What Are the Methods to Improve Steviol Glycoside Bitter Flavor?

Stevia (also known as stevioside) is a natural sweetener extracted and refined from stevia. It is known for its high sweetness (200-300 times that of sucrose), low calorie (1/300 of sucrose), zero calories and stability under light, heat and acid-base conditions, it is well known and has become the third largest sugar source after sucrose and beet sugar [1]. Stevia is a white crystal or powder that is easily soluble in water and is widely used in the food and beverage industries [2]. Stevia is not easily absorbed by the human body after consumption, so it can be used as a sweetener for people with diabetes or obesity.

It also has the characteristics of anti-hypertension, anti-oxidation, anti-cancer, anti-inflammatory, antibacterial and improving kidney function [3], and can be used as a substitute for health products and medicines, with good application prospects. However, the slight aftertaste of bitterness and the licorice flavor of stevia have become limiting factors for its further development [4]. Therefore, we analyzed the causes of the undesirable flavor of stevia and proposed solutions, with the aim of providing a reference for solving the problem of the undesirable flavor of stevia.

1 Causes of the undesirable flavor of stevia

1.1 The immature extraction process of stevia

Stevia is often used as the raw material for the extraction of steviol glycoside . The extraction process includes steps such as resin adsorption and concentration, solvent washing, recrystallization, and purification with ion exchange resins, and finally pure stevia is obtained by spray drying [5]. However, in addition to the sweet component stevioside, stevia also contains bitter components such as tannins, flavonoids and volatile oils. These bitter components are not completely removed during the extraction and purification process, which can result in bitter impurities remaining in the stevia product and affect the taste of the final product.

There are several reasons for the bitterness produced during the extraction of stevia [6-7]. First, during the resin adsorption and concentration stage, if the resin is not selected and used properly, stevia cannot be effectively adsorbed, and the corresponding bitter components will also be retained. Second, the purpose of solvent cleaning is to remove grease and some bitter substances. If the solvent is incorrectly selected, the bitter components cannot be effectively removed. Third, recrystallization is a key step in improving the purity of stevia, and if this step is not performed properly, it may lead to the concentration of the bitter components rather than their removal; fourth, ion exchange resins are used to remove soluble impurities, and if the resin is not of high quality or the operating conditions are not suitable, the bitter components may not be effectively removed. Fifth, although spray drying is the final step, if the operating conditions (such as temperature, pressure, etc.) are not properly controlled, it may also affect the flavor of the product.

To reduce the bitterness of stevia, the extraction process needs to be carefully optimized, including improving the resin adsorption and solvent washing processes, optimizing the recrystallization conditions, selecting the appropriate ion exchange resin, and adjusting the spray drying parameters.

1.2 Structural differences in steviol glycosides

Following stevia, other steviol glycosides have been isolated from stevia, with 10 common types. They have a similar tetracyclic diterpene chemical skeleton, all with steviol as the aglycone (Figure 1), with the R1 and R2 positions being substituted with glucose, xylose or rhamnose groups of different lengths, thus forming various glycosides with very different organoleptic and physicochemical properties (Table 1). In this structure, steviol itself has strong hydrophobicity and exhibits a certain bitterness; in taste perception, bitterness comes slightly after sweetness, so as the concentration increases, the aftertaste of bitterness is also enhanced. On the other On the other hand, the slight structural changes of stevioside affect its sweetness and taste quality. The difference in the C-13 (R1) position, the substitution of pyranose, and the length of the substituent group at the C-19 (R2) position are the key factors affecting the bitterness of stevioside. It is speculated that this phenomenon may be related to the activation of bitter taste receptors hT2R4 and hT2R14 [8].

Table 1 Structure, Sweetness and Mouthfeel of Steviol Glycosides

①N/A indicates that it is not applicable.

2 Solutions to improve the undesirable flavor of stevia

2.1 Optimization of stevia production process

2.1. 1 Emerging extraction technologies In order to further improve the extraction efficiency of stevia, many studies on the extraction of glycosides have been carried out at home and abroad, including hot water extraction, enzyme-assisted extraction, ultrasonic extraction and other emerging extraction technologies [6]. The development of these results provides ideas for process optimization of the stevia production line, which can improve the total stevioside content (usually greater than 95%) while reducing the residual bitter components such as tannins, flavonoids and volatile oils, thereby improving the aftertaste of stevia.

