How to Extract Reb A Steviol Glycosides?

Currently available artificial sweeteners such as sodium saccharin, acesulfame and sodium cyclamate on the market can hardly fully satisfy consumer demand. Steviol glycosides therefore stand out from the crowd as a healthy natural sweetener. Steviol glycosides are a kind of glycoside with low calories and high sweetness. They are extracted from the leaves of the stevia plant, which is native to the leaves of the stevia plant native to northeastern Paraguay in South America. Steviol glycosides are 200 to 300 times sweeter than sucrose, but only about 1/250 as high in calories[1]. They are a natural non-nutritive high-potency sweetener that can prevent and treat obesity, high blood pressure, and tooth decay.

As early as the early 1970s, a Japanese company considered using it as a sugar substitute in food, replacing some sucrose or all of the synthetic sweeteners such as saccharin [2]. In late 2008, Steviol Glycosides was approved for use by the US Food and Drug Administration (FDA) and is known as the world's world's “third sugar source”. The sweet component in stevia was discovered in 1909, and steviol glycosides were extracted from stevia leaves by chemical means in 1931. Its chemical structure was confirmed as a diterpene derivative in 1952 (Figure 1) , with the main components being stevioside (ST) and rebaudiosides A–E (Reb A–E), steviol glycosides (BIO) and dulcoside (DA), a total of 8 dihydrotetracyclic glycosides [3].

Steviol Glycosides, as a natural sugar substitute, meets the requirements of the gradual development of low sugarization in food and beverage and consumers' awareness of healthy eating. It also has great market potential because it can reduce production costs. As a functional sweetener, it is bound to have good development prospects. However, ST, as an important component of Steviol Glycosides, has a bitter taste, which greatly hinders the practical application of Steviol Glycosides [4]. Rebaudioside A (Reb A) (Figure 2) is the component of Steviol Glycosides with the closest taste to sucrose. Its sweetness is 300 to 450 times that of sucrose, while its caloric value is only 1/300 of that of sucrose [5]. It does not participate in metabolism in the human body and is non-accumulative and non-toxic. It is a purely natural, low-calorie, high-sweetness sweetening ingredient [6]. Therefore, the level of Reb A in steviol glycosides has become the key to measuring its quality, providing a new direction for the development of the steviol glycosides industry.

Steviol glycosides have been industrially produced for more than 30 years and have now developed into the third generation of products. The first generation of products is stevioside, the second generation of products is mainly composed of ST and Reb A, and the third generation of products is mainly composed of Reb A [7]. It can be seen that the extraction and purification process of Reb A has become a research hotspot in the steviol glycosides industry in recent years. This paper reviews the extraction and purification processes of Reb A and their progress based on related research. The extraction of Reb A, which is also the extraction of steviol glycosides, mainly uses methods such as maceration, decoction, reflux, fermentation, and continuous countercurrent extraction. There are three main methods for refining Reb A: using the differences in solubility and polarity between Reb A and other components of steviol glycosides and the adsorption properties of macroporous adsorption resins (MARs) to refine Reb A. Membrane separation technology, as an emerging technology, is also mentioned in this article. Among the existing refining processes, recrystallization and resin methods are commonly used in industrial production, and researchers have done a lot of work in improving and perfecting related processes.

1 Extraction process

Reb A is the main component of the new natural sweetener steviol glycosides, but its content only accounts for about 25% of the total mass of steviol glycosides [8]. The main components of steviol glycosides, ST, Reb A and RC, have the same glycoside and similar structures and molecular polarities, which complicates the process of refining Reb A from crude steviol glycosides. Therefore, researchers have been exploring a set of simple steps with high product purity for refining Reb A. At present, the basic steps for producing Reb A are pretreatment, separation, purification and refinement [9], and refinement is a key step in obtaining high-purity Reb A.

The amount of glycosides contained in the leaves of different stevia varieties varies greatly, and the growth period also has a significant impact on the quality of stevia leaves. It has been reported that the genetic characteristics of different genotypes themselves affect 80% of crop yield and quality [10]. Therefore, there are two factors that affect the high or low content of Reb A in the extracted Steviol Glycosides: the quality of Stevia itself and the process of extracting Steviol Glycosides.

