organic acids such as malic acid and tartaric acid can improve its bitter aftertaste.

3. 2. 2 Modification of glycosides

As mentioned above, the glycosides at the C-13 and C-19 positions have a significant effect on the sweetness and sweetness of the steviol glycosides. Therefore, many scholars have focused their research on modifying the glycosyl groups. Currently, the main methods for modifying the glycosides of steviol glycosides are enzymatic catalysis and microbial transformation. By modifying the glycosides, the astringent aftertaste of steviol glycosides can be largely improved, and the sweetness is also improved to a certain extent.

There are two main mechanisms: (1) the introduction of a better-tasting sugar group (ifose, glucose) and an increase in the proportion of sugar groups in the molecule; (2) the biocatalytic reaction has a two-way effect, catalyzing the linking of sugar groups while also catalyzing the hydrolysis of glycosides. The resulting sugar (e.g., glucose) can form a “compound” with the newly produced glycoside.

Many scholars have studied the modification of glycosides using the cyclodextrin glucanotransferase (CGTase) method [47-49], which is currently the most in-depth method for studying the improvement of stevia taste quality by enzymatic catalysis. The results of many studies have shown that this method can improve sweetness and sweetness to a certain extent. However, no matter how the influencing factors such as optimized strains, enzyme source, substrate type and concentration and other influencing factors, the glycosylation results all show low selectivity.

For example, as the substrate concentration changes during the reaction, the introduction of new glycosides will also change, mainly including monosaccharides, disaccharides and trisaccharides. For example, when a new glycosyl group is introduced, the selectivity for the hydroxyl group of St is poor. Some scholars have also used the glucosidase transfer method to modify glycosides [50]. This method has been studied less than the CGTase method. It allows some of the C chains in the stevioside molecule to be re-glycosylated with new glucose moieties.

Although this method can directly obtain glucose-linked stevioside, and the stevia taste quality has also been improved to a certain extent, but the catalytic activity is low and the yield is low. At the same time, some of the glucose moieties linked at the C-13 and C-19 positions may also be enzymatically hydrolyzed. Overall, these two methods have poor selectivity, low yields, and a wide range of by-products, and there have been few reports on the structures of these products.

Although the above-mentioned enzymatic conversion methods have not achieved the desired results, the research results of the galactosidase method, β-fructofuranosidase method, and microbial transformation method have shown that the improvement of stevioside by biological enzyme catalysis still has promising prospects.

Danieli et al. [51] and Zhu Haixia et al. [47] used the galactosidase method to modify the glycosyl group. This method can highly selectively transfer galactose to the C-13 glucose residue of stevioside. However, this method has a long reaction time, requires the addition of other coenzymes to produce donors, and the taste of ST glycosylated with galactose is slightly worse than that of other glycosylated products. Therefore, Zhu Haixia et al. [47] used the fact that the glucose moiety is a good acceptor in the catalytic transglycosylation reaction of the CG-Tase, while the galactose moiety is not. The galactosidase method was combined with the CGTase method, with the galactosidase enzyme to catalyze the glycosylation reaction, attaching the galactose group to the glucose group at position 19, and then using this product as the substrate for CGTase catalysis, so that the glycosylation reaction can selectively occur at the glucose group linked to 13-OH.

Li Yu et al. [52] used the β-furanosyl fructosidase method (FFase) to introduce a fructosyl group to stevioside and rebaudioside A, which allowed the fructosyl group to be linked to the 6-OH of the 19-O-β-glucosyl group by a β-2,6 glycosidic bond. This method, the optimal catalytic conditions are: pH 6.5, reaction temperature 40 °C, and it is stable at pH 6 to 8 and below 40 °C. The molar ratios of stevioside and steviol glycoside to sucrose molar ratio of 0.0005 and 0.0012, enzyme amount of 15 U/mL, and reaction time of 15 h.