In traditional production, hot water extraction is commonly used to extract stevioside. This method is energy intensive, time consuming, has a low total stevioside extraction rate, and the finished product has a dark color. During the extraction process, water acts as a medium to dissolve impurities such as organic acids, proteins, and polyphenols. Therefore, the main improvement to this method is targeted at subsequent impurity removal. Zhang Menglei et al. [9] used chitosan flocculation and precipitation combined with reverse-phase chromatography to remove impurities, reducing costs and shortening the time required for impurity removal. Kovacevic et al. [10] used pressurized hot water to extract and effectively recover thermally unstable and polar impurities in stevia leaves, which provides good prospects for industrial production.

Enzyme-assisted extraction improves the extraction efficiency of stevioside at low temperatures by adding cellulase to break cell walls and reduce energy consumption. However, enzymes are expensive to use, and existing enzymes cannot completely break down cell walls. Puri et al. [11] optimized the extraction conditions using a response surface method and demonstrated that enzyme-assisted extraction is more efficient than traditional solvent extraction. It can be seen that enzyme-assisted extraction is expected to become an effective alternative to solvent extraction for stevioside extraction.

Ultrasonic extraction uses the cavitation effect of ultrasound to quickly break the cell walls of plants and accelerate the release of bioactive substances. This method has high extraction efficiency, short extraction time, low solvent consumption, and is easy to operate. However, ultrasonic extraction may cause unknown impurities to be mixed in the solution, which will bring challenges to the subsequent separation process. Subsequent process optimization involves adjusting multiple experimental parameters [12], such as ultrasonic frequency, power, processing time, solvent type and concentration, and solid-liquid ratio, to find the optimal extraction conditions. By optimizing these parameters, extraction efficiency can be maximized while minimizing energy consumption and potential chemical degradation.

In addition to the above-mentioned traditional extraction methods, some new extraction technologies have emerged. For example, Miao Qing et al. [13] used natural eutectic solvents (NADES) instead of traditional solvents, which is not only environmentally friendly but also highly efficient. Jentzer et al. [14] optimized the automatic extraction conditions of stevia using accelerated solvent extraction. These new technologies provide new perspectives and possibilities for the extraction of stevia glycosides.

2.1.2 Blending with other sweeteners

The blending method synergizes stevia with other sweeteners. The flavor of the compound sweetener is closer to that of sucrose, and the aftertaste of stevia is eliminated. For example, combining stevia and erythritol in an eutectic mixture can retain the original functions and characteristics of both, while erythritol can change the sweetness onset curve of stevia and eliminate some of the slight aftertaste.

Combining stevia with allulose and monk can be combined to promote the browning caused by the Maillard reaction between the three, and is often added to carbonated drinks; combining stevia, erythritol, allulose and monk fruit sweetener can bring out the advantages of the four sweeteners and neutralize their respective defects. The refreshing taste of allulose and erythritol can improve the unpleasant taste of stevia and monk fruit sweeteners, while allulose can reduce the crystallinity of erythritol and allow it to participate in the Maillard reaction [15]. This solution is easy to operate, can restore the natural sweetness, and at the same time, the total amount of sweetener used is relatively small, which is less costly. However, stevia still cannot match sucrose in terms of multiple senses such as sweetness and aroma, just by relying on the assistance of other sweeteners.

2.1.3 Chemical and enzymatic modification

Some studies have shown that when stevia is hydrolyzed, it produces steviol glycosides, which have a lower sweetness and a weaker aftertaste. Inspired by this, the carbohydrate part can be chemically or enzymatically modified [16] to reduce the corresponding aftertaste of stevia. Chemical modification refers to changing the taste quality of stevia by chemically reacting to alter the sugar group attached to the steviol glycoside.

However, due to the complicated and demanding chemical modification process, few people currently use this method. Enzyme modification technology refers to the hydrolysis of the acceptor substrate into glucose or other sugar ligands with different chains under the catalytic action of an enzyme. These products are further catalyzed by enzymes to transfer the C-13 or C-19 of the sugar ligand to the acceptor, thereby forming a variety of stevioside derivatives [17].