The common method of extracting Steviol Glycosides from Stevia leaves is to use water or food-grade alcohol as an extractant to extract a crude product from the Stevia leaves, and then to remove impurities and decolorize to obtain Steviol Glycosides. Process research shows that Steviol Glycosides are mainly extracted using methods such as maceration, decoction, reflux, fermentation, and continuous countercurrent extraction. Zhao Yongjin et al. [11] found through comparative research that the cooking method is suitable for industrial production; Yu Jun et al. [12] found that tertiary continuous countercurrent extraction is more suitable for industrial production; Hu Huanrong et al. [13] reported a new industrial production process for Steviol Glycosides using low-temperature continuous countercurrent ultrasound extraction, a new industrial production process using special macroporous resin adsorption to extract Reb A, and Steviol Glycosides with a Reb A content of more than 60% was obtained. Through continuous exploration, the content of Reb A in Steviol Glycosides has been continuously improved. This is the first stage in obtaining high-purity Reb A and is also a key step in extracting Reb A, which can further affect the purity of the refined Reb A product. The Reb A content in the extracted Steviol Glycosides is one of the important indicators for testing the quality of Steviol Glycosides.

2. Purification process

The second stage in obtaining high-purity Reb A is to further purify it by methods such as solvent crystallization, chromatographic separation and adsorption on an adsorbent to obtain high-purity Reb A. At present, there are three main methods for refining Reb A reported in the literature: one is to extract and refine Reb A by exploiting the difference in solubility between Reb A and other components of steviol glycosides. The representative method is recrystallization, and the key lies in the selection of the recrystallization solvent; the second is to refine Reb A by exploiting the difference in polarity between Reb A and other components of steviol glycosides . The representative method is high performance liquid chromatography (HPLC), and the choice of stationary phase and flow affect the efficiency of separating Reb A from steviol glycosides. The third method is to use the adsorption characteristics of a high-molecular-weight polymer macroporous adsorption resin (MARs) to purify Reb A. This is also an emerging industrial method for purifying Reb A in recent years. In addition, membrane separation technology is also an emerging process for purifying Reb A.

2. 1 Purification of Reb A by exploiting the solubility difference between Reb A and other components

The solubility of different components of steviol glycosides in specific organic solvents is different. Steviol glycosides can be crystallized using this characteristic to obtain a single component Reb A with high purity. A large number of documents report the purification of Reb A by exploiting the solubility difference between Reb A and other components [14]. Studies have shown that the recrystallization method is a common method for refining Reb A using this principle, and it is also a common method for refining Reb A industrially. Low-grade alcohols are often used as the crystallization solvent. However, the method of refining Reb A by solvent extraction and crystallization is only suitable for experimental research due to its limitations.

2. 1. 1 Recrystallization method

The recrystallization method is used to separate and enrich Reb A, the main component of stevioside, and provides a simple method for purifying Reb A. The solvent used is generally an alcohol solvent with a carbon number of no more than 3, such as methanol, ethanol, and isopropanol [15], or an alcohol solvent is used in combination with other solvents to improve the purity of Reb A after crystallization. Methanol was the first solvent used for the crystallization and purification of Reb A, and it has been used ever since, with a certain advantage in terms of crystallization yield.

As early as 1999, Payzant et al. [14d] reported the use of methanol as a recrystallization solvent to purify Reb A. During the same period, Zhang Yaxiong et al. [16] also reported that secondary crystallization of methanol can obtain steviol glycosides with a higher Reb A content. Steviol glycosides can be recrystallized in one or two different proportions of methanol, isopropanol, and water to obtain Reb A with a higher purity [15a]. Later, researchers found that ethanol recrystallization can further improve the purity of Reb A [10a, 17]. Ethanol-water as a crystallization solvent can crystallize high-purity Reb A at lower temperatures, and is commonly used as a solvent for crystallizing refined Reb A because it is cheap and environmentally friendly [17b, 17c, 18]. In addition, the use of lower alcohols in combination with other organic solvents (e.g., ethanol and acetone) has also been found to be able to refine Reb A as a crystallization solvent, but the yield is low [3].

