What Is Steviol Glycoside Rebaudioside A?

High sugar intake in the daily diet is one of the factors that cause obesity. Cardiovascular diseases caused by obesity, such as hypertension, hyperglycemia, and diabetes, are a major public health crisis that affects human health worldwide [1]. Stevioside is a zero-calorie sweetener extracted from the leaves of the stevia plant, and is known as one of the “world's three major sugar sources” along with cane sugar and beet sugar[2]. The stevioside in the leaves of the stevia plant is a mixture of molecules with different structures, mainly including stevioside (St), rebaudioside A (Reb A), rebaudioside D (Reb D) and rebaudioside M (Reb M)[3]. Reb A), rebaudioside D (Reb D), and rebaudioside M (Reb M), etc.[3]. The sweetness of stevioside is 250 to 350 times that of sucrose, and it can replace sucrose as a new generation of zero-calorie natural sugar source[4].

Since the 1990s, food and drug regulatory organizations in many countries in Europe and the United States have evaluated the safety of stevioside and unanimously approved high-purity (≥95%) stevioside as a safe sweetener[5]. In recent years, some clinical trials have shown that stevioside can not only be used as a sweetener[6] , but also has a variety of health benefits such as anti-diabetes, lowering blood pressure, cardiotonic, anti-inflammatory, antibacterial, anti-tumor and so on[7] .

These health-promoting properties have given stevioside a broader market demand. Researchers have currently developed a variety of new methods for extracting and synthesizing stevioside, which have greatly promoted the production and application of stevioside [8⁃10]. This paper outlines the safety and market demand of stevioside, summarizes the application of stevioside in the fields of food and health products, and provides an outlook on the plant extraction and biosynthesis of stevioside, laying a theoretical foundation for in-depth research and industrial production of stevioside.

1. The safety of stevioside and market demand

In the late 1980s, the safety of stevioside was widely questioned, so countries launched research on the safety of stevioside. In 1995, the US Food and Drug Administration (FDA) approved stevia for use in dietary supplements [5]. In 2008, 2013 and 2014, Reb A, Reb D and Reb M were successively recognized as generally recognized as safe (GRAS) by the FDA [11⁃12]. Subsequently, the safety of steviol glycosides was also successively recognized by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) [13], the Food Standards Australia New Zealand (FSANZ) [14], the European Food Safety Authority (EFSA) [15], and the Food Safety and Standards Authority of India (FSSAI) [16]. FSANZ) [14], European Food Safety Authority (EFSA) [15], and Food Safety and Standards Authority of India (FSSAI) [16]. In 2011, the relevant departments in China officially approved the use of stevioside as a food additive in beverages, preserved fruits, condiments, pastries and other foods [3].

China is the world's largest producer of stevia glycosides. According to the China Stevia Association, in 2009, China's stevia cultivation area was about 16,667 hectares, mainly distributed in Xinjiang, Gansu, Inner Mongolia, Hebei, Jiangsu, Anhui, Heilongjiang and other places, with an annual output of about 40,000 tons of stevia raw materials[17]. Stevia glycosides production has been fully promoted in more than 27 provinces and cities. About 80% of the stevioside produced in China is exported to more than 20 countries, including Japan, South Korea, the United States, and Malaysia. China is currently the world's largest exporter of stevioside[18]. In addition, countries and regions such as Russia, India, Canada, South Africa, and Kenya also have different cultivation areas for the promotion of cultivation[19⁃21].

Currently, stevioside has become the world's most widely used natural high-intensity sweetener with zero calories. Stevioside can improve energy balance and assist in weight control. According to statistics from the International Stevioside Association, in 2016, about 3,000 food and beverage products containing stevioside were launched globally, and the number of consumers has exceeded 4 billion[22]. In 2017, the number of food products using stevioside as a sweetener additive exceeded the number of those using aspartame as a sweetener. products, and the number of people consuming them has exceeded 4 billion[22]. In 2017, the number of food products using stevia glycosides as a sweetener surpassed the number of products using aspartame as a sweetener[23]. It is estimated that by 2027, the worldwide consumption of stevioside powder will reach 10,254.93 tons at an annual growth rate of 7% to 8% [24].

