What Is the Use of Stevia Glycoside in the Food Field?

Stevia is a member of the Asteraceae family, native to South America and now cultivated worldwide. It has been found to contain more than 40 different steviol glycosides, including rebaudioside A (RebA), rebaudioside B (Reb B), rebaudioside C (Reb C), Reb E, Reb F, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, stevioside, and dulcoside A. The total content can be as high as 20% of the dry leaf weight [1]. Stevioside is a diterpene compound with steviol as the aglycone (Figure 1). Different amounts of glucose, rhamnose, xylose, fructose and deoxyglucose are linked to its C-13 or C-19 position via 1,2-, 1 , 1,4-, 1,6-a or b-glycosidic bonds at their C-13 or C-19 positions, forming different types of steviol glycosides [2]. Pure stevioside is a white powder with the chemical formula C38H60O18. It is easily soluble in water, methanol and ethanol, but insoluble in organic solvents such as benzene, ether and chloroform.

Stevia glycoside is a natural active substance that is heat-resistant, stable, not easily decomposed by light, non-toxic and harmless, and does not participate in reactions in the human body. It is 250 to 300 times sweeter than sucrose and is a natural sweetener [3]. Thirteen steviol glycosides, including stevia glycoside, rebaudioside A and dulcoside, have been included in the Food Safety National Standard Food Additives Steviol Glycosides (GB 1886.355-2022). Studies have found that stevioside has a variety of biological functions, including antioxidant, hypoglycemic, hypotensive, anticancer, improving gastrointestinal function and treating amnesia. Therefore, this paper introduces the application of stevia glycoside in the food industry in recent years and the progress of research on its biological functions, providing a reference for the further development and utilization of stevia glycoside.

1. Application of steviol glycosides in the food industry

Sucrose can provide sweetness and improve the texture and color of foods. Replacing sucrose partially or completely is the strategy used in most foods to reduce sugar. Steviol glycosides have important advantages. Their zero energy value and high health safety make them superior to sucrose. More importantly, studies have shown that steviol glycosides do not accumulate in the body and have many health-promoting properties. However, whether the texture and taste of foods can be guaranteed is a problem that most food manufacturers face today [4-5]. Steviol glycosides have been used as natural sweeteners in a variety of foods such as beverages, condiments, yogurt and bread [6-8].

1.1 Steviol glycosides in beverages

Steviol glycosides is highly stable in beverages and has a long-lasting aftertaste, which can reduce the sweetness and greasiness of sucrose [9]. After stevia glycoside was approved as a food additive in the European Union, countries such as the United Kingdom and France added stevioside and sucrose-based compound sweeteners to Sprite, replacing the original sucrose [10]. Fruit tea drinks and milk drinks on the Chinese market also use stevioside to replace some of the sucrose. Tang Jin et al. [11] used stevioside in different ratios to replace sucrose in a myrica rubra juice drink. They found that when stevioside replaced sucrose by 40%, the drink had a good taste, was less bitter, had a long aftertaste and had the best beverage characteristics. Buniowska et al. [12] added stevioside to a compound beverage made from papaya juice, orange juice, mango juice and oat drink, and used a response surface method with a central composite design. It was found that when the stevia concentration was 2.5% (V/V), the beverage had the highest antioxidant properties and the best taste.

1.2 Application of stevia glycoside in condiments

The use of steviol glycosides to replace 20% to 30% of sucrose in the processing of soy sauce and vinegar can improve the flavor [13], shorten the marinating time, extend the shelf life, reduce the occurrence of fermentation in the product, avoid browning reactions, and increase the pass rate. Lee Kum Kee Oyster Sauce and Kewpie Sweet Salad Dressing both use steviol glycosides. Pan Jiahui et al. [14] added a compound sweetener consisting of 0.07% monk fruit extract, 14.00% erythritol and 0.03% steviol glycosides to the teriyaki sauce instead of 40% sucrose, achieving “low sugar” while making the teriyaki sauce taste close to the teriyaki sauce with added white sugar.

