Stevia Leaf Extract Research Articles

Batch Adsorption of Tannins from Stevia Leaf Extract using Activated Carbon: An Investigation into Adsorption Isotherms and Adsorption Energy

Stevia leaf extract (Stevia rebaudiana Bertoni) could have been utilized as an alternative natural sweetener because it possessed a sweetness level 300 times that of sucrose sugar. However, stevia leaf extract still contained a bitter taste attributed to the presence of tannins. The method that can be used to reduce tannin content is batch adsorption using activated carbon. This study aimed to investigate the batch adsorption process for the tannin content of stevia leaf extract using activated carbon with variable adsorption times ranging from 5 to 180 minutes, which included the appropriate adsorption isotherm model and adsorption energy. Stevia leaves were extracted through maceration with 70% ethanol. The results of the stevia leaf extract were adsorbed in batches using activated carbon treated with 1M NaOH. The analysis was carried out by testing the tannin content (mg/g TAE) using a UV-Vis spectrophotometer at a wavelength of 735 nm and calculating the adsorption isotherm and adsorption energy. The results showed that the tannin content before adsorption was 0.910 mg/g TAE, and the lowest tannin content was 0.040 mg/g TAE at 100 minutes. The tannin content decreased between 5–90 minutes, remained constant, reached equilibrium at 95–120 minutes, and increased again at 125–180 minutes. The suitable adsorption isotherm model was the Freundlich isotherm with a value of R2 = 0.9998; KF = 25.293 [(mg/g)(L/mg)1/nf], and adsorption energy = -8.03 kJ/mol. The adsorption that occurred was classified as physical adsorption.

Integration of Antioxidant Activity Assays Data of Stevia Leaf Extracts: A Systematic Review and Meta-Analysis.

Stevia rebaudiana Bertoni, a no-calorie natural sweetener, contains a plethora of polyphenols that exert antioxidant properties with potential medicinal significance. Due to the variety of functional groups, polyphenols exhibit varying solubility depending on the nature of the extraction solvents (water, organic, or their mixtures, defined further on as hydroalcoholic extracts). In the present study, we performed a systematic review, following PRISMA guidelines, and meta-analysis, synthesizing all available data from 45 articles encompassing 250 different studies. Our results showed that the total phenolic content (TPC) of hydroalcoholic and aqueous extracts presents higher values (64.77 and 63.73 mg GAE/g) compared to organic extracts (33.39). Total flavonoid content (TFC) was also higher in aqueous and hydroalcoholic extracts; meta-regression analysis revealed that outcomes in different measuring units (mg QE/g, mg CE/g, and mg RUE/g) do not present statistically significant differences and can be synthesized in meta-analysis. Using meta-regression analysis, we showed that outcomes from the chemical-based ABTS, FRAP, and ORAC antioxidant assays for the same extract type can be combined in meta-analysis because they do not differ statistically significantly. Meta-analysis of ABTS, FRAP, and ORAC assays outcomes revealed that the antioxidant activity profile of various extract types follows that of their phenolic and flavonoid content. Using regression meta-analysis, we also presented that outcomes from SOD, CAT, and POX enzymatic antioxidant assays are independent of the assay type (p-value = 0.905) and can be combined. Our study constitutes the first effort to quantitatively and statistically synthesize the research results of individual studies using all methods measuring the antioxidant activity of stevia leaf extracts. Our results, in light of evidence-based practice, uncover the need for a broadly accepted, unified, methodological strategy to perform antioxidant tests, and offer documentation that the use of ethanol:water 1:1 mixtures or pure water can more efficiently extract stevia antioxidant compounds.

Effect of the proportion of stevia leaf extract (Stevia rebaudiana B) on the chemical characteristic properties of functional pudding

Functional pudding, a popular dessert, has attracted attention in recent years due to its potential as a vehicle for delivering health-promoting ingredients. This study investigates the impact of different proportions of stevia leaf extract (Stevia rebaudiana B) on the physicochemical properties of functional pudding. Stevia leaf extract, known for its natural sweetness and negligible impact on blood sugar levels, holds promise as a low-calorie alternative to traditional sweeteners. Through systematic experimentation, various concentrations of stevia leaf extract are incorporated into pudding formulations, and their effects on water content, ash content, fat content, protein content, and carbohydrate content are evaluated. The results indicate significant alterations in the physicochemical characteristics of the pudding samples, with variations observed in water content, ash content, fat content, protein content, and carbohydrate content. Specifically, sample F6, containing the highest proportion of stevia leaf extract, exhibits notable changes, including a high-water content of 88.31%, low ash content of 0.49%, moderate fat content of 0.81%, elevated protein content of 5.35%, and reduced carbohydrate content of 5.02%. These findings underscore the potential of stevia leaf extract as a functional ingredient in pudding formulations and provide insights for the development of healthier dessert options.