The co-use of lower alcohol solvents can also effectively improve the crystallinity of Reb A. It has been reported that the co-use of methanol and isopropanol for multiple crystallizations of Reb A can achieve a purity of Reb A of more than 80% [15c, 19].

The recrystallization method is simple to operate, but after crystallization, a considerable amount of sugar in the mother liquor cannot be crystallized, resulting in a low crystallization yield [15a, 15c, 20]. In addition, it requires a lot of time and organic solvents, which leads to defects in practical applications. Therefore, the purification process of Reb A needs to be further developed. Recently, Gasmalla et al. [21] reported that isopropanol as a crystallization solvent assisted by ultrasound can effectively remove colored impurities and further improve the purity of Reb A, thus improving the recrystallization process.

2. 1. 2 Dissolution and crystallization method

Zhao Hao et al. [22] reported that the dissolution and crystallization method also uses the solubility difference between Reb A and other components to refine Reb A. This process has high energy consumption, slow crystal growth, and is time-consuming. There is also the problem of organic solvent residue, so it is not suitable for industrial production.

2. 2 Refining Reb A using the polarity difference between Reb A and other components

Using the polarity difference between Reb A and other components in steviol glycosides is an efficient method for purifying Reb A. The literature reports high-performance liquid chromatography (HPLC), column chromatography, thin-layer chromatography (TLC), high-speed countercurrent chromatography (HSCCC), and capillary electrophoresis (CE), etc. These methods are characterized by high efficiency, high sensitivity, fast separation speed, etc., and the purity of the Reb A obtained by separation is very high. Due to its small processing capacity, it is only suitable for laboratory purification of Reb A. The stationary phase and mobile phase used also affect the purity of the Reb A obtained by separation.

2. 2. 1 HPLC method

It uses high-pressure liquid as the mobile phase, which flows into a chromatographic column containing a stationary phase at different times, so that the components are separated, and then enter the detector for detection, collect the desired components, so as to achieve the separation of a certain substance. Several components of steviol glycosides have similar polarities and similar molecular sizes, so HPLC is often used to separate them. Kolb et al. [23] developed an improved HPLC separation method using an NH2 column, acetonitrile-water as the mobile phase, and acetic acid to adjust the pH of the mobile phase to 5 to achieve the separation of Reb A. Liu Chao et al. [20] and Li Aifeng et al. [24] successively reported the use of HPLC to separate ST and Reb A. The experiment used a Kromasil NH2 column with acetonitrile-water as the mobile phase. Magomet et al. [18a] also reported that acetonitrile-water gradient elution can separate Reb A with a purity of up to 90% to 91%. Berg s  et al. [25] reported an innovative chromatographic method for the two-step purification of Reb A by HPLC. The mobile phase for the first step was water, and the mobile phase for the second step was acetonitrile-water, to obtain high-purity Reb A. HPLC purification of Reb A can obtain a higher purity product and is easier to operate, but its yield is low, the amount of separation is small, and it is not suitable for industrial production [15c].

2. 2. 2 Column chromatography

Kovylyaeva et al. [26] reported that Reb A was separated using column chromatography with a silica gel impregnated with boric acid as the stationary phase, and further purified by recrystallization to obtain high-purity Reb A. Chiang et al. [27] reported in 2015 that Reb A can also be separated using water and ethanol as the eluent in column chromatography.

2. 2. 3 TLC method

It is a very important experimental technique for the rapid separation and qualitative analysis of small amounts of substances. It is also used to track the progress of reactions. Thin-layer chromatography is commonly used to separate substances with similar polarities. In the steviol glycosides system, the polarity of ST is lower than that of Reb A. Using this characteristic, combined with TLC, ST and Reb A can be effectively separated. Shi Rongfu et al. [28] reported that Reb A can be separated in a developing system of chloroform: methanol: water = 30: 20: 4 (volume ratio, same below). Teng Xiangjin et al. [29] reported that ST and Reb A can also be separated when the developing system is n-butanol: acetic acid: ether: water = 9: 6: 3: 1. Antonio et al. [30] successfully separated Reb A and ST using a developing system of chloroform: methanol: water = 40: 20: 2. Subsequently, Vikas et al. [31] reported that Reb A was easily separated in an ethyl acetate: ethanol: water = 80: 20: 12 developing system.

