What Is Steviol Glycoside and What Is Its Use?

Stevia is a perennial herb in the Asteraceae family and the genus Stevia. It is considered the world's third-largest source of sweetness. Currently, China ranks first in the world in stevia cultivation, stevioside production and stevioside exports[1]. Steviol glycoside is a natural sweetener extracted from the leaves of stevia. The European Food Safety Authority (EFSA) and the Codex Alimentarius Commission (CODEX) both approved steviol glycosides as a food additive in 2011. Steviol glycosides are a natural, safe, low-calorie, high-intensity sweetener with a wide range of applications in food, medicine, cosmetics and household chemical products worldwide[2].

According to data from the International Market Analysis Research and Consulting Group (IMARC), the global market value of stevia is expected to reach nearly 818 million US dollars by 2024 [3]. Studies have found that in addition to being used as a sweetener, steviol glycoside also has a variety of physiological functions such as antioxidant, anti-inflammatory, improving blood sugar and blood pressure, antibacterial, anti-tumor, anti-diarrhea, diuretic and immune regulation [4]. Studies in recent years have shown that adding appropriate steviol glycoside to livestock and poultry feed can help improve their production performance [5-6], increase feed utilization [7-8] and regulate the intestinal microbiota [9], indicating that steviol glycoside has great potential for use in livestock and poultry production. Therefore, this paper mainly summarizes the digestion, metabolism and biological functions of ssteviol glycoside, aiming to provide a reference for the rational development and application of stevioside in livestock and poultry production.

1 Chemical structure, extraction method and taste evaluation of steviol glycoside

Steviol glycoside (C38H60O18) is the main sweetening component of stevia. It is a white powdery substance that is a type of tetracyclic diterpene glycoside [10]. It is mainly distributed in the roots, stems and leaves of stevia, with the highest content (up to 14%) in the leaves. Studies have shown that steviol glycoside is stable under various processing and storage conditions, does not react with water-soluble vitamins, organic acids, sweeteners, or caffeic acid; is thermally stable and does not participate in the Maillard reaction; and does not ferment during long-term storage [11]. Steviol glycoside has the advantages of being hygroscopic, low in heat, highly sweet, stable and non-toxic. Its sweetness is 50 to 300 times that of sucrose and it can be used as a substitute for sucrose [1, 12].

Sixty-four steviol glycosides have been identified from the leaves of Stevia rebaudiana, including stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D , Rebaudioside E, Rebaudioside F, Stevioside, D-Glucopyranosyl-alpha-L-fucopyranoside, and Rebaudioside M, etc. 11 kinds of relatively high content [13]. The main difference in the chemical structure of different types of stevioside lies in the difference between the R1 and R2 groups (Table 1), with the R1 and R2 groups attached to the C13-hydroxy and C19-carboxyl groups, respectively [14].

At present, the main methods for extracting steviol glycoside include hot water extraction and solvent extraction [15-17]. Among them, the hot water immersion method is currently the main extraction process for the industrial production of steviol glycoside. This process does not require high equipment and technical requirements, and is easy to industrialize. However, this method is time-consuming and labor-intensive, and the yield of steviol glycoside is low and contains a large number of impurities, such as proteins, polyphenols and organic acids, which increases the difficulty of subsequent decontamination [18]. Therefore, continuous optimization of the subsequent decontamination process to more efficiently extractsteviol glycoside has become a key issue in this process.

Zhang Menglei [19] used chitosan flocculation and precipitation combined with reverse phase chromatography to remove impurities from hot water extracts. Compared with existing industrial production processes, it has the advantages of short process time, being more environmentally friendly and lower cost. advantages. Bursa Kovaevi et al. [15] effectively recovered thermally unstable and non-polar to polar components from Stevia leaves through pressurized hot water extraction optimization.

In addition, water and ethanol are often selected as the extraction agents in solvent extraction methods, and mainly the immersion method and the decoction method are used for extraction. Although the immersion method can effectively extract stevioside, the production cycle is long and the process is complicated. In contrast, the decoction method is more concise, the production cycle is short, and a high-quality steviol glycoside product can be obtained [20]. Chen Hu et al. [21] used a press extraction method to obtain steviol glycoside, and the advantages of this process are short extraction time and high efficiency. It can be seen that with the advancement of extraction process optimization technology, this will bring good application prospects for the industrialized large-scale production of steviol glycoside.