Benefits of Stevia (Stevia rebaudiana B.) and Sirih (Piper betle L.) Leaves Combination Againts Dental Care

Oral health is the most important part, to improve its hygiene by maintaining teeth. Cavities are a common problem. Betel leaf as an antibacterial. Stevia leaves as bacteriostatic and bactericidal also eliminate the cause of tooth decay and gingivitis. The purpose of the study was to determine the antibacterial activity of toothpaste combined with betel leaf extract with stevia leaves against the growth of Streptococcus mutans bacteria. The study used an experimental method consisting of making betel leaf and stevia leaf extracts using the maceration method. Preparation of toothpaste with a combination of betel leaf extract with stevia leaves at a concentration of 15% with the ratio of betel leaves and stevia leaves, namely formula 1 (5%: 10%), formula 2 (7.5%: 7.5%), formula 3 (10%: 5%). Testing antibacterial activity by measuring the clear zone using the disc paper method with blood agar media. Data analysis using ANOVA and Paired T test. The results of the study obtained formula (5%: 10%) clear zone of 16.42 mm, formula (7.5%: 7.5%) of 17.24mm, formula (10%: 5%) of 17.94mm, positive control of 18.66mm, negative control of 2.75mm. Conclusion 10:5 combination toothpaste has antibacterial activity.

UJI EFEKTIVITAS ANTIOKSIDAN DARI KOMBINASI EKSTRAK DAUN STEVIA (Stevia rebaudiana (bertoni) bertoni) DAN DAUN TEH HIJAU (Camellia sinensis (L.) Kuntze) DENGAN MENGGUNAKAN METODE DPPH (1,1- Difenil-2-Pikrilhidrazil)

The stevia plant is known to have various benefits in the pharmaceutical field and is used as a health medicine because of its antioxidant, antifungal and non-carcinogenic effects. Green tea is a plant that contains various antioxidant compounds that are beneficial for health. The aim of this research is to determine whether the combination of stevia leaf extract and green tea leaves is effective as an antioxidant. Ethanol extract of stevia leaves and green tea leaves is made by maceration with 96% ethanol solvent, combination extract has blackish green. The ethanol extract of stevia leaves and green tea leaves was screened for phytochemical compounds. Antioxidant effectiveness was tested using the DPPH (1,1 Diphenyl-2-Picrylhydrazyl) method, with varying concentrations of 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm. Then Antioxidant activity test by DPPH measured using a UV-Vis spectrophotometer. The results of screening for phytochemical compounds show that stevia leaf extract contains alkaloids, saponins, flavonoids and tannins. Meanwhile, the results of screening for phytochemical compounds in green tea leaf extract contain alkaloids, steroids, saponins, flavonoids and tannins. The research results showed that antioxidant levels in the combination of Stevia leaf extract and green tea leaves with concentrations of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm with a ratio of (1:1) had an IC50 analysis result of 10 ppm of 19.90 mg/L, 20 ppm is 12.90 mg/L, 30 ppm is 9.60 mg/L. 40 ppm is 6.80 mg/L. 50 ppm is 6.60 mg/L where a concentration of 50 ppm is classified as a very strong antioxidant. The antioxidant effect of the combination of stevia leaf extract and green tea leaves is still higher than vitamin C. The combination of stevia leaves xtract and green tea leaves has antioxidant effectiveness.

Effectivity of Irrigation Solution from Stevia rebaudiana Bertoni Leaf Extract on the Growth of Enterococcus faecalis Bacteries

