How to Get Enzymatically Modified Stevia Glucosyl Stevia?

1 Preface

For nearly half a century, scientists have been committed to finding sweeteners with a similar taste to sucrose but with a lower energy value to replace sucrose in order to reduce the occurrence of obesity and the three highs (high blood pressure, high blood sugar, high blood lipids). Currently, artificial sweeteners include acesulfame, alitame, aspartame, cyclamate, sucralose, neotame, etc. With the continuous improvement of living standards, people have begun to pursue natural and green foods. Stevia stands out from natural sweeteners. Its sweetness is 250 to 400 times that of sucrose. Stevia is extracted from the Stevia rebaudiana Berton herb of the Asteraceae family. Its main components are the secondary metabolites stevioside (ST) and rebaudioside A (RA) accumulated in the leaves of the plant (see Figure 1 for chemical structure). such as the secondary metabolites stevioside (ST) and rebaudioside A (RA) (see Figure 1 for chemical structure).

Stevioside research began at the beginning of the last century. To date, there have been more than 1,000 scientific papers and patents on stevioside worldwide, most of which focus on stevioside extraction techniques, taste improvement and safety and toxicology studies based on in vivo samples. In the past, the safety of stevia consumption was controversial due to incomplete human toxicology and metabolism data, and it was only used in large quantities in South Korea and Japan. After years of comprehensive research by various countries, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) clearly stated in the report of its 69th session in June 2008 that stevia has no side effects on the human body when the daily intake is below 4mg/kg of body weight. In December of the same year, the US Food and Drug Administration (FDA) approved the application for the safety and reliability (GRAS) certification of high-purity stevia A glycosides (RA content above 95%) products on the US market with a “no objection” opinion. This marked the beginning of the widespread use of stevia in the food industry markets in Europe and the United States. Stevia, a natural sweetener with high sweetness and low calories, will be a good substitute for sucrose [1-3].

2 Technological progress

After more than 30 years of industrial production, stevia has developed into its third generation: the first generation of stevia is stevioside, the second generation is a mixture of ST and RA, and the third generation is mainly is RA. The sweetness and its characteristics are shown in Table 1. The preparation process of stevia mainly includes the following steps: drying and crushing stevia leaves, liquid phase extraction, impurity removal, resin treatment, spray drying and refinement.

2.1 Extraction

Stevia leaves at the initial bud stage have the highest stevia content. After the leaves at this stage are dried and crushed, polar solvent liquid-phase extraction is usually used to extract stevia, such as hot water or alcohol. Supercritical carbon dioxide extraction has not yet been applied in industry. Non-polar substances such as oil, chlorophyll and essential oils in the leaves can be removed with chloroform or hexane before extraction [4-5]. There are two extraction methods: tank batch extraction and countercurrent continuous extraction [6]. Stevia extract is obtained by removing broken leaves and other solid impurities by centrifugation, plate and frame filter pressing, and membrane filtration [7-8].

2.2 Impurity removal

Separation and purification of stevia: The stevia extract contains a large amount of impurities such as protein, organic acid, tannin, saponin, pigment, and inorganic salt. The content of these impurities is 5-7 times that of the glycoside content [9]. Column chromatography or membrane filtration is commonly used to separate and purify stevia from the stevia extract [10-25].

When using column chromatography to separate and purify stevioside, the stevia extract must first be pretreated. Otherwise, on the one hand, impurities such as pigments in the liquid feed will contaminate the resin, causing the resin to adsorb a large amount of pigment and reduce the adsorption of glycosides, shorten the adsorption cycle, increase the loss of alcohol and glycosides, reduce the extraction rate,   raising production costs. On the other hand, some metal ions in the liquid can cause permanent poisoning of the resin, reducing its service life and affecting the normal progress of production. Thirdly, when the product is used in beverage production, problems such as sedimentation and foaming during filling can occur, affecting its widespread use.