1.3 Stevia glycoside in yogurt

Stevia glycoside has long been used as a sweetener in yogurt and ice cream products. Studies have found that the addition of stevioside to probiotic yogurt does not affect the growth of probiotics and can maintain a corresponding functional level after 28 days of storage [15]. RIBERIO et al. [16] compared stevioside at different concentrations with a standard sample containing a mixture of sucrose and sucralose. Stevioside 1 (75% rebaudioside A + stevioside), 5% stevioside 2 (9 5% rebaudioside A) and 40% stevioside 3 (50% rebaudioside A) were most similar to the sensory characteristics of the standard sample, and there was no significant difference in consumer acceptance, which promoted the use of stevioside in yogurt. Yan Liping et al. [17] used stevioside instead of sucrose in apple sauce yogurt. By measuring the acidity and whey precipitation rate of the yogurt during a 20-day storage period, they found that when stevioside replaced 30% of the sucrose, the resulting yogurt had the highest sensory score, a smooth surface, and a delicate texture.

1.4 Stevia glycoside in baked goods

Steviol glycosides can be used as a substitute for sucrose in baked goods and improve the sensory and nutritional properties of the products. RUIZ et al. [18] compared wheat bread containing steviol glycoside extracts at different concentrations as a substitute for sucrose with traditional wheat bread. Compared to the control group, the bread with stevia extract was softer and had slower microbial growth during the shelf life. In terms of nutritional contribution, the bread with stevia extract has a higher dietary fibre content and a lower digestible carbohydrate content, so it can significantly reduce calorie intake. Replacing 20% of the sucrose with stevia and replacing all the cocoa powder with cocoa dietary fibre had no negative effect on the quality parameters and consumer acceptance of the muffins [19]. Since steviol glycosides are not prone to caramelization, adding steviol glycosides to baked goods will only make the product sweeter without affecting the color.

1.5 Steviol glycosides in jelly and jam

Jelly and jam are products that most people consume on a daily basis. The sugar content of their formulations is about 60%, and sugar not only provides sweetness but also increases the total soluble matter content. With people's increasing awareness of health, food developers have begun to study ways to reduce the sugar content in jellies and jams. PARK et al. [20] added different proportions of stevia powder instead of sucrose to Schisandra chinensis jellies. As the amount of stevia powder added increased, the hardness and chewiness decreased. Combining multiple indicators such as hardness, transparency and sensory characteristics, it was found that the quality of the jelly made with 1.0% stevia leaf powder instead of sucrose was the best. SUNA et al. [21] added stevioside as a sweetener to persimmon jam, developing a jam with antioxidant activity that extended the shelf life of the product. The texture and mouthfeel of jams and jellies are affected to some extent by the addition of steviol glycosides, so improving the quality of low-calorie jams and jellies is an issue that needs to be addressed at this stage.

2 Research progress on the biological activity of stevioside

2.1 Antioxidant activity

When the body is under oxidative stress, a large number of free radicals are produced. Excessive free radical levels can lead to cell and tissue damage, which may trigger diseases such as asthma, Alzheimer's disease, and cancer. Natural products such as polysaccharides and phenolic substances extracted from plants have radical scavenging activity and metal chelating properties, and are strong antioxidants. Supplementing the diet with natural product supplements or natural medicines is important for maintaining homeostasis [22-23]. Like other plants, stevia's antioxidant effect comes from its phenolic compounds and flavonoids. Stevia leaves soaked in water contain higher levels of antioxidants and higher levels of alanine, aspartic acid, leucine and proline than dried stevia leaves [24].

SHIVANNA et al. [25] found that feeding Stevia whole leaf powder to streptozotocin-induced diabetic rats protected the rats from the harm of streptozotocin-induced diabetes, reduced the concentration of malondialdehyde (MDA) in their livers, increased the content of antioxidant enzymes, and reduced the risk of oxidative stress. BENDER et al. [26] found that stevia leaf extract had antioxidant activity and that the antioxidant activity was higher than that of stem extract, as measured by in vitro oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA). Zhang Tongtong et al. [27] studied the in vitro antioxidant activity of stevioside and found that its scavenging abilities for DPPH free radicals and hydroxyl radicals were 24.8% and 13.8%, respectively. The scavenging ability of stevioside against DPPH radicals is related to its structure, while the scavenging ability against hydroxyl radicals is not related to its structure. It is thus found that stevia leaves or their crude extracts can not only be used as a natural sweetener, but also as a resource for food preservatives.