Steviol glycoside, as a new type of sugar substitute, can be perceived by taste receptors as having a sweet taste and a slight bitter taste. Studies have found that stevioside can activate the human taste receptor T1R2 and T1R3 to perceive sweetness, and can also activate the taste receptor T2R4 and T2R14 to perceive bitterness [26]. The perception of steviol glycosides is related to structural features such as the length of the sugar chain, pyranose substitution and the C16 double bond. The fewer glucose units there are on C⁃19 in the carbon skeleton, the longer the aftertaste of bitterness, while the more glucose units there are on C⁃13, the faster the sweetness sets in and the stronger the sweetness. For example, compared to Rebaudioside A and Rebaudioside C, which have a weak sweetness and a strong bitterness, Rebaudioside M and Rebaudioside D have a strong sweetness and a weak bitterness, although their natural content in stevia is very low. However, stevioside and rebaudioside have a strong aftertaste of bitterness and a weak sweetness [27].

Studies have shown that the most direct way to eliminate or reduce the bitter taste of stevioside is to add flavor enhancers or taste modifiers to the product to mask its bitterness, such as erythritol, maltitol and xylitol [28], or spray drying technology can be used to encapsulate maltodextrin and inulin to minimize the bitter taste of steviol glycosides [29], or the hydrophilicity of steviol glycosides can be enhanced through enzymatic reactions to mask their bitter taste [30]. Studies have found that modifying the stevioside backbone structure directly with the Kamiya⁃8 group can also improve the affinity of stevioside for sweet taste receptors T1R2 and T1R3 and reduce its affinity for bitter taste receptors T2R4 and T2R14, thereby improving the palatability of stevioside [3].

2 Hydrolytic metabolism of steviol glycoside in the body

Current research shows that the hydrolytic metabolic sites and metabolic processes of steviol glycoside in humans and rats are not the same. Both in humans and rats (Figure 1), steviol glycoside enters the digestive tract after oral administration. Since it cannot be degraded by gastric juice and digestive enzymes, it cannot be normally hydrolyzed and absorbed in the intestine [31-32].

Studies have shown that the colonic flora is involved in the hydrolysis and absorption of steviol glycoside, and that the genus Bacteroides can hydrolyze stevioside to steviol [32-34]. After entering the body, stevioside is first broken down by intestinal microorganisms into steviol, which is then further metabolized in bile and the kidneys via the enterohepatic cycle. After entering the kidneys, steviol is metabolized by cytochrome P450 enzymes to produce monohydroxy and dihydroxy metabolites [32]. Studies have confirmed that the peak concentration of steviol in the blood of rats occurs 15 minutes after oral administration. Further research using the Caco⁃2 cell model found that the absorption and transport of steviol by intestinal epithelial cells is rapid, which is consistent with the results of the rat body model [34]. In addition, stevioside was not detected in human feces after continuous oral administration for 3 days, and only steviol was detected [35-36].

Cardoso et al. [37] studied the distribution and metabolism of stevioside in vivo by intravenous injection of 131I-labeled stevioside into Wistar male rats. They found that the highest concentrations of radioactive material were in the liver and small intestine of rats 10 and 120 min after injection. Due to the differences in the digestive systems, the metabolic processes of stevioside in different animals also differ, and may involve organic anion transport in the kidney [38]. Studies have shown that the main metabolites of stevioside in rats are excreted in the feces via bile [39].

Steviol glycoside is mainly metabolized by the human body through the kidneys and then excreted in the urine. After 72 hours, 62% of stevioside will be found in the urine in the form of steviol glucuronide [35-36, 40], which is related to the difference in organic anion excretion. The molecular weight of steviol glucuronide is 494 u. Organic anions with a molecular weight of 600 u or less are mainly excreted through the kidneys in the human body, while organic anions with a molecular weight of 600 u or more are mainly excreted by the body through the bile. [41]; on the contrary, compounds with a molecular mass of 325 u or greater are mainly excreted from the body through the bile in rats. [41]. However, there is currently relatively little research on the metabolic patterns of steviosides in livestock and poultry.