Reb A is the most widely used stevioside on the market. From January 2015 to February 2017, the price of high-purity Reb A (≥95%) rose from 73,000 USD/ton to 77,300 USD/ton [23]. In 2017, global market reached 417 million US dollars. With the advancement of technology and the development of a new generation of natural sweetener Reb M, it is expected that by 2024, the market sales of stevia will grow at an annual rate of 8.2% to 721 million US dollars [25]. According to relevant market research reports, the global stevioside market was worth 620.8 million US dollars in 2020, and is expected to reach 1.14 billion US dollars by 2028, with a compound annual growth rate (CAGR) of 8.0%. billion US dollars[24] , and the compound annual growth rate could reach 8.5% by 2030, with a market size of 16.428 billion US dollars[26] .

2 Steviol glycosides applications

2.1 Steviol glycosides as sweeteners

Steviol glycosides can be used in food at different proportions as a food additive[27] because they still maintain a good taste when used in combination with other sweeteners. With the safety of steviol glycosides being recognized, steviol glycosides have been widely used in the market. Common forms of steviol glycosides are powders, tablets and liquids, which can be used as natural sweeteners in foods such as beverages, beer, bakery products, delicatessen, preserved foods, processed fruits and vegetables, condiments, frozen foods, sauces, snacks and cereals [6].

In 2013, Coca-Cola began producing drinks containing stevia, reducing their calorie content by 30% [28]. In 2018, Coca-Cola launched a stevia cola that completely replaces white sugar with stevia glycosides, changing the sweet and greasy feeling brought by white sugar and replacing it with a sweet and delicious sweetness. While satisfying the need for sweetness, it also achieves low glycation [29]. Stevioside can reduce the viscosity of the substrate in some application scenarios, such as changing the viscosity of fruit wine to make it more palatable, and enhancing the foaming effect of beer to make the foam dense and long-lasting [30].

The use of steviol glycosides instead of sucrose in baked goods can reduce the fermentation rate of the dough and the volume of the bread, and improve the nutritional value of the bread by reducing the glycemic index and energy content [31]. Oat biscuits prepared with different proportions of stevia extract to replace sucrose were evaluated for their sensory properties such as appearance, taste, smell and texture. The results showed that biscuits prepared with 25% and 50% sucrose replaced with stevia extract were the most popular with consumers [27].

In the production of jelly and jam, the addition of sugar not only affects the final sweetness, but also helps to increase the total soluble solids [32]. The sugar in jelly or jam gels with water or a hydrogel. When steviol glycosides are used to completely replace sucrose, the jelly does not set properly and the appearance is affected. When sucralose, stevioside and agar are used together, the jelly sets as desired and the calorie content of the jelly and jam can be reduced [33-34].

Stevia glycosides can be used to replace sucrose in sugar-free candies made from yam, dragon fruit, papaya, guava and kiwi fruit, as well as nuts such as walnuts, peanuts and pine nuts. While providing mineral nutrition, they also satisfy consumers' desire for sweetness [35]. Replacing the sucrose in chocolate with steviol glycosides not only produces sugar-free chocolate with a taste similar to the original, satisfying people's demand for taste, but also reduces the risk of possible weight gain and sugar metabolism diseases [36]. In addition, steviol glycosides are not utilized by microorganisms. Replacing sucrose with steviol glycosides in pickles and pickled vegetables can control the proliferation of microorganisms and prevent spoilage during fermentation[37].

2.2 Health-promoting functions of steviol glycosides

Stevioside not only acts as a sweetener, but also has a variety of biological activities and health benefits, such as anti-diabetic, anti-cardiac fibrosis, anti-fatty liver, anti-inflammatory, antibacterial, and anti-tumor properties[7]. Since the final metabolite of different steviosides is all steviol, steviol does not accumulate in the body, making steviosides safe to use as health products without causing any toxic side effects[38].