Therefore, calcium hydroxide and iron sulfate are often used as flocculants to help remove impurities from the stevia liquid. Calcium salts can not only form insoluble calcium salts with organic acids such as citric acid, tartaric acid, succinic acid, malic acid, ascorbic acid and oxalic acid to precipitate, but also with polyphenols, proteins, polysaccharides, pigments such as chlorophyll, carotene and lutein to form insoluble calcium salts and precipitate. Iron salts can precipitate tannins and metal salts such as copper, zinc, aluminum and calcium. After pretreatment to remove some of the colored substances, proteins and foreign substances in the stevia extract, the stevioside in the extract is selectively adsorbed by macroporous resin, and then eluted with water or an organic solvent. The salts, most of the pigments and other polar impurities in the eluate are removed by anion and cation exchange resins to obtain a stevioside-enriched solution [26].

2.3 Resin treatment

Research on the purification of stevia liquid using resin in China began in 1982. In 1984, Chen Dechang and others established the first factory in China to purify stevia using resin in Zhenjiang City, Jiangsu Province.

The commonly used cation exchange resins are Amberlite IR-120B, 001×14.5, etc., and the anion exchange resins are Amberlite IRA-93, D-280, etc. The adsorption resins can be Amberlite XAD-2, AB-8, Diaion Hp-20, ADS-7,D107, D108, etc. [27-30].

When stevioside is separated and purified using membrane filtration, filtration through ceramic micropores, ultrafiltration membranes, and nanofiltration membranes can remove ionic compounds and organic molecules with molecular weights that differ greatly from those of stevioside from the stevia liquid, yielding a stevioside-enriched liquid [31]. In 1998, the National Research Council of Canada developed a method for purifying stevia using membrane filtration, which was patented in the United States [32].

2.4 Spray drying

The concentrated liquid can be subjected to cooling crystallization, concentration crystallization or spray drying to obtain a crude stevia product with a dark color and the main components ST and RA. Activated carbon can be used to decolorize the product before crystallization or spray drying to obtain a white product. Most stevia manufacturers in China produce this type of product [3].

2.5 Refining

Refined stevia rich in RA or ST: Due to the difference in solubility between ST and RA in hydrophilic solvents, the crude stevia can be dissolved in a solvent such as methanol or ethanol to a saturated concentration, and then ST or RA crystals can be selectively precipitated by methods such as concentration and cooling. To ensure high purity RA, the crystals can be re-dissolved in an ethanol-water solution, filtered to remove insoluble impurities, and then spray-dried or concentrated to crystallize the product.

3 Analysis methods

The qualitative and quantitative analysis of stevioside can be carried out using colorimetric, precipitation, gas chromatography, liquid chromatography, thin-layer chromatography and other methods. China's national standard GB8270-1999 stipulates the testing standards and methods for ST content. However, there is no official standard for the detection of RA. The Food Chemicals Codex (FCC) published a public discussion draft in 2008, which standardized the determination of RA and ST by high performance liquid chromatography. Amino columns or sugar analysis columns were used as the chromatographic column, and acetonitrile-water mixed solution was used as the mobile phase [34].

4 Flavor improvement

Stevioside is mainly used as a low-calorie sweetener in beverages, yogurts, desserts and table-top sweeteners [35]. ST has a certain degree of bitterness and menthol taste, and its sweetness characteristics are not perfect. RA's sweetness characteristics are closer to sucrose than ST. However, because it is more complicated to purify RA, stevia containing 97% RA still has a certain aftertaste of ST. There are three main ways to improve the taste of stevia.

4.1 Add other flavour enhancers to create a compound sweetener

A compound sweetener is formed by mixing ST with other taste masking agents and sweetening enhancers. By taking advantage of the synergistic effect between various sweeteners and physiological characteristics, it can reduce unpleasant tastes, shorten the time it takes for the taste to develop, and improve the stability and richness of sweetness. It can maintain the special functions of a single sweetener, bring out new flavors, and reduce costs [36-40].

4.2 Increasing the RA content of stevia

Botanists have studied more than 220 species of plants in the Asteraceae family in South America in an attempt to find plants with higher sweetener content or new sweeteners. However, only one plant, Stevia phlebophylla, was found to contain ST like Stevia rebaudiana Bertoni. This plant was discovered in 1899 and is now extinct.