3 Biological functions of steviol glycoside

3.1 Antioxidant effect

Studies have confirmed that steviol glycoside has strong antioxidant activity. Stevioside significantly increased the activities of superoxide dismutase (SOD) and catalase (CAT) in the livers of diabetic rats, which has a significant protective effect against oxidative damage [42]. Stevioside can inhibit oxidative stress in the kidneys of mice by inhibiting extracellular regulated protein kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NF-κB), thereby reducing the toxic effects of free radicals produced in various secondary reactions [43].

In vitro studies have also confirmed that stevia and its extracts have good antioxidant activity. Jahan et al. [44] measured the maximum absorbance of the 80% ethanol extract of stevia to be 1.0 71 nm, while the maximum absorbance of ascorbic acid as a standard substance was 1.374 nm, indicating that stevia ethanol extract has a higher reducing power than ascorbic acid (vitamin C). The aqueous extract of Stevia rebaudiana Bertoni leaves has a strong scavenging effect on superoxide anions, hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH) [45]. Studies have also found that treating streptozotocin-induced diabetic mice with stevia leaves can significantly increase glutathione peroxidase (GSH-Px) activity and reduced glutathione (GSH) levels [46]. In addition, the addition of 200 mg/kg stevia residue extract can significantly improve the antioxidant status and liver fat accumulation of mice induced by high-fat high-fructose diet (HFFD) [47], while the addition of 200 and 400 mg/kg stevia residue extract can also improve the intestinal structure and intestinal antioxidant status of mice with hyperuricemia [48]. Studies have shown that the good antioxidant activity of stevia extract makes it possible to use it as a preservative in salmon pastes and other seafood products [49].

3.2 Anti-inflammatory effect

Studies have shown that organic solvent extracts of stevia have good anti-inflammatory effects. For example, ethanol extracts of stevia leaves can effectively relieve ear edema inflammation in the CF⁃ 1 mouse model of inflammation induced by arachidonic acid (AA) and furobol 12 - myristate 13 acetate (TPA) [50]. Both the chloroform extract and the methanol extract of stevia significantly inhibited carrageenan-induced rat foot swelling [51].

Stevioside can downregulate Toll-like receptor 2 (TLR2), NF-κB and mitogen-activated protein kinase (MAPK) pathways to reduce the levels of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in mice with mastitis model [52]. α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), which are proinflammatory factors, in a mastitis model mouse [52]. Stevia glycosides can also significantly inhibit the levels of TNF-α and IL-6 in mice with colitis models and inhibit the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins [53]. Similarly , a study found that stevioside can downregulate the expression of proinflammatory factors such as IL-6, TNF-α, IL-1β, COX-2, high mobility group box 1 protein (HMGB1) and inducible nitric oxide nitric oxide synthase/nitric oxide (iNOS/NO) and other proinflammatory factors, and upregulates the expression of the anti-inflammatory factors transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10)[54]. Other It has also been found that stevioside and steviol are non-toxic to Caco⁃2 cells and can block the nuclear factor inhibitor protein α/nuclear factor-κB (IκBα/NF⁃κB) signaling pathway, which is induced by lipopolysaccharide and leads to the production of TNF⁃α, IL⁃1β and IL⁃6 [55].

3. 3. Hypoglycemic and antihypertensive effects

Steviol glycosides, as a low-calorie, high-sweetness sugar substitute, can improve blood glucose levels. Shivanna et al. [42] showed that stevia leaf powder can enhance insulin secretion by pancreatic β cells in type 1 diabetic rats, and the insulin sensitivity and glucose tolerance of type 2 diabetic rats can also be improved.

In addition it has been found that stevioside can significantly improve the insulin and glycogen levels in diabetic rats, while significantly reducing their fasting blood glucose and glycosylated haemoglobin levels [56-57]. When the When the blood glucose level is high, there is a direct dose-response relationship between insulin secretion and rebaudioside A [58]. Stevioside can improve blood glucose by activating calcium ions (Ca2+) to activate taste receptors and cation channels (TRPM5) on pancreatic β cells to enhance glucose-induced insulin secretion [59-60]. Some studies studies have found that steviol can significantly reduce glucose accumulation in the intestine, which is due to steviol's ability to inhibit intestinal absorption of glucose by reducing the adenosine triphosphate (ATP) content of the intestinal mucosa and changing the intestinal morphology and structure [61].