2. 2. 4 HSCCC method

This is a liquid-liquid chromatography separation technique that can eliminate the irreversibility of traditional chromatographic columns in adsorbing samples. It also has the unique advantages of high efficiency, high recovery, and ease of upscaling [32]. Huang et al. [33] successfully isolated and purified Reb A from Stevia rebaudiana leaves using HSCCC in 2010. They reported the optimal HSCCC separation conditions to be a solvent system of hexane: n-butanol: water = 1.5:3.5:5, a flow rate of 1.0 mL/min, and a sample concentration of 10 mg/mL. This process has high equipment requirements, and optimizing the solvent system is time-consuming and laborious. It is still a long way from practical application.

2. 2. 5 CE method

This is a new liquid phase separation technique that uses a capillary as the separation channel and a high-voltage electric field as the driving force. Mauri et al. [15b] used sodium tetraborate and sodium dodecyl sulfate as the buffer solution, and used CE separation technology to achieve good separation of ST and Reb A in a short time. Combining with semi-preparative HPLC, Reb A was finally obtained. Shao Hanjuan et al. [34] used CE to successfully separate the main components of steviol glycosides in 5 min.

2. 3 Adsorption-selective purification of Reb A using MARs

MARs adsorption separation technology is a relatively new separation method. Due to its high adsorption capacity and selectivity, as well as its low cost, easy regeneration and good stability, it has obvious advantages in industrial production [35]. MARs has a large pore network structure and a large specific surface area, which allows it to selectively adsorb through adsorption, thereby separating a substance.

Chen Tianhong et al. [36] synthesized a series of MARs and studied their separation effect on Reb A. They found that MARs containing pyridine and ketone groups had the best effect. Hu Jing et al. [37] found that D107 and D108 resins have selective adsorption of Reb A. Combined with column chromatography, D107 and D108 resins can effectively purify Reb A when used as the stationary phase and 50% ethanol as the mobile phase.

Subsequent studies have shown that a mixture of MARs can effectively improve the efficiency of refining Reb A from Stevia rebaudiana extract (Steviol Glycosides). Liu Yongfeng et al. [35c] reported that ST and Reb A can be effectively separated when the mass ratio of HPD750, LSA-40, 07C and LX-68M is 2:3:3:2. Li Jie et al. [38] continued to study on this basis and found that HPD750, LSA40, LSA30 and DS401 have a mass ratio of 3.75:2.5:0.05:0.45, which has a better effect on refining Reb A. Only one adsorption/desorption cycle is required to achieve a Reb A purity of 97%. 5: 0.05: 0.45. The refined Reb A has a better effect, and only one adsorption/desorption cycle is required to achieve a purity of 97%. Chen Zhenbin et al. [4] found that LZ-1 + LZ-20 + LZ-30 + LZ-37 + LZ-36 refined Reb A also showed good results, and a single cycle of adsorption/desorption could produce high-purity Reb A.

Ye Faying et al. [39] have successively reported the application of D392, DA-1M and SD-2 in the separation of ST and Reb A since 2012. D392 has a stronger adsorption capacity for ST than Reb A, and can obtain Reb A with a purity of 88.4%. DA-1M is a functionalized product with a benzeneboronic acid group, which is prepared by modifying poly(ethylene-co-vinyl acetate) (DA-1) with 3-aminobenzeneboronic acid via an amidation reaction. It has a better adsorption capacity for ST in steviol glycosides than DA-1 1 has better adsorption properties than DA-1 and can preferentially adsorb ST. In a column with DA-1M as the stationary phase, Reb A can be purified using water as the mobile phase [40]. SD-1 is a new type of amino-based adsorbent, which is prepared by amination of hexamethylenetetramine with chloromethylated styrene-co-divinylbenzene copolymer (SD-0). SD-2, which contains a phenylboronic acid group, is prepared by modifying SD-1, and the purity of the refined Reb A can reach 98% [41].