The cultivation of stevia with a high RA content has been carried out for decades, and Japan is one of the best countries for research on stevia variety improvement and application. In addition to some stevia varieties cultivated by our country itself, the varieties of stevia used in China in recent years mainly come from Japan. The Japanese variety Morita, with its relatively low price and excellent quality characteristics, occupies a large market share of the stevia planting sector in China. However, the variety is degenerating seriously, which has a negative impact on the stability and development of the source of China's stevia industry. The Nanjing Botanical Research Institute, Qingdao Chuangsheng Company, Shandong Huaxian Stevia Co., Ltd., Yancheng Xiaguang Stevia Trading Co., Ltd., and other units in China have also developed varieties with higher glycoside content. Table 2 lists the stevioside content in the dried leaves of some stevia varieties [41-43].

In addition to breeding new varieties to increase the RA content in plants, chemical methods for converting ST to RA have also been reported [44]. The difference between ST and RA lies in the number of glucose units attached to the C13 position of the steviol glycoside (see Figure 1). The key to converting ST to RA is the selective hydrolysis of the β-sophoroside and terminal sugar ether linkage of the stevioside molecule by the enzyme “Takadiastase Y” extracted from the microorganism Aspergillus oryzae, to obtain the product Rubusoside. glycoside molecule β-sophoroside and the terminal glycoside ether linkage are selectively hydrolyzed to obtain the product stevioside, Rubusoside. Rubusoside is another natural diterpene glycoside that can be extracted from the Rosaceae plant Rubus suaviss mus. After the glucose group at the C19 position is removed by alkaline hydrolysis, a phenylaldehyde is used to form an acetal to protect the hydroxyl groups C4 and C6 of the glucose group at C13. and C19-carboxylic acid hydroxyl groups, and then the hydroxyl groups of C2 and C3 of the glucose attached to C13 and the carboxylic acid hydroxyl group of C19 are condensed to add a glucose group, and then barium hydroxide is used to remove the protective group in a methanol solution to obtain RA.

4.3 Enzymatic modification

Enzymatic modification is another commonly used method to improve the taste of stevia. By studying the mechanism of sweetness production and using the catalytic effect of enzymes, specific groups such as glucose (Glc) or fructose (Fru) units are added to ST and RA to make the taste closer to that of sucrose. Enzyme-modified stevia is about 100 times sweeter than sucrose [45–47].

(1) Using α-1,4-glucosidase (e.g., cyclodextrin glucanotransferase), glucose units are added to the stevioside via an α-addition reaction to improve the aftertaste of stevia. However, as the enzymatic reaction proceeds, the 4th hydroxyl group of the glucose bound to the stevioside will further polymerize with the new glucose group according to the α-1,4-addition reaction, forming a long chain of glucose. The sweetness of the resulting α-glucosyl stevioside is reduced by about 45% compared to before modification. The sweetness of the modified α-glucopyranosyl stevioside is reduced by about 45%. β-amylase can be used to cut and shorten the long glucose chains and improve the quality of the product [48-49].

(2) Using β-1,4-galactosyltransferase, the disaccharides (e.g., lactose, α-D-galactose, etc.) that are composed of galactose and glucose can be used as β-1,4-galactosyl sources to combine a glucose group at the 13-position of the steviol skeleton (called 13-G1), as well as two glucose groups at the 1 3 -G1, and the two glucose units bound to 13 -G1 (called 13 -G2, 13 -G3), and the four glucose units bound to the 19th position of the steviol skeleton (called 19 -G1), are modified by binding one galactose unit with a β -1, 4 bond to one to three glucose units, respectively. In September 2007, Dainippon Ink and Chemicals Inc (Dic Fine Chemicals, Inc) used this method to prepare ST and RA derivatives GST and GRA, and obtained a US patent. GRA and GST both maintain a high sweetness, but also have a mellow sweetness similar to sugar, a good aftertaste, and reduce the bitter aftertaste of ST [50-51].

(3) β-fructofuranosidase is used to hydrolyze sucrose under the action of phosphoric acid, introducing a fructose group to ST and RA. The fructose group is linked to the 6-OH of the 19-O-β-glucosyl group by a β-2,6 glycosidic bond.

Enzyme-modified stevia can effectively reconcile the aftertaste of ST, but it increases sugar intake and is not suitable for diabetics.