Steviol glycosides steviol, steviol glucoside, stevioside, rebaudioside A, stachydrine and Reb A were used to feed streptozotocin-induced type 2 diabetic mice. Compared with untreated type 2 diabetic mice, type 2 diabetic mice fed with stevioside derivatives had significantly increased insulin secretion, enhanced glucose metabolism in the liver, accelerated hepatic glycogen was accelerated, blood glucose concentration and glycated hemoglobin (HbA1c) levels were reduced, and diabetes was improved in mice [39].

After 6 weeks of feeding fatty liver rats with 75 to 150 mg/kg St, the degree of fatty liver in the experimental group of rats was significantly reduced, indicating that St can lower blood lipids [40]. St was used to feed mice with ulcerative colitis induced by sodium dextran sulfate, mice with lethal shock induced by lipopolysaccharide (LPS), and rats with adjuvant arthritis induced by Freund's complete adjuvant (FCA). It was found that St can inhibit the release of pro-inflammatory factors, enhance the production of anti-inflammatory cytokines, and significantly reduce inflammatory responses [41]. ( Freund's complete adjuvant, FCA) induced adjuvant arthritis rats, it was found that St can inhibit the release of proinflammatory factors, enhance the production of anti-inflammatory cytokines, and significantly reduce the inflammatory response [41⁃43].

Stevioside has obvious efficacy in anti-cardiac fibrosis, antibacterial and antitumor. After isoproterenol-induced myocardial fibrosis in mice was treated orally with st for 40 days, the mice's myocardial hydroxyproline levels and heart weight index decreased, and the degree of myocardial fibrosis was significantly reduced [44]. 20 mg/mL st can inhibit the growth of Escherichia coli. Adjusting the concentration of st can inhibit the growth of Bacillus subtilis, Aspergillus niger, Rhizopus oryzae, etc. inhibitory effect [45]. Reb A can self-assemble into micelles. Formulating Reb A and honokiol (HK) into self-assembled micelles Reb A⁃HK can improve the oral bioavailability of HK and enhance its antitumor activity. Therefore, stevioside has great potential in the delivery of hydrophobic antitumor drugs [46]. Steviol glycosides can also scavenge reactive oxygen species such as hydroxyl and superoxide radicals, and can be used to treat diseases such as hypertension, type 2 diabetes, atherosclerosis and tumors.

3 Preparation methods for steviol glycosides

3. 1 Plant extraction of steviol glycosides

Traditional methods for extracting steviol glycosides from plants include hot water extraction, solvent extraction, maceration, and adsorption on macroporous resins [47⁃50]. These methods are not only time-consuming and laborious, but also relatively inefficient, and they consume excessive solvents and energy [51]. With the development of modern biotechnology, researchers have developed a variety of methods for extracting steviol glycosides from natural stevia.

Microwave-assisted extraction (MAE) can use microwave energy to promote the transfer of stevioside to the solvent. Compared with the traditional maceration technique, this method has a lower optimal operating temperature and the optimal extraction time is shortened to 1/7 of the maceration method [49]. Supercritical fluid extraction (SFE) is not only more efficient than traditional maceration techniques, but also reduces carbon dioxide emissions and solvent consumption [52].

Ultrasound⁃assisted extraction (UAE) mainly uses ultrasonic vibration to rupture cells and release intracellular substances. Compared with other extraction techniques, this method has a milder extraction temperature [53]. Reb A and St extracted using rapid solid-liquid dynamic extraction (RSLDE) are colorless and transparent liquids, while the product extracted using the traditional maceration method is dark yellow. The RSLDE method not only increases the production of stevioside, but also reduces the subsequent product purification steps, thereby reducing the production cost of stevioside [54]. The above extraction methods are still in the laboratory research stage and further research is needed to reduce the cost of industrial application [49, 53].

The contents of steviol and rebaudioside A in Stevia leaves are the highest, accounting for 5% to 10% and 2% to 4% of the dry weight of the leaves, respectively [55]. Therefore, the main components of the products obtained by the existing extraction techniques are steviol and rebaudioside A. Reb D and Reb M are present in Stevia leaves at extremely low levels, accounting for only 0.4%–0.5% of the dry weight of the leaves, so it is inefficient and costly to directly extract Reb D and Reb M from Stevia [9, 56].