Some studies Studies have shown that after 2 years of taking 500 mg/d stevioside, the systolic and diastolic blood pressures of female hypertensive patients were significantly reduced [62]. et al. [63] also found that after only 7 days, the systolic and diastolic blood pressures of female hypertensive patients aged 28 to 75 years who took 0.25 g of stevioside 3 times a day were significantly reduced. The study concluded that the good blood pressure lowering effect of stevioside may be related to its strong inhibition of angiotensin-converting enzyme activity [64].

3. 4. Antibacterial and anticancer effects

Puri et al. [65] found that stevioside purified by extraction with organic solvents such as ethanol, methanol and chloroform can inhibit the growth of Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus subtilis and Salmonella typhimurium. and Salmonella typhimurium. In particular, the antibacterial activity against Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa showed a significant positive dose-dependent change. It is that the amount of plaque accumulated in the mouth after an adult gargled with rebaudioside was 82% lower than that after gargling with sucrose [66], and that stevioside is widely used in the traditional treatment of mouth ulcers and gum disease due to its antibacterial properties [67].

Through research has found that stevioside can also inhibit the proliferation of bladder cancer cells by selectively inhibiting their proliferation and inducing apoptosis to inhibit their growth [68].

Stevioside glycosides can also reduce the migration and metastasis potential of breast cancer cells MDA⁃MB⁃231 [69], reduce their invasiveness, and the main component of stevioside, steviol, has a strong cytotoxic effect on MCF⁃7 breast cancer cells, and can inhibit the proliferation of MCF⁃7 cells by G2/M cycle arrest [70 . Chen et al. [71] showed that steviol had an antiproliferative effect on six cancer cell lines, including human gastric MKN-45, MGC-803 and HCG-27, and colorectal HCT116, HCT-8 and Caco-2. The inhibition of cell viability was dose-dependent and gastric cancer cells are more sensitive than colorectal cancer cells. Further analysis of the cell cycle confirmed that steviol can cause MKN⁃45, HGC⁃27, HCT⁃ 116 and Caco⁃2 cells to block in the G1 phase, while MGC⁃803 and HCT⁃8 cells block in the G2 phase.

4 Stevioside Application of stevioside in animal production

4.1 Application of stevioside in livestock production

Studies have found that stevioside can affect the production performance of taste-sensitive livestock such as pigs, cattle, and sheep, increase daily weight gain, and help reduce diarrhea in young livestock [5, 72]. Research showed that adding 167 mg/kg stevioside to the feed after the second week of weaning significantly increased the daily weight gain of weaned piglets, but adding different proportions (0 .083 3%, 0. 167% or 0. 334%) of stevioside had no significant effect on the feed intake and feed conversion ratio of weaned piglets [72].

When When 300 mg/kg stevioside was added to the feed, the average daily weight gain of 28-day-old weaned piglets was the highest, the average daily feed intake showed a linear increase, and the feed conversion ratio showed a linear decrease. In addition the addition of 0.3% stevioside to the feed can significantly increase the daily weight gain and feed utilization rate of Duroc pigs, enhance immunity, and improve carcass traits such as backfat thickness [7]. Overall in general, the addition of stevioside to the feed may help improve the growth performance of pigs by increasing feed utilization and enhancing immunity.

 At present, there are relatively few studies on how stevioside affects the changes in the intestinal flora of livestock animals, and whether the improvement of growth performance in pigs is related to the regulation of the intestinal flora remains to be further revealed. However, been confirmed that the addition of stevia residue (containing stevioside, chlorogenic acid, flavonoids and other substances) to the feed of pregnant sows can regulate the composition of the intestinal flora, significantly promote the relative abundance of beneficial intestinal bacteria ( such as the family of Fusobacteriaceae and the family of Ruminococcaceae, and reduce the relative abundance of harmful bacteria such as Clostridium perfringens. It can also significantly increase the average daily feed intake of nursing sows [73].