Disinfection of Enterococcus faecalis bacteria in dental root canal treatment using NaOCL 0.5-2.5% is still a challenge. There is still very little data on the effectvity of Stevia rebaudiana Bertoni leaf extract irrigation solution against the growth of Enterococcus faecalis. This study evaluated the effectiveness of Stevia rebaudiana Bertoni leaf extract irrigation solution against Enterococcus faecalis ATCC 29212 through MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericide Concentration) values. A number of 100 µl of E. faecalis suspension was inoculated into 96 microtitration wells, each containing 100 µl of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.56% of Stevia leaf extract irrigation solutions, positive (NaOCL 2.5%) and negative (distilled water) control. Incubation was carried out for 24 hours at 37°C then optical density was measured with a microplate reader at 600 nm. Five µl of 10-9 dilution of each group was dripped and spread onto nutrients agar plate. The number of Enterococcus faecalis colonies was counting manually. There was no significant difference in average colony count of Enterococcus faecalis between the 100% and 50% Stevia leaf extract irrigation solution groups and the positive control (p>0.05). However, the 25%, 12.5%, 6.25%, 3.125%, and 1.56% Stevia leaf extract irrigation solution groups showed significant differences (p<0.05). Stevia leaf extract irrigation solution suppressed growth at MIC 6.25% by turbidimetry and MIC 1.56% by total plate count method using nutrient agar and than killed Enterococcus faecalis at MBC 50%. The bactericidal ability of 100% and 50% Stevia leaf extract irrigation solution is equal to NaOCL 2.5%.

EFFECT OF DIFFERENT SWEETENERS ON GLYCOGEN CONTENT OF BLOOD LEUKOCYTES AND DIFFERENTIAL LEUKOCYTE COUNTS IN C57BL/6 MICE

We studied the eff ect of stevia leaf extract, saccharinate and cyclamate in various dosages on the glycogen content of blood leukocytes in mice, as a necessary substrate for the realization of phagocytosis. An increase of glycogen content was found in all experimental groups which obtained sweeteners at a dosage of 10 mg/g of body weight. The eff ect of sweeteners on the diff erential leukocyte counts was found only for mice treated with stevia leaf extract at a dosage of 10 mg/g of body weight. They demonstrated increase of neutrophil-to-lymphocyte ratio. New data expand our understanding of the metabolism of sugar substitutes in the organism and their eff ect on physiological systems, in particular the hematopoietic and immune systems.

Effects of stevia (Stevia rebaudiana Bertoni) leaf extracts on the quality and shelf life of refrigerated catla (Gibelion catla) fillets

The effects of stevia leaf extracts (SLE) on the quality and shelf life of catla (Gibelion catla) fillets were assessed during refrigerated storage for 20 days. The fish fillets were treated with aqueous ethanolic (80%) extracts of stevia leaf at various concentrations (1% SLE, 2% SLE, and 3% SLE). The control (distilled water only) and SLE-treated fillets were preserved at 4±1 °C, and chemical (pH, peroxide value (PV), free fatty acid (FFA), thiobarbituric acid reactive substances (TBARS), and total volatile basic-nitrogen (TVB-N)), bacteriological and sensory evaluation was performed periodically for quality assessment. During refrigerated storage, 2% SLE-treated fillets exhibited significantly (p < 0.05) the lowest pH, PV, FFA, TBARS, and TVB-N values compared to other SLE-treated and control fillets. Aerobic plate counts (APC), psychrotrophic bacterial counts, Enterobacteriaceae, lactic acid bacteria, H2S-producing bacteria and Staphylococcus counts of 2% SLE-treated fillets were significantly lower than those of other SLE-treated fillets and control fillets. The maximum APC were 7.22, 7.29, 6.85, and 7.17 log CFU/g for control, 1% SLE, 2% SLE, and 3% SLE-treated fillets at the 12th, 16th, 20th, and 20th days of the storage period, respectively. However, sensory evaluation results revealed that 2% SLE- and 3% SLE-treated fillets had better sensory attributes up to 16th days followed by 1% SLE (12th days) and control (8th days) fillets. Pearson's correlation test and principal component analysis confirmed the relationship among chemical, bacteriological and sensory attributes of refrigerated catla fillets. Based on the chemical, bacteriological, and sensory evaluation results, 2% SLE could be used as a natural additive for catla fillets preservation during refrigerated storage.