3. 2 Biosynthesis of stevioside

Direct extraction of stevioside from plants is often affected by the content of stevioside and the plant growth cycle. In recent years, researchers have used techniques such as genome sequencing and expressed sequence tags (ESTs) to reveal the main synthesis pathways of steviosides in natural stevia [10, 57], laying the foundation for the heterologous production of steviosides using synthetic biology. Studies have shown that the main difference between different types of steviol glycosides lies in the number and position of the sugar groups, which results in different sweetness and mouthfeel [58]. The trisaccharide St and the tetrasaccharide Reb A are 250 to 300 times sweeter than sucrose, with a slight aftertaste [59]; the pentasaccharide Reb D and the hexasaccharide Reb M are 350 times sweeter than sucrose, with almost no aftertaste and a better taste [9].

The main synthetic pathway of Reb D and Reb M in stevia is as follows: steviol is catalyzed by the glycosyl transferases SrUGT85C2 and SrUGT74G1 to transfer the glucose group of the sugar donor uridinediphosphate glucose (UDP-glucose) to the C13-hydroxyl group of the steviol skeleton via a β-D-glucoside bond. glucose (uridinediphosphate glucose, UDPG) to the C13-hydroxyl and C19-carboxyl functional groups of the steviol skeleton via a β-D-glucoside bond to form rubusoside (Rub) [ 60]; the C13-glycosyl group of rubusoside forms a 1,2-β-D-glycosidic bond under the catalysis of the glycosyl transferase SrUGT91D2 to form St[61]; the C13-glucosyl group of St forms a 1,3-β-D-glycosidic bond under the catalysis of the glycosyl transferase SrUGT76G1 catalyzes the formation of a 1,3-β-D-glycosidic bond to generate Reb A[56]; the glucosyl group at the C19 position of Reb A is successively catalyzed by SrUGT91D2 and SrUGT76G1 to generate Reb D and Reb M (Figure 1) [61].

The biotransformation of stevioside is currently the most cost-effective way to achieve industrial production of stevioside. The biotransformation of stevioside mainly includes: (1) constructing a stevioside synthesis pathway in microorganisms by overexpressing glycosyltransferase genes to heterologously synthesize stevioside using glucose as a carbon source; (2) synthesizing stevioside using biocatalysis.

3. 2. 1  The de novo synthesis of stevioside

Construct the mevalonic acid (MVA) production pathway in Escherichia coli, and then introduce a terpene module containing the genes for geranyl diphosphate synthase, cyclopentapropanoyl diphosphate synthase and kaurene synthase, a cytochrome P450 module containing the genes for diaglycolate oxidase, kaurene 13α-hydroxylase and cytochrome P450 reductase, and 13α-hydroxylase and cytochrome P450 reductase, as well as a glycosyltransferase module composed of the glycosyltransferases SrUGT85C2, Sr UGT91D2w, SrUGT74G1 and SrUGT76G1, the obtained strain SSY10 pSY447 can synthesize 10.03 mg/L of Reb A from scratch within 5 days[61].

A synthetic pathway from steviol to Reb M has been successfully constructed in Saccharomyces cerevisiae (Figure 1). The mutant UGT76G1 Leu257Gly produced four times as much Reb D as UGT76G1, and the mutants UGT76G1 Lys337Pro and UGT76G1Thr55Lys both produced about 4 times as much Reb M as UGT76G1. was 4 times that of UGT76G1. The mutant UGT76G1 Lys337Pro and UGT76G1Thr55Lys had an enhanced ability to produce Reb M by about 20%, and the production of by-products such as Reb G and Reb Q was almost eliminated [9].

Compared with traditional plant extraction and chemical synthesis methods, the de novo synthesis of artificially designed steviol glycosides can produce specific steviol glycosides in the expected route in a shorter time and in an environmentally friendly manner. However, because the de novo synthesis of steviol glycosides by microorganisms involves many catalytic reaction steps, the expression levels of enzymes in microorganisms for some key steps are low, and the activity is poor, resulting in generally low yields of steviol glycosides.