2.2 Hypoglycemic activity

Diabetes is a metabolic syndrome characterized by hyperglycemia, manifested in the body as insulin resistance, decreased insulin secretion, or both. 90% to 95% of diabetic patients have type 2 diabetes (T2D). People with long-term diabetes are prone to complications such as ketoacidosis, retinopathy, nephropathy, and cardiovascular, cerebrovascular, and peripheral vascular diseases. Therefore, early prevention and effective treatment of diabetes is of paramount importance. MYINT et al. [28] found that stevioside and steviol increased glucose uptake by the heart and brain and reduced glucose accumulation. KUJUR et al. [29] found that stevioside had a certain hypoglycemic effect on diabetic rats and was time-dependent by feeding diabetic rats with stevia water, ether and methanol extracts. The hypoglycemic effect of stevioside may be related to antagonizing the glucotoxicity of pancreatic β cells, and may also be related to inhibiting the secretion of glucagon by pancreatic α cells. Therefore, non-nutritive sweeteners, including steviol glycosides, are recommended dietary supplements for patients with T2D.

2.3 Blood pressure-lowering activity

Arterial hypertension is a pathological state involving an inappropriate relationship between vascular resistance and blood volume, affecting about 20% of the world's adult population [30]. Hypertension causes pathological changes in small and medium-sized arteries, which are characterized by thickening of the vessel wall and narrowing of the vessel diameter. This results in the heart needing to work harder to pump enough blood to meet the needs of all the tissues, thereby increasing the risk of heart attack or stroke. Lifestyle changes and dietary therapy are the two most important ways to prevent and treat hypertension. In animal experiments, stevia has been shown to lower blood pressure.

HSU et al. [31] found that an oral administration of 50 mg·kg-1 of stevioside significantly lowered blood pressure in rats with sensitive hypertension, renal hypertension, and spontaneous hypertension after anesthesia. SCHMANDKE [32] found that intravenous or intraperitoneal injection of stevioside at certain concentrations can lower the blood pressure of spontaneously hypertensive rats blood pressure, and in isolated rat aortic rings, stevioside can cause vasodilation by inhibiting calcium release into the blood vessel. LIU et al. [33] also confirmed that stevioside is an effective natural antihypertensive substance, and inferred from in vitro experiments on rat A7r5 cells that its antihypertensive mechanism may be related to the inhibition of Ca2+ inward flow. A large number of human studies have now confirmed that oral administration of crude stevioside solution is safe and can lower blood pressure in hypertensive patients.

2.4 Antitumor activity

Cancer causes mental and physical suffering and financial stress for people. Therefore, various cancer treatments have been developed, including various anticancer drugs, but these methods all have problems such as serious side effects and drug resistance. To address these problems, it is important to develop chemopreventive agents for cancer and improve cancer treatments. Among the many stages of carcinogenesis, the tumor-suppression stage is considered to be one of the most effective preventive methods [34]. In view of the above, many people have been studying cancer preventatives in natural products and identifying their tumor-suppressing and tumor-preventive effects. It has been reported that stevioside, which is extracted from Stevia rebaudiana, has antitumor effects.

LOPEZ et al. [35] confirmed that stevioside can inhibit the proliferation of cervical cancer, pancreatic cancer and colon cancer cells by studying the effect of stevioside on cyclin D1-CDK4, and found that stevioside is an effective apoptosis inducer. PAUL et al. [36] found that stevioside can promote induce apoptosis by promoting the production of intracellular reactive oxygen species, thereby inducing changes in the transmembrane potential of mitochondria, inducing the mitochondrial-mediated apoptosis pathway, and promoting apoptosis of breast cancer cells. KHARE et al. [37] found that stevioside compounds can inhibit the proliferation of MDA-MB-231 and SKBR3 cells and have antitumor activity against breast cancer cell lines. Therefore, stevioside is a bioactive compound with high anti-cancer activity.