PENGARUH PEMBERIAN EKSTRAK DAUN STEVIA DALAM MENJAGA KESTABILAN KADAR GULA DARAH PENDERITA DIABETES

Background. Diabetes Mellitus is an example of a type of disease that is not contagious and attracts a lot of attention. DM sufferers are required to control blood sugar levels by maintaining dietary rules so that blood sugar levels remain controlled. Blood sugar levels will increase drastically after eating foods that contain carbohydrates or sugar in them.Stevia is a natural sweetener substitute for sugar that comes from the stevia plant. Stevia leaves are hypoglycemic which functions to lower blood glucose levels for people with diabetes mellitus.&#x0D; Purpose. This study aims to determine the effect of stevia sugar on the stability of blood sugar in diabetes sufferers.&#x0D; Methods. The method used is a systematic literature review. Data collection is carried out by recording all articles that have similar research in the research report. The articles used in this search are national and international journals taken from Google Scholar, Crossref, Semantic Scholar and Scopus databases in 2019-2023 using the Publish or Perish application.&#x0D; Result. Based on the results of the literature review, it was found that stevia has an effect on reducing blood glucose levels in DM sufferers. Consumption of stevia leaf extract plays an important role in reducing levels of glucose, cholesterol, total fat and other blood characteristics.&#x0D; Conclusion. Based on the results of research that has been carried out, it can be concluded that stevia leaf water extract can maintain and even reduce blood glucose levels in diabetes patients because serum insulin and adiponectin levels increase dramatically.

Effect of Stevia Leaf Extract Substitution on Functional Pudding Characteristics

Extracted stevia leaves can be natural sweetener in pudding. This research aimed to obtain formulation of functional pudding with stevia leaf extract as a substitute for granulated sugar. This research used a simple randomized block design consisting of three replications. The treatments added were the proportion of sugar and stevia leaf extract, which consisted of six treatments, namely 170 g and 0 g, 136 g and 0.17 g, 102 g and 0.34 g, 68 g and 0.51 g, 34 g and 0.68 g, 0 g and 0.85 g. Variables of the tests carried out are rate fiber food, syneresis test, texture test, color test, pH test, and organoleptic test. The best treatment was pudding sugar 0 g and extract stevia leaves 0.85 g with average rate fiber 0.21 %, syneresis test 2.7167 mg min-1, gel strength 50.1 N, intensity color: brightness (L) 31.26, redness (a) 0.33, yellowish (b) 1.13, organoleptic: color 3.50 (fair), aroma 2.5 (no smell), taste 3.47 (enough delicious), texture 3.47 (enough soft), pH 7.41. Differences in the proportion of stevia leaf extract did not have a significant effect on the redness level and organoleptic variables of the pudding aroma but has a real effect on other variables, i.e rate fiber, syneresis, gel strength, brightness level, yellowish level, pH, color, taste, and texture.

Effects of extraction techniques on antioxidant and antibacterial activity of stevia (Stevia rebaudiana Bertoni) leaf extracts

The effects of extraction techniques on the antioxidant and antibacterial activity of stevia (Stevia rebaudiana Bertoni) leaf extracts were assessed. The stevia leaf extracts contained appreciable amounts of total phenolic contents (7.8–21.63 mg GAE/g of dry extract), and total flavonoid contents (31.37–70.41 mg QE/g of dry extract). The EC50 values of DPPH and ABTS radical scavenging activity were 0.41–1.10 and 0.82–2.96 mg/ml, respectively. The ECA0.50 values of cupric reducing antioxidant capacity ranged from 0.01 to 0.21 mg/ml. Extracts obtained by shaker showed comparatively higher phytochemical contents and antioxidant activity than the extracts obtained by soxhlet extraction technique, regardless of the solvent used. An aqueous ethanolic (80 %) extract obtained by shaker exhibited a higher zone of inhibition against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa than other solvent extracts. The phytochemical contents of stevia leaf extracts were correlated with antioxidant activity. Phytochemical constituent profile of 80 % aqueous ethanolic extracts obtained by shaker was assessed by HPLC-ESI-Q-TOF-MS, and several phenolic acids, flavonoids, and steviol glycosides were identified. The results of this study demonstrated that 80 % aqueous ethanolic extracts of stevia leaf obtained by shaker showed significantly the highest antioxidant and antibacterial activity, which could be applied in food preservation and pharmaceutical use.

Antioxidant Activity of Leaf Extracts from Stevia rebaudiana Bertoni Exerts Attenuating Effect on Diseased Experimental Rats: A Systematic Review and Meta-Analysis.