5 Domestic development

The development of edible stevia in China began in the 1970s at the Nanjing Zhongshan Botanical Garden. In 2000, China's stevia extract exports were less than US$1 million. Since 2006, exports have accelerated, and in 2009, exports were already 84 times that of 2000, with an export volume of about 2,500 tons (equivalent to the sweetness of 675,000 tons of sucrose). It is exported to more than 10 countries, with an annual output value of more than 600 million yuan. China has become the world's leading producer and exporter of stevia glycosides[52]. Domestic stevia manufacturers are mainly concentrated in Shandong, Jiangsu, Anhui, Fujian, Henan and other places.

6 Application

More than 500 years ago, the Paraguayans already used stevia to make sweet tea. Stevia was approved as a food additive in South America and some Asian countries such as China, Japan and South Korea in the last century. The US Food and Drug Administration (FDA) issued a Generaly Recognized as Safe (GRAS) notification in 2008, confirming that they “have no objection” to the use of RA as a zero-calorie sweetener with a minimum purity of 95%.

France, Australia, New Zealand, and Switzerland have also issued RA food additive licenses. In 2008, JECFA announced a RA ADI of 0–4 mg/kg body weight/day. Stevia rebaudiana is not broken down by enzymes in the digestive tract. Stevia rebaudiana enters the colon through the stomach and small intestine and is fermented by intestinal microorganisms to produce short-chain fatty acids. The caloric value of stevia is indirectly generated by short-chain fatty acids and is approximately 6.3 kJ/g. Stevia is a low-calorie sweetener that is used in many countries to replace sugar in order to reduce the calories in dairy products, ice cream, candy, and baked goods.

Moreover, the indigestibility of stevioside means that its intake does not cause an increase in blood glucose levels, and it does not promote an increase in blood insulin levels, making it suitable for use by diabetics. In addition, stevia has great potential medicinal value, with functions such as lowering blood sugar, preventing tooth decay, strengthening the stomach, and relieving fatigue. It has a supporting effect on patients suffering from obesity, diabetes, heart disease, hypertension, and atherosclerosis.

The main components of stevia, ST and RA, have good thermal stability to acid and alkali, and are very stable to light, whether in powder or solution form. They do not go mouldy or deteriorate during long-term storage, and the finished product does not brown after heat treatment, as with sucrose. It can be used in carbonated drinks, tea, wine, coffee, biscuits, sweets, chocolate, dairy products such as ice cream, fruit and vegetable juices, seafood, meat, medicine, cosmetics and other daily commodities [53].

The world's two largest beverage companies, Coca-Cola and PepsiCo, have already launched stevia-sweetened products: Coca-Cola has teamed up with the agricultural company Cargill to develop Truvia, and PepsiCo has teamed up with the company Global Sweetener to develop PureVia, in order to promote the launch of low-calorie beverages and increase product sales.

7 Outlook

Compared with traditional sugar-free sweeteners such as aspartame, cyclamate, acesulfame and saccharin, stevia has the advantages of being natural, stable and safe. Stevia cultivation has good economic value. The yield of dried stevia leaves is 250 to 300 kg per mu, and the price of dried stevia leaves varies according to the glycoside content. In the south, stevia can be harvested twice a year; in the north, farmers can plant stevia together with winter wheat in different seasons, which can effectively increase their income. Using stevia to replace sucrose costs only 20% to 40% of the cost of sucrose. Generally, stevia is more than 90% pure and is 250 times sweeter than sucrose in a 2% sucrose solution and 200 times sweeter than sucrose in a 5% sucrose solution.

Stevia has been used in Japan and South Korea for more than 30 years and is widely used in beverages, food and medicine. In recent years, the use of stevia in Japan has increased by 20% to 30%. South Korea is not suitable for growing sugar beets and sugar cane, and completely depends on imports of sucrose, so there is a strong demand for sucrose substitutes. Since its introduction in 1973, stevia has captured 40% of the Korean sweetener market, most of which is used in traditional Korean soju. In addition, the amount of stevia used in ice cream, chewing gum, candy, kimchi, beverages, sauces, etc. is also increasing. Stevia is in line with today's consumer concepts of “natural, green, safe, and health care.” At present, the world's top 500 companies such as Coca-Cola, Cargill, and PepsiCo are investing in stevia on a global scale, which will greatly promote the development of the stevia industry. As the most promising alternative to sucrose, stevia will further gain market recognition.

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