The enzyme commonly used is cyclodextrin glucanotransferase (CGTase), which catalyzes the transfer of glucose groups from starch and cyclodextrin to the sugar groups of steviol glycosides, thereby introducing new sugar moieties to the steviol glycosides [18] to form glucose-based steviol glycosides. This product can reduce the bitterness, increase solubility, improve the sweetness of stevia, and has the characteristics of a good aftertaste and a mellow flavor. It is often used in combination with sugar alcohols in low-sugar yogurt, where the flavors complement each other. It has been approved for use as a food flavor. However, the above enzymes are relatively expensive, and the use of corresponding sugar donors also increases costs. Further research is needed to develop and screen steviosides with better cost performance and taste.

2.2 Developing a new generation of high-yield Reb D and Reb M

In addition to stevia, the second-generation steviol glycoside Reb A has the highest content. High-purity Reb A has a sweetness similar to sucrose, but its subsequent bitterness is still obvious [19]. The third-generation steviol glycosides Reb D and Reb M have emerged through continuous improvement and optimization. Compared with Reb A, Reb D and Reb M exhibit excellent properties. They have a higher sweetness and taste, no aftertaste and a licorice-like flavour. They have attracted widespread attention as an ideal new generation of sweeteners. However, Reb D and Reb M are rare and low in yield, and only a small amount is contained in conventional stevia plants, making large-scale commercial production difficult. Therefore, the development of new methods to isolate and prepare high-purity Reb D and Reb M is currently a research hotspot in the field of stevia.

2.2.1 Breeding method

The breeding method refers to the use of agricultural technology to scientifically cultivate and grow plants, optimize the best growing conditions, and thus cultivate high-yielding varieties of Reb D and Reb M. Compared with ordinary stevia, its yield is increased by nearly half [20]. The advantage of this method is that it can cultivate natural steviol glycosides, which maintain sweetness without a bitter aftertaste. However, it requires more human and material resources, and the planting environment and conditions need to be optimized, which may require a lot of research and experimentation.

2.2.2 Bioconversion method

The biotransformation method involves starting with the most abundant component, Reb A, and using specific biological enzymes to convert it into Reb D and Reb M [21]. However, this method requires specific enzymes, which are relatively expensive, which increases production costs. The availability of enzymes is also a consideration.

2.2.3 Fermentation method

Fermentation refers to the use of enzymes produced by genetically engineered yeast to ferment stevia extract into Reb D and Reb M [22]. Steviol glycosides fermented in this way have a similar texture to sucrose and a refreshing taste, without changing the original texture of the food. The main raw materials are glucose and sucrose, which can ensure mass production, with the advantages of low cost and scalability. Although the initial research and development costs are high, large-scale production can be carried out when conditions are ripe, reducing costs. It is currently the best performing, most popular and most promising solution.

3 Conclusion

In summary, as people's demand for their own health increases, reducing sugar has become a trend and trend in current social development. People pay more attention to diet and physical health, so there is a need to develop a new generation of perfect sugar substitutes that are equivalent in sweetness, have zero calories and do not affect blood sugar. Currently, there are relatively few known natural sweeteners, and stevia itself meets the above conditions, so the demand for stevia has increased dramatically. However, its subsequent bitter and licorice-like taste limits its large-scale use.

Therefore, the optimization of the stevia extraction process and the development of a new generation of steviol glycosides are urgent. The causes of the slightly bitter aftertaste of stevia were analyzed, and existing solutions were summarized, namely the development of new extraction processes and the construction of a new generation of high-yield stevia glycosides without aftertaste. The development of new extraction methods has become the mainstream of current research on the optimization of extraction rates. Among them, enzyme-assisted extraction methods have good prospects, but there are also challenges with high technical barriers.

Other extraction condition optimization methods, such as ultrasonic extraction, are relatively easy to achieve at the current level of technology and have great advantages for industrial production. The development of a new generation of sweeteners, however, requires rigorous food safety assessments and regulatory approval, which can take a long time. Maintaining the stability of Reb D and Reb M during food processing and storage, as well as ensuring the shelf life of the final product, are also technical issues that need to be addressed during the development process. These challenges need to be overcome through interdisciplinary research and collaboration, combined with modern biotechnology and engineering methods. In future developments, these approaches can be combined, for example, using genetic engineering to assist in breeding and promote the mass production of new high-yielding strains. When the shortcomings of stevia are remedied, it is bound to set off a new wave of research and applications.

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