2.5 Treating memory loss

Population ageing has become a global phenomenon, and dementia, which is accompanied by memory loss and intellectual decline, is one of the most common diseases among the elderly. Alzheimer's disease (AD) is the most common form of dementia, accounting for 60% to 70% of all cases [38]. Its most prominent feature is the loss of cholinergic neurons in the basal nuclei of the large cortical cells, which is also the main cause of memory loss [39]. To date, cholinesterase inhibitors such as galantamine are the main drugs for the treatment of Alzheimer's disease, but most current drugs only relieve the symptoms of dementia and cannot stop the progression of dementia, and they have certain side effects. Therefore, the development of a drug that can inhibit the progression of dementia is urgent.

In recent years, there has been a great deal of evidence that a moderate increase in circulating glucose levels can enhance the formation of new memories in rodents and humans [40]. Glucose can enhance memory processes by increasing the synthesis and release of acetylcholine in the hippocampus. SHARMA et al. [39] used scopolamine-induced amnesia rats as a model to evaluate learning and memory ability using a water maze test. Cholinergic system activity was evaluated by measuring brain acetylcholinesterase (Acetylch olinesterase (AChE) activity, to evaluate the activity of the cholinergic system, and to measure the levels of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) to evaluate the degree of oxidative stress. The results showed that oral administration of stevioside (250 mg·kg-1) in rats significantly reversed the learning and memory impairment induced by scopolamine, while inhibiting the increase in brain acetylcholinesterase activity and the level of oxidative stress in the brain induced by scopolamine. This shows that stevioside can protect the memory of rats with cognitive impairment through multiple actions.

2.6 Prevention of dental caries

Dental caries is a chronic oral disease that causes the loss of calcium and phosphorus from the surface of the tooth enamel, resulting in tooth decay. One of the causes of dental caries is the intake of carbohydrates such as sucrose, which can be fermented into acids that cause tooth demineralization [41]. Therefore, an important way to prevent tooth decay is to find a substitute for sucrose. Several studies have shown that stevioside can be used as a substitute for sucrose. DE SLAVUTZKY et al. [42] rinsed plaque with a 10% sucrose solution and a 10% stevioside solution for 4 times within 5 days, and measured the plaque index to obtain the amount of plaque accumulation after rinsing with stevia. The amount was reduced by 57.82% compared to when rinsing with sucrose. COCCO et al. [43] investigated the effect of daily consumption of snacks containing stevia on dental caries in 271 children (6-9 years old) at high risk of dental caries.

The results showed that compared with sugary snacks, short-term consumption of snacks containing stevia can reduce the acidity of dental plaque and reduce the production of cariogenic bacteria, thereby reducing the possibility of future new dental caries. BRAMBILLA et al. [44] compared the effects of stevioside and sucrose on Streptococcus mutans biofilm formation and intracellular pH. The results showed that compared to sucrose, rinsing with stevioside reduced the formation of Streptococcus mutans biofilms, and the intracellular pH was not reduced, indicating that stevioside is not only non-cariogenic but also non-acidogenic. These research results all show that stevioside can reduce the formation of dental caries. Long-term human studies are needed to investigate the potential role of stevia or its derivatives in dental caries, as well as their mechanism of action and long-term safety.

3 Conclusion

With people paying more and more attention to healthy living and nutritious diets, the application of steviol glycosides in the food industry and research into their biological activities have become current research hotspots. In addition to being added to foods as a natural food additive, steviol glycosides are also widely used in many industries such as medicine, cosmetics and health products. At present, the extraction, separation and purification processes of stevioside need to be optimized, the most suitable stevioside needs to be found, the bitter taste needs to be removed, and the taste in foods needs to be improved. In addition, the verification of the biological activity of stevioside is currently only applied to cells or animals such as mice, and the actual application in the human body remains to be explored. There has been little research on the safety of stevioside at home and abroad. In China, the latest national standard for food safety, Food Additives – Stevioside (GB 1886.355–2022), requires stevioside to be at least 90% pure, indicating that impurities in stevioside products that have not been highly purified may pose potential hazards.

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