Stevia (Stevia rebaudiana Bertoni) is an aromatic plant known for its high sweetening power ascribed to its glycosides. Stevia also contains several bioactive compounds showing antioxidant, antiproliferative, antimicrobial, and anti-inflammatory activities. Since inflammation and oxidative stress play critical roles in the pathogenesis of many diseases, stevia emerges as a promising natural product that could support human health. In this study we set out to investigate the way stevia affects oxidative stress markers (e.g., SOD, CAT, GPx, GSH, MDA) in diseased rats administered stevia leaf extracts or glycosides. To this end, we performed an inclusive literature search, following PRISMA guidelines, and recruited multivariate meta-analysis and meta-regression to synthesize all available data on experimental animal models encountering (a) healthy, (b) diseased, and (c) stevia-treated diseased rats. From the 184 articles initially retrieved, 24 satisfied the eligibility criteria, containing 104 studies. Our results demonstrate that regardless of the assay employed, stevia leaf extracts restored all oxidative stress markers to a higher extent compared to pure glycosides. Meta-regression analysis revealed that results from SOD, CAT, GSH, and TAC assays are not statistically significantly different (p = 0.184) and can be combined in meta-analysis. Organic extracts from stevia leaves showed more robust antioxidant properties compared to aqueous or hydroalcoholic ones. The restoration of oxidative markers ranged from 65% to 85% and was exhibited in all tested tissues. Rats with diabetes mellitus were found to have the highest restorative response to stevia leaf extract administration. Our results suggest that stevia leaf extract can act protectively against various diseases through its antioxidant properties. However, which of each of the multitude of stevia compounds contribute to this effect, and to what extent, awaits further investigation.

Measurement of Antioxidant Activity Combination of Robusta Coffee (Coffea canephora), Matoa Leaves (Pometia pinnata) and Stevia Leaves (Stevia rebaudiana) with Various Solvent Extractions

Antioxidants are chemical compounds that can donate one or more electrons to free radicals, so that these free radicals can be be suppressed. The human body does not have excess antioxidant reserves, so if there is excessive exposure to radicals, the body needs antioxidants. There is concern about the possibility of unknown side effects from synthetic antioxidants, causing natural antioxidants to become a much-needed alternative. The development of coffee drinks, growing rapidly and increasingly diverse. Some of them like the taste of pure coffee without any additives and many of them choose to add herbal plants, which have antioxidant properties to enhance their coffee drinks and also add natural sweeteners instead of sugar. This study aims to study the antioxidant activity of a combination of robusta coffee extract (Coffea canephora), matoa leaf (Pometia pinnata) and stevia leaf (Stevia rebaudiana), with various solvents, using the DPPH (1,1-Diphenyl-2-pikrihidrazil) method. The results showed that the ethanol extract of robusta coffee had antioxidant activity with an IC50 value of 18.96 ppm, the ethanol extract of matoa leaf had an IC50 value of 5.46 ppm, and the stevia leaf extract of ethyl acetate had an IC50 value of 17.54 ppm. The value of antioxidant activity in the combination of the three in comparison (4:4:2), has an IC50 value of 9.94 ppm. Robusta coffee, matoa leaves and stevia leaves are beverage that have very strong antioxidant activity. The results of the ANOVA test on the extraction various solvents and samples showed a value (p &lt;0.01) indicating that there was a significant difference.

Organoleptic evaluation and chemical tests of various formulations of kencur drink (Kaemfelia galanga L.): efforts to find the best formulation to increase body immunity

Kencur drink is one of the traditional drinks that has been known to be popular and has become an icon for daily healthy drinks. Kencur drink is very useful in preventing and treating diarrhea in Indonesia, can prevent colds and flu. Kencur can also prevent cancer, stop vomiting, prevent and treat fungi, lower cholesterol and improve appetite. Therefore, various ways are done so that this kencur drink remains a healthy daily drink, such as giving a mixture of other ingredients, one of which is the provision of stevia leaf extract. There is also the addition of lemon, brown sugar, and others. Methods in the study with the addition of stevia extract were increased from groups one to six. While other compositions remain such as kencur, lemon and brown sugar. From the results of the study it was obtained that the addition of stevia leaf extract was the highest most preferred and contained vitamin C which was more abundant with other groups. It was concluded that giving stevia leaf extract as much as 24.24% became a favorite drink and had the highest vitamin C content

New aspects on nutraceutical formulations of whey protein concentrate sweetened with steviol glycosides for patients with non-communicable diseases