3. 2. 2 Biocatalytic synthesis of stevioside

Biocatalytic synthesis of stevioside refers to the process of using enzymes or enzyme-producing microorganisms as catalysts for the synthesis of stevioside. Stevia UGT76G1 can catalyze the production of Reb A from St[9] . The glycosyltransferase UGT76G1 is expressed on the surface of Pichia pastoris GS115 using the anchor protein Gcw61p. The recombinant strain uses St as a substrate, UDPG as the glycosyl donor, whole-cell catalysis to generate Reb A , the conversion rate is about 70.37% [62]. UDPG is expensive, in order to reduce the cost of catalysis, researchers co-expressed the Arabidopsis sucrose synthase AtSUS1 and the sorghum U GT76G1 was co-expressed in E. coli. The crude enzyme solution of AtSUS1 catalyzed the conversion of sucrose and uridine diphosphate (UDP) to UDPG, and the recombinant enzyme SrUGT76G1 catalyzed the synthesis of Reb A using St as a substrate[58]. This method uses inexpensive UDP and sucrose as substrates to synthesize UDPG in situ, further reducing the cost of industrial production of stevioside (Figure 2) [11, 58].

Stevia rebaudiana UGT91D2 can catalyze the production of Reb D from Reb A [10]. However, there is currently no research on the recombinant enzyme SrUGT91D2. Researchers expressed the tomato glycosyltransferase UGTSL2 in E. coli, and the recombinant enzyme was able to catalyze the production of Reb D and Reb M2 from Reb A as a substrate[63]. Reb M2 is an isomer of Reb M, and its safety has not been verified[16].

Further saturation mutagenesis of UGTSL2 resulted in the mutant Asn358Phe, which had a 21.9% increase in catalytic activity, but a small amount of Reb M2 was still present in the product [64]. 9%, but a small amount of Reb M2 was still present in the product [64]. The glycosyltransferase EUGT11 from rice (Oryza sativa) was expressed in Escherichia coli and Pichia pastoris to obtain recombinant strains XE⁃3 and BL21 (pET28a~ⅣOsEUGT 11). The recombinant enzyme EUGT11 expressed by XE⁃3 has the highest activity in catalyzing the production of Reb D at 45 °C in a sodium phosphate buffer at pH 6.0–6.5 with UDPG as the sugar donor. The recombinant enzyme EUGT11 expressed by BL21 (pET28a-OsEUGT11) has the highest catalytic activity at 35 °C in Tris HCl buffer at pH 8.5 [65].

The recombinant EUGT11 expressed in Pichia pastoris has higher acid resistance and thermal stability than the recombinant enzyme expressed in Escherichia coli [65]. A two-step temperature control strategy developed using an orthogonal design was used to optimize the production of Reb D in the XE⁃3 strain. The XE⁃3 recombinant strain was cultivated in BMMY medium containing 0.75% methanol and pH 5 BMMY medium ~ (buffered methanol⁃complex medium) at 28 ° C for 3–4 d to obtain a target protein of approximately 790 mg/L OsEUGT11. Subsequently, Reb A and the sugar donor UDPG were added to the bacterial culture, and the whole cells of the XE⁃3 recombinant strain catalyzed the specific production of Reb D from Reb A at 35 °C for 4 d, with a yield of 93.47%.

This method simplifies the steps of protein separation and purification (Fig. 3) [65]. In order to discover new glycosyltransferases with higher activity, researchers combined bioinformatics methods, such as homology sequence comparison, domain analysis and tertiary structure simulation, to screen for glycosyltransferases CaUGT from Capsicum annuum and StUGT from Solanum tuberosum. They were expressed in Escherichia coli, Both recombinant enzymes, CaUGT and StUGT, can use UDPG as a sugar donor to catalyze the conversion of Reb A to Reb D. However, the catalytic product of recombinant enzyme CaUGT contains the by-product Reb M2, while recombinant enzyme StUGT can specifically catalyze the conversion of Reb A to Reb D with a yield of 97% [66].