Under the optimized reaction conditions, the conversion rates of the two glycosides can reach 69.4% and 72%, respectively. In addition to the above method of improving the taste of stevioside by enzymatic catalysis, Kusakabe et al. [53] used actinomycetes to transfer a glucose group to the 2-glucosyl-β-glucoside at the 13-carbon position of stevioside, successfully converting stevioside to rebaudioside A. The conversion rate was about 20%, but it only catalyzed the production of one kind of product, with high specificity. Ishikawa et al. [54] used a combination of microbial fungi and β-fructofuranosidase to transfer β-fructofuranose to the 13-carbon position of stevioside and rebaudioside A. The above method used by the researcher is selective, the product is relatively simple, and it has potential application value.

Enzymatic modification of stevia has long been a research hotspot for removing its bitter aftertaste. Although after more than 20 years of research by numerous scholars, no industrial production route has been found, the conclusion that enzymatic transglycosylation and microbial transformation are effective way to improve the sweetness of stevioside. With the deepening of research on the mechanism of enzymatic sugar transfer, it is hoped that stevia can be directed and modified at the molecular level to obtain a more perfect stevioside sweetener.

3. 2. 3 Improving the aglycon part

Modifying the aglycon part is a way to fundamentally improve the quality of stevioside taste. There have been relatively few reports on this, and they mainly include methods such as improving the structure of the aglycone and physically enclosing the aglycone. Lee Thomas et al. [55] modified the aglycone and the sugar moiety of stevioside by transferring the double bonds at C-15 and C-17 on the aglycone to C-15 and C-16, and chose glucose, rhamnose or pyranose as the sugar moiety, thereby improving the sweetness and astringency.

Wang Deji et al. [56] aimed at the aglycone part, the main source of the bitter taste of stevioside glycosides. Through analysis and calculation, cyclodextrin was added to the extract and concentrate to physically enclose the aglycone part of the stevioside molecule. The resulting product had the same sweetness as before but the bitter aftertaste disappeared. Although eliminates the bitter aftertaste of stevioside, but at the expense of the excellent low-carbon properties of stevia.

4 Conclusion

In recent years, as people's awareness of healthy eating has increased, sweeteners such as sucrose and glucose have gradually been unable to meet people's needs due to their high calorie content, their tendency to cause tooth decay in young children, and their unsuitability for people with diabetes. Stevia's the main sweetening component of stevia, is an emerging natural sweetener that has the advantages of being low in calories and high in sweetness, stable in physical and chemical properties, and safe with no side effects. It also has important biological activities such as lowering blood sugar, blood lipids and blood pressure, as well as anti-cancer, antibacterial and antiviral properties, making it known as the “most promising new sugar source”.

However, the stevia's relatively serious aftertaste of bitterness seriously affects its quality, and is also one of the important reasons why stevia currently occupies a relatively small proportion of the market. In order to address this defect, many scholars at home and abroad have conducted research from the aspects of compounding, refining, and structural modification, in the hope of obtaining a perfectly refreshing-tasting stevia.

Among the many methods, the more successful one is the method of refining Rebaudioside A by recrystallization and adsorption on macroporous resin, which has already been applied in production. The compound method The sweetening effect of the compound method is limited. Although enzymatic catalysis and microbial conversion have greatly improved the sweetness, they are costly, and the structure and biological activity of the products are unknown. Some of these methods achieve the purpose of improving sweetness at the expense of Stevia's natural advantages of low calories and heat, such as the compound method using cyclodextrin and glucose, which have high calories and heat, and the physical encapsulation method using cyclodextrin as an encapsulating agent.

Developing stevia as the perfect sweetener has been a feasible, but winding path. Although the use of purified steviol glycosides has improved the quality of stevia products to a certain extent, the aftertaste of stevia products has not been fundamentally improved because steviol glycosides themselves also have a certain bitter aftertaste. Therefore, the author believes that we should accelerate the structural identification, sweetness evaluation, and safety research of new products structure identification, sweetness evaluation, and safety research of the new products generated during the process, with the aim of promoting the optimization of this type of stevioside to industrial production. At the same time, if a product with a sweetness that is superior to that of rebaudioside A can be discovered, it will provide a new and clear research direction for improving the quality of stevia products in the future.

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