The current paper aimed to highlight new aspects of nutraceuticals with proven health benefits for patients with non-communicable diseases (NCDs). Four nutraceutical formulations were structured scientifically according to the Indian Standard (IS: 8220/1976) for “Specification for Protein-Rich Concentrated Nutrient Supplementary Foods”. High-quality milk derivatives with considerable protein content (10g protein/serving) rich in essential amino acids with valuable branched chain amino acids (BCAAs) reaching about 18% of total amino acids content and calcium content 325 mg/serving were included in whey protein concentrate (WPC) and skim milk powder (SMP). Zero-calorie artificial sweetener steviol glycosides (SGs) from a natural source (stevia leaf extract) were added with vitamins, minerals, and natural flavors. One of the new aspects is the valuable nutrient-nutrient interaction, as in the synergistic effect between WPC and SGs. Also, the presence of natural flavors (vanilla-cinnamon-coffee-raw cocoa) has great effects with their contents of bioactive components and antioxidants. Valuable applications of these nutraceuticals were discussed here to show their medicinal benefits with regular consumption by patients with NCDs and those with special food needs, such as in malnourished people, pregnant and lactating women, adolescents, and athletes to improve their physical activity, mentality, and overall performance summed up in the term of improving quality of life (QoL). Research design was carried out by applying evidence-based approaches. Well-structured scientific publications were examined thoroughly to collect updated information about using these nutraceuticals as adjuvant therapy in the treatment protocols for patients with NCDs.

Characteristics of Instant Chocolate Drink with the Addition of Natural Sweeteners Sugar Stevia Leaf Extract (Stevia Rebaudiana Botani)

Instant chocolate drink is a type of processed chocolate product that has many devotees, the use of sucrose as a sweetener for flavour in the process of making instant chocolate drinks can have side effects on health, namely it can cause heart attacks, allergies, diarrhoea, increase the risk of bladder cancer, hypertension and migraines. Stevia leaves contain glycosides which have a sweetness level of 250 higher than sucrose. The purpose of this study was to determine the effect of stevia sugar as a natural sweetener in the manufacture of instant chocolate drinks. The method used is a laboratory study using a single factor at 3 levels of treatment, namely the concentration of instant chocolate and stevia sugar used as much as 11g and 7g; 9g and 9g; as well as 7g and 11g. Data analysis used two methods, namely the Chi-square method for organoleptic data and the One Way ANOVA method with a 95% confidence level followed by Duncan's New Multiple Test (DMRT). The results of this study are samples with concentrations of cocoa powder and stevia sugar as much as 11g and 7g are the best treatment by having a colour parameter value of 4.13 (like), taste parameter of 3.80 (somewhat like), aroma parameter of 4.13 (like), the viscosity parameter is 4.33 (like), the water content parameter is 4.14%, the solubility parameter is 90.15%, the pH parameter is 6.4 and the total dissolved solids parameter is 9.03.

Non-Nutritive Sweetener Intake Is Low in Preschool-Aged Children in the Guelph Family Health Pilot Study.

There is limited research on the intake of non-nutritive sweeteners (NNS) among preschool-aged children. Canada’s Food Guide suggests limiting intake of NNS for all population groups and Health Canada recommends that young children (<2 years) avoid consuming beverages containing NNS. The aim of this study was to investigate the frequency and type of non-nutritive sweetener (NNS) intake in preschool-aged children participating in the Guelph Family Health Study pilots. Parents (n = 78 families) completed 3-day food records (n = 112 children; n = 55 females, n = 57 males; 3.6 years ± 1.3). Nineteen children (17%) reported consumption of foods or beverages containing NNS. Food sources with NNS included: freezies, oral nutritional supplements, flavored water, carbonated drinks, sugar free jam and protein powder. The majority of NNS contained in these foods were identified as stevia leaf extract, acesulfame K, sucralose, monk fruit extract and aspartame. Future research should continue to study NNS intake patterns longitudinally in children and examine the association of NNS intake with diet quality and health outcomes.

Safety of the proposed amendment of the specifications for enzymatically produced steviol glycosides (E 960c): Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract.