Apart from SrUGT76G1, no other glycosyltransferase has been found to catalyze the conversion of Reb D to Reb M. Recombinant SrUGT76G1 expressed in Escherichia coli can catalyze the conversion of Reb D to Reb M with a conversion rate of 72.2% [67]. The mutant SrUGT76G1T284S increased the conversion of Reb D to Reb M by about 50% [68]. SrUGT76G1 expressed in E. coli is prone to form inclusion bodies, which affects the efficient production of glycosyltransferases [65]. The researchers fused a short acidic peptide tag to the C-terminus of SrUGT76G1 to obtain four acidic-tail fusion enzymes, which improved the soluble expression level, thermal stability and catalytic activity of SrUGT76G1 in E. coli. The acidic C-terminal fusion enzyme has 202.46% of the activity of the wild type when catalyzing the production of Reb M using Reb D and UDPG as substrates in a glycine-sodium hydroxide buffer at pH 9.0 [69].

In order to catalyze the direct production of the high-value stevioside from cheaper substrates, shorten the reaction time, and reduce production costs, researchers have developed a multi-enzyme cascade system. When OsEUGT11, SrUGT76G1, and the Arabidopsis sucrose synthase AtSUS3 are co-expressed, the recombinant bacteria can directly catalyze the production of Reb M from Reb A, UDP, and sucrose as substrates [68]. Mutating the gene encoding SrUGT76G1 in the recombinant bacterium so that the threonine at position 284 is mutated to a serine increases the ratio of the whole-cell catalyzed products Reb M and Reb D from 1:3.9 to 7:1, reduces the proportion of the intermediate product Reb D, and increases the yield of Reb M [68]. Enzyme immobilization technology can increase the reusability of enzymes and reduce the cost of enzymatic reactions.

Using glutaraldehyde as a cross-linking agent and chitosan as a carrier, OsEUGT11 and SrUGT76G1 expressed in Escherichia coli were covalently bound to chitosan microspheres, respectively, which can improve the storage stability and reusability of the recombinant enzymes. However, the production of Reb M is limited by the expensive Reb D substrate. In order to directly generate Reb M from the cheap Reb A in one step, the researchers constructed a cascade reaction by simultaneously immobilizing OsEUGT11 and SrUGT76G1 on chitosan. The obtained co-immobilized enzyme uses UDPG as a sugar donor. In a sodium phosphate buffer at pH 7.0 with 3 mmol/L MgCl2, Reb A can be directly catalyzed to generate Reb M, which is 3.2 times more active than the mixed system of individual immobilization (Figure 4). sodium phosphate buffer with 3 mmol/L MgCl2 added, the Reb A can be directly converted to Reb M by enzymatic catalysis, and the activity is 3.2 times higher than that of the mixed system alone (Figure 4), successfully reducing the production cost of Reb M [63].

In summary, biocatalytic technology is simple to operate, has high catalytic specificity, and produces few by-products, which facilitates the subsequent separation and purification of the product, thereby facilitating the industrial production of steviol glycosides [70].

4 Summary and outlook

As a new type of zero-calorie natural sweetener, the food and medicinal value of stevioside is also being continuously explored. In 2018, Reb D and Reb M, which are even sweeter, received widespread attention and are expected to replace Reb A as a new generation of natural sweeteners. However, the content of Reb D and Reb M in stevia is extremely low, and the cost of extracting and purifying them from plants is high, which limits their application and development.

The biosynthesis of stevioside is an important way to promote the efficient production of Reb D and Reb M. In the future, the biosynthesis efficiency of stevioside can be improved in the following ways: (1) The number and activity of existing glycosyltransferases are limited. Bioinformatics can be used to discover new types of highly high-efficiency glycosyltransferase, and use molecular modification technology to improve the expression level, catalytic efficiency and thermal stability of the recombinant enzyme; (2) use synthetic biology technology to construct pathways for synthesizing Reb D and Reb M from glucose in microbial cells such as Escherichia coli, and improve the yield of Reb D and Reb M by regulating related metabolic networks, developing and optimizing gene regulatory elements, etc., to increase the yield of R e b D and R e b M ; (3) developing immobilization, whole-cell catalysis, multi-enzyme cascades and coenzyme regeneration technologies to reduce production costs, thereby accelerating the market application of stevioside.

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