Currently there is relatively little research on the use of stevioside as a feed additive in cattle and sheep diets. Han et al. [74] found that adding 400–800 mg/kg stevioside to the diet of Shandong black goats significantly increased the amount of dry forage eaten and the total amount of feed eaten. In feed 0.3% stevioside in the feed can improve the final weight, weight gain and crude protein content of the carcass of Han cattle, significantly reduce drip loss and shear force, and increase the meat color redness value of the longissimus dorsi muscle, thereby improving carcass quality [75]. However, further research is needed on the specific mechanism of stevioside added to the feed to regulate the production performance of cattle and sheep.

4. 2. Steviol glycoside application in poultry production

Studies have found that stevioside has the effect of improving poultry production performance and immune function. However, birds are not sensitive to sweetness due to the lack of the T1R2 sweet taste receptor, so birds mainly affect their production performance through non-T1R2-dependent pathways [6].

For example , for example, the addition of 0.3% stevia extract can significantly increase the daily weight gain and feed utilization of 3-week-old green-footed chickens, increase serum albumin levels, and improve organ indices such as the bursa of Fabricius, spleen and thymus [8, 76]. Jiang et al. [6] also found that adding 250 mg/kg stevioside to the feed of Ross 308 broilers significantly increased their body weight, average daily weight gain and average daily feed intake. Recent research shows that stevioside may promote the increase in broiler feed intake by regulating the hypothalamic neuroactive ligand-receptor interaction pathway and changing the intestinal microbial composition [9].

Due to the antioxidant and anti-inflammatory effects of stevioside, stevioside significantly improved the antioxidant capacity of broiler serum and jejunum, significantly reduced the mRNA expression of NF-κB in the ileal mucosa, and had a tendency to reduce the mRNA expression of IL-1β in the jejunum and γ-interferon (IFN-γ) mRNA expression tended to decrease, and significantly increased the mRNA expression of Nrf2, CAT and SOD1 in the jejunum and ileum mucosa of broilers [77]. In addition the addition of 80 mg/kg stevia sweetener (containing 0.5% stevioside) to the feed can significantly increase the body weight and daily weight gain of Cobb broilers after the 42nd day of the experiment [78].

The above results show that the addition of stevioside to poultry feed can improve feed utilization, antioxidant capacity and immune function, and regulate the composition of intestinal microorganisms, thereby helping to improve poultry growth performance. However, , it has also been found that the addition of 667 mg/kg stevioside has no significant effect on the feed intake, weight gain and feed conversion rate of broiler chickens [79]. It should be be noted that a high dose of stevioside (3,200 mg/kg) had no significant effect on the average daily weight gain, average daily feed intake, feed conversion ratio and immune organ index of Arbor Acres broilers, but significantly lowered their blood glucose levels, inhibited glucose synthesis, and increased the apparent digestibility of calcium and phosphorus [80]. The above There are some differences in the results reported in different studies, which may be related to factors such as animal breed, the effective activity and added dose of stevioside, treatment time and feeding management.

Stevia glycosides added to the feed The addition of stevioside to the feed may also have a maternal and intergenerational effect. Jiang et al. [81] found that the addition of 250 mg/kg stevioside to the feed of broiler breeders improved the development of their chicken embryos development and improved intestinal morphological integrity and intestinal immune function in the offspring broilers. It was also found that stevioside can also alleviate the damage to the intestinal mucosa of the offspring broilers caused by lipopolysaccharides by regulating the intestinal microbiota [82]. In addition , feeding laying hens a diet supplemented with 1% stevia extract can not only increase daily weight gain and feed efficiency, improve gastrointestinal function, enhance immunity, but also enhance the yolk color of eggs and the total amount of essential amino acids and protein in eggs [83]. At present , and there have been many reports on the application of stevioside as a feed additive in broilers and laying hens, while there have been relatively few studies on its application in other poultry.

5 Summary and outlook

In summary, stevioside, as a natural sweetener, has good biological functions such as anti-oxidation, anti-inflammation, improving blood sugar and blood pressure, antibacterial, anticancer and immune regulation. Research have shown that the addition of stevioside to the feed has the effect of increasing feed intake and promoting growth in livestock and poultry, as well as improving the antioxidant capacity of livestock and poultry, regulating the immune function of the body and intestines, and having a certain effect on improving the quality of livestock and poultry meat and eggs. Therefore, stevioside has broad application prospects as a new feed additive in livestock and poultry production. However,  

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