The EFSA Panel on Food Additives and Flavourings (FAF Panel) provides a scientific opinion on the safety of a proposed amendment of the specifications of enzymatically produced steviol glycosides (E 960c) with respect to the inclusion of rebaudioside D produced via enzyme‐catalysed bioconversion of purified stevia leaf extract. Rebaudioside D (95% on dry basis) is produced via enzymatic bioconversion of purified stevia leaf extract using uridine diphosphate (UDP)‐glucosyltransferase (UGT) and sucrose synthase enzymes produced by the genetically modified yeast K. phaffii UGT‐A, that facilitates the transfer of glucose to purified stevia leaf extract via glycosidic bonds. The same enzymes from K. phaffii UGT‐A may be used in the manufacturing process of the food additive, rebaudioside M produced via enzyme modification of steviol glycosides from stevia (E 960c(i)). The Panel considered that separate specifications would be needed for this food additive produced via the manufacturing process described in the current application, aligned with those already established for E 960c(i). The Panel concluded that there is no toxicological concern for Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract using UDP‐glucosyltransferase and sucrose synthase produced by a genetically modified strain of the yeast K. phaffii. However, based on the available data, the Panel could not exclude the possibility that some residual amount of DNA coding for the kanamycin resistance gene could remain in the final product. Should this gene propagate in microbiota due to the presence of recombinant DNA in the final product, this would be of concern. Therefore, the Panel concluded that the safety of Rebaudioside D produced via this enzymatic bioconversion was not sufficiently demonstrated with the available data given that the absence of recombinant DNA was not shown.

The Potential of Cold Plasma and Electromagnetic Field as Stimulators of Natural Sweeteners Biosynthesis in Stevia rebaudiana Bertoni.

Stevioside (Stev) and rebaudioside A (RebA) are the most abundant steviol glycosides (SGs) responsible for the sweetness of Stevia rabaudiana Bertoni. As compared to Stev, RebA has a higher sweetening potency, better taste and therefore is the most preferred component of the stevia leaf extracts. The aim of this study was to determine the effect of pre-sowing seed treatment with abiotic stressors cold plasma (CP) and electromagnetic field (EMF) on the amount and ratio of RebA and Stev in the leaves of stevia. Additionally, the effect on total phenolic content, flavonoid content and antioxidant activity was investigated. Seeds were treated 5 and 7 min with cold plasma (CP5 and CP7 groups) and 10 min with electromagnetic field (EMF10 group) six days before sowing. The germination tests in vitro demonstrated that all treatments slightly increased germination rate and percentage. HPLC analysis revealed that CP and EMF had strong stimulating effect on SGs accumulation. All treatments increased RebA concentration approximately 1.6-fold; however, the ratio of RebA/Stev decreased from 8.5 in the control to 1.9, 2.5 and 1.1 in CP5, CP7 and EMF10 groups respectively, since the concentration of Stev increased more than RebA, 7.1, 4.6 and 11.0-fold, respectively, compared to control. However, treatments had opposite effect on total phenolic content, flavonoid content, and antioxidant activity. We have demonstrated for the first time that short time pre-sowing treatment of stevia seeds with CP and EMF can be a powerful tool for the enhancement of biosynthesis of RebA and Stev, however it can have negative impact on the content of other secondary metabolites.

Histomorphological Changes in a Rat Model of Polycystic Ovary Syndrome and the Contribution of Stevia Leaf Extract in Modulating the Ovarian Fibrosis, VEGF, and TGF-β Immunoexpressions: Comparison with Metformin.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of fertile females. It has been reported that stevia leaf extract (SLE) has antidiabetic and antihyperlipidemic properties. Therefore, the current study hypothesized and investigated the role and mechanistic aspects of a natural sweetener; SLE in treating a rat model of letrozole-induced PCOS and to compare it with metformin. Thirty-five female Wistar albino rats were divided into 5 groups: control, PCOS-induced group (letrozole, 1 mg/kg/d, for 21 days), SLE, metformin, and combination-treated groups (300 mg/kg/d, for the next 28 days in SLE and metformin-treated groups). Vaginal smears were done. The levels of glucose, lipid, and hormonal profiles were measured in the serum meanwhile, malonyl dialdehyde (MDA), superoxide dismutase (SOD), and tumour necrosis factor (TNF-α) were measured in the ovary. Ovarian sections were subjected to hematoxylin and eosin, Masson, and immunohistochemical identification of VEGF and TGF-β followed by morphometric analysis. PCOS rats showed altered hormonal and lipid profiles, in addition to hyperglycemia. Also, the ovarian tissue levels of MDA and TNF-α were elevated, and SOD was decreased. Numerous cystic follicles, decrease/absence of corpora lutea, interstitial fibrosis with positive VEGF and TGF-β immunoreactivity were evident. SLE improved all altered parameters. SLE showed potential therapeutic merits in letrozole-induced PCOS via anti-inflammatory, antioxidant, anti-fibrotic, and angiogenesis regulating mechanisms. Its effects were almost comparable to metformin, and the combination of both has no further synergistic effect.