It has been newly reported that Reb A can be effectively separated with a purity of up to 99% using a one-dimensional chromatography method with acetonitrile/water as the mobile phase in a column with 860021 as the stationary phase [42].

2. 4 Application of membrane separation technology

Membrane separation technology (MST) was first used in the purification of steviol glycosides. Later, it was found that it could be effectively used to purify Reb A from steviol glycosides. The selective permeability of the membrane was used to effectively improve the quality of Reb A. Das et al. [43] reported that Reb A was separated using a 30 kDa polyethersulfone ultrafiltration membrane to obtain an ideal color, clarity and purity.

From more than 30 years of research on the industrial production of steviol glycosides, it can be concluded that the recrystallization method and the resin method are commonly used methods for refining Reb A and have been effectively applied in industrial production. The recrystallization method is simple and suitable for quantitative production, but it is time-consuming and uses organic solvents, which causes pollution and therefore needs to be improved; the resin method can effectively improve the purity of Reb A, and the MARs can be regenerated. It is currently an effective method for refining Reb A. As an effective method for refining Reb A, improving the reusability of MARs to reduce production costs is the direction of future research; membrane separation technology, as a new process for refining Reb A, also has its own unique advantages and is expected to be applied to the industrial production of Reb A in the future.

3 Prospects

With the progress of society, the development of technology and the improvement of living standards, people are paying more and more attention to diet and health. In order to reduce the problems caused by sucrose and artificial sweeteners, the use of high-potency sweeteners that are calorie-free and safe for the human body as sugar substitutes is bound to become a trend. Stevia, as a sugar plant, is highly adaptable to the environment and can be cultivated on a large scale. Steviol Glycosides, the leaf extract refined from Stevia, meets this market demand. Reb A is the ingredient in Steviol Glycosides that tastes most closest to sucrose, with zero calories, stable physicochemical properties, and non-fermentable.

It can be widely used in the food and pharmaceutical industries. Not only does it taste good and is inexpensive, but it can also prevent the hidden dangers of obesity and diabetes associated with consuming sucrose. The investment in stevia by Coca-Cola, Cargill, PepsiCo and other Fortune Global 500 companies will greatly promote the development of the stevia industry. Therefore, the process of extracting and refining high-purity Reb A is bound to be the current global direction of development for the stevia industry. In addition, stevioside can be converted to Reb A by enzymatic methods [44] or new high-yielding varieties with Reb A as the main component can be selected [45] to improve the yield and taste of Reb A. Therefore, the development and utilization of Reb A has broad prospects.

4 Conclusion

With the recent safety approval of Reb A in stevia by the US FDA and JECFA, Reb A shows good prospects for development and application as a new high-potency sweetener or as a substitute for sucrose. Reb A is expected to develop into a major high-potency sweetener in the natural food market in all regions of the world in the future. Reb A is the safest natural sweetener and can be widely used in the food, beverage, and pharmaceutical industries to replace sucrose and artificial sweeteners, but its content only accounts for about 25% of the total mass. Therefore, the refining technology for obtaining high levels of Reb A from stevia plant extracts has become a hot research topic at home and abroad, with potential commercial value. Research and development of high-purity Reb A is an important future development direction.

At present, the purification methods for Reb A include recrystallization, thin-layer chromatography, and resin methods. The industrial methods mainly include crystallization and resin methods. In the former method, the solubility of Reb A in different solvents is the key to the crystallization research, which requires a lot of solvents and time , the process is cumbersome, the production cost is high and the yield of Reb A is low; the latter is a method for refining Reb A that emerged in the late 1990s and is currently a hotspot for research on the separation of Reb A. The reusability of macroporous adsorption resins is the key to reducing costs. The current process for refining Reb A has improved the quality of steviol glycosides products to a certain extent and effectively increased the purity of Reb A. However, there are still limitations, such as the low yield of Reb A and the low reusability of the resin, which leads to an increase in production costs. Therefore, in future research, a method for refining Reb A with high purity, which is simple in process, low in cost and high in yield, should be found, with a view to promoting it for industrial production. At the same time, if a product with a sweetness that is superior to Reb A can be discovered, it will provide a new and clear research direction for improving the quality of Reb A